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LA1512

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protein - bound lys with its free -amino group is considered the amino acid that is most susceptible to react with other compounds present in ingredients , foods , and feeds during thermal processing . one example is the reaction between amino groups and reducing sugars ( maillard reaction ) , resulting in the formation of maillard reaction products . this reaction renders lys unavailable for protein synthesis and concomitantly reduces the level of bioavailable lys in foods and feeds . analyzing lys using conventional amino acid analysis provides an inaccurate estimate of bioavailable lys , as early maillard reaction products can revert back to lys under the strong acidic conditions used to hydrolyze protein during amino acid analysis . a number of methods have been developed that can determine lys possessing a free -amino group , i.e. reactive lys , by reacting the latter group with a chemical reagent . in 1935 , greenstein reported that the chemical reagent o - methylisourea ( omiu ) was specific for the -amino group of lys in a guanidination reaction , which was corroborated in a number of subsequent studies . the guanidination reaction with omiu results in the conversion of lys to homoarginine , an acid stable amino acid which can be quantified using conventional amino acid analysis , thereby allowing the omiu - reactive lys content to be determined . the guanidination method for determining reactive lys has been shown to accurately predict lys availability in feed ingredients for growing pigs and has been extensively used to determine standardized ileal digestibility of reactive lys for different foods and feeds such as wheat , soybean meal , heated skim milk powder , breakfast cereals , and cat foods . however , in 1967 kimmel stated that the reaction of omiu is specific for the -amino group if the -amino group is blocked , suggesting that omiu might be able to bind to the -amino group of amino acids under certain conditions . evidence for the nonspecificity of the guanidination reaction has been observed in the binding of omiu to the free -amino group of gly and to a lesser extent of met , ser , val , leu , phe , glu , and ala when omiu is used to enhance maldi mass spectra of peptides . in addition , the omiu - reactive lys content in diets containing crystalline l - lys hcl was recently reported to be underestimated when analyzed using the guanidination reaction . the authors hypothesized that omiu had reacted with the free -amino group of crystalline l - lys hcl under the specific conditions of the assay . nonspecificity of omiu for the -amino group of lys may have implications when determining reactive lys if foods , feeds , ingredients , and ileal digesta contain appreciable quantities of free and n - terminal lys . since it has been hypothesized that omiu also binds to the -amino groups of amino acids in addition to the -amino group of lys , the current study investigated the specificity of omiu for the -amino group of crystalline l - lys and the binding of omiu to -amino groups of selected crystalline amino acids . reaction conditions ( omiu to lys ratio , reaction time , and ph of the omiu solution ) for the specificity of omiu to react with the -amino group of crystalline l - lys were investigated . practical implications of the results are assessed by examining the free lys content of several food / feed ingredients and ileal digesta . the current study focused on ingredients used in feeds , but implications also account for food ingredients . barium hydroxide octahydrate , crystalline l - lys , l - arg , l - phe , l - val , l - ile , l - thr , and gly were obtained from sigma - aldrich ( castle hill , australia ) and crystalline l - lys hcl ( 78% l - lys ) from bdh laboratory supplies ( poole , england ) . the omiu sulfate salt was obtained from sigma - aldrich ( st . louis , mo ) . free lys was determined after extraction with 0.1 m hcl and precipitation of coextracted nitrogenous macromolecules by sulfosalicylic acid followed by centrifugation , separation and detection using ion - exchange chromatography employing postcolumn ninhydrin or o - phthalaldehyde derivatization . total lys was determined after acid hydrolysis in 6 m hcl for 24 h at 110 c followed by separation and detection using ion - exchange chromatography employing postcolumn ninhydrin or o - phthalaldehyde derivatization . reactive lys was determined as being equivalent to the molar amount of homoarginine quantified after incubation of the sample with omiu followed by acid hydrolysis with 6 m hcl for 24 h at 110 c . a 0.6 m omiu solution was prepared according to the procedure described by moughan and rutherfurd except that 6 g of barium hydroxide octahydrate , instead of 4 g , was added to approximately 16 ml of boiled distilled deionized water , which had been boiled for at least 10 min to remove carbon dioxide , in a centrifuge tube . the solution was cooled for 30 min at room temperature before being centrifuged at 6,400 g for 10 min . the supernatant was retained and the precipitate was washed with approximately 2 ml of boiled distilled deionized water and centrifuged again . both supernatants were combined and the ph was determined to ensure it was above 12 . thereafter , the ph was adjusted to 10.6 by adding 6 m hcl and made up to 20 ml with boiled distilled deionized water . the binding of omiu to amino groups of seven crystalline amino acids was investigated . lysine , arg , and phe were selected because these amino acids have been reported to have the highest browning activity , i.e. most likely to react with sugars during processing . valine , ile , and thr were selected because these amino acids are acid stable and frequently added in crystalline form to pig diets . glycine was selected because it was previously reported to react with omiu via its -amino group . the following guanidination procedure was used in the present study since it was reported to be optimal for diets and ileal digesta and used by hulshof et al . : each amino acid ( 0.0006 mol ) was separately incubated in 0.6 m omiu ( omiu to amino acid ratio of 1000:1 ) at 25 2 c in a shaking water bath for 7 days . the samples were reduced to dryness under vacuum ( savant speedvac concentrator sc250exp , savant instruments inc . farmingdale , ny ) , subsequently dissolved in citric acid buffer ( ph 2.2 ) , and analyzed in duplicate using a cation - exchange hplc system ( shimadzu corp . , kyoto , japan ) employing postcolumn o - phthalaldehyde derivatization . each unreacted amino acid ( 0.0006 mol ) was also analyzed in duplicate to determine the amino acid content in non - omiu incubated samples . separate solutions of crystalline l - lys and crystalline l - tyr ( 0.0006 m ) were incubated with omiu for 3 days at room temperature in a shaker using an omiu to amino acid ratio of either 10:1 , 100:1 or 1000:1 . tyrosine was chosen as a model amino acid , in addition to lys , because of its relatively high mw ( 181.19 g / mol ) and low polarity , which both favor rp - lc detectability . samples were analyzed by an acquity ultrahigh - performance liquid chromatography ( uplc ) system ( waters , milford , ma ) using an acquity uplc beh c18 column ( 2.1 150 mm , 1.7 m particle size ) with an acquity beh c18 vanguard precolumn ( 2.1 50 mm , 1.7 m particle size ) . eluent a was 1% ( v / v ) acetonitrile containing 0.1% ( v / v ) trifluoroacetic acid in millipore water and eluent b was 100% acetonitrile containing 0.1% ( v / v ) trifluoroacetic acid . samples ( 1 l ) were injected into the column maintained at 40 c . the analysis was conducted using the following elution profile : for omiu incubated crystalline l - lys , isocratic elution with 99.9% eluent a and 0.1% eluent b ; for omiu incubated crystalline l - tyr , 0 to 2 min isocratic 99.9% eluent a , from 2 to 15 min linear gradient from 99.9% to 50% eluent a , from 15 to 20 min linear gradient from 50% eluent a to 99.9% eluent b , from 20 to 25 min isocratic at 99.9% eluent b , then re - equilibration to the initial conditions . the photodiode array detector was operated at a sampling rate of 40 points / s in the range 200400 nm , resolution 1.2 nm . the synapt g2si mass spectrometer was operated in positive ion mode , capillary voltage 3 kv , sampling cone 30 v , source temperature 150 c , desolvation temperature 500 c , cone gas flow ( n2 ) 200 l / h , desolvation gas flow ( n2 ) 800 l / h , acquisition in the full scan mode , scan time 0.3 s , acquisition range 1502000 m / z . the ms data were processed using the software masslynx v 4.1 ( waters , milford , ma ) . the influence of omiu to lys ratio and reaction time on the guanidination of crystalline l - lys was assessed using a 4 3 factorial arrangement with four omiu to lys ratios and three reaction times . the omiu to lys ratios were 1.5:1 ( optimal to convert crystalline l - lys to homoarginine ) , 10:1 ( reported to be optimal for casein ) , 100:1 , and 1000:1 . the reaction times were 1 , 3 , and 7 days with the remaining reaction conditions as described above . the influence of ph of the omiu solution and omiu to lys ratio on guanidination of crystalline l - lys was assessed using a 7 2 factorial arrangement with seven ph levels and two omiu to lys ratios . the ph values ranged from 8.6 to 11.0 with 0.4 increments , with ph 9.0 and 10.6 being the pka values for the - and -amino groups of lys , respectively . a reaction time of 3 days was used with the remaining reaction conditions as described above . l - lysine hcl ( 78% l - lys ) , i.e. a form of crystalline l - lys that is supplemented to diets , and an equimolar mixture of the other six selected crystalline amino acids ( i.e. , arg , phe , val , ile , thr , and gly ) were analyzed using an omiu to amino acid ratio of 10:1 and 1000:1 , an omiu ph of 10.6 and a reaction time of 3 days . unreacted and omiu - incubated solutions of crystalline l - lys hcl and the mixture of six crystalline amino acids were analyzed in duplicate using the hplc system as described above . data on the free lys as percentage of total lys for 44 different food / feed ingredients were obtained from ajinomoto eurolysine s.a.s . the free and total lys contents were determined by ajinomoto eurolysine s.a.s . using the procedures described above . samples of ileal digesta were selected based on the protein source present in the experimental diets and the method used to collect the digesta during animal trials with growing pigs or broilers previously conducted at the riddet institute ( palmerston north , new zealand ) and animal nutrition group of wageningen university ( wageningen , the netherlands ) . with regard to the growing pig trials diets contained soybean meal or rapeseed meal as the sole protein source and were each fed to seven ( steered ileo - cecal valve ) cannulated growing pigs ( n = 14 ) . crystalline l - lys hcl was added to the rapeseed meal diet . in the second experiment ( h. chen , wageningen ur livestock research , personal communication ) , a protein - free diet was fed consisting of corn starch , dextrose , arbocel ( fiber source from j. rettenmaier & shne group , rosenberg , germany ) , soy oil and vitamins / minerals / marker . in the same study , soybean meal or rapeseed meal was added as the sole protein source to the experimental diets at the expense of corn starch . each diet was fed to three growing pigs and ileal digesta was collected at slaughter ( n = 9 ) . in the third experiment ( s. m. rutherfurd , riddet institute , personal communication ) , a protein - free diet ( corn starch , sugar , cellulose , soybean oil , vitamins / minerals / marker ) was fed to growing pigs and ileal digesta collected at slaughter . the ileal digesta of four pigs was pooled based on freeze - dry matter content ( n = 1 ) . in the fourth experiment ( s. m. rutherfurd , riddet institute , personal communication ) , a protein - free diet ( wheat starch , sucrose , cellulose , soybean oil and vitamins / minerals / marker ) or a 15% gelatin - based diet was fed to growing pigs and ileal digesta was collected at slaughter . one pooled ileal digesta sample was obtained for the protein - free diet by combining samples of two pigs based on the freeze - dry matter content ( n = 1 ) . two pooled ileal digesta samples were obtained for the gelatin diet by combining samples of two and four pigs based on the freeze - dry matter content ( n = 2 ) . in the fifth experiment ( s. m. rutherfurd , riddet institute , personal communication ) , diets contained one of two whey protein concentrates or a whey protein isolate as the sole protein source and were fed to growing pigs . the ileal digesta of three , five and four pigs for the two whey protein concentrate diets and the whey protein isolate diet , respectively , were pooled based on the freeze - dry matter content ( n = 2 for whey protein concentrate and n = 1 for whey protein isolate ) . samples from broilers were obtained from two experiments . in the first experiment , maize ( 30% ) and rapeseed meal ( 35% ) were the main protein - containing ingredients in the experimental diet and ileal digesta samples were collected at slaughter . the ileal digesta from six cages containing 11 broilers were pooled based on freeze - dry matter content ( n = 2 by pooling samples per three cages ) . in the second experiment , wheat ( 65% ) and soybean meal ( 28% ) were the main protein - containing ingredients in two experimental diets and ileal digesta samples were collected at slaughter . the ileal digesta of six cages per experimental diet containing eight broilers were pooled based on the freeze - dry matter content ( n = 4 by pooling samples per four and two cages per experimental diet ) . the samples were analyzed for free lys and total lys content using the methods described above . the contribution of endogenous or dietary free lys to the total free lys content in ileal digesta was determined by comparing ileal digesta from growing pigs fed protein - free or protein - containing diets . the free lys content in ileal digesta collected at slaughter from growing pigs or broilers fed protein - containing diets was also compared . the recovery of amino acids after omiu incubation was calculated using eq 1:1for crystalline l - lys , the difference between lys in the non - omiu incubated sample ( 100% recovery ) and the sum of the recovery of unreacted lys ( i.e. , lys having a free - and -amino group ) , and homoarginine ( i.e. , lys with omiu bound to the -amino group ) , was attributed to lys with a cn2h3 ( i.e. , omiu ) bound to the - and -amino group ( i.e. , lys that could not be recovered by hplc analysis ) . for the other crystalline amino acids , the difference between the recovery of the amino acid in the non - omiu incubated sample ( 100% recovery ) and the recovery of the amino acid in the omiu incubated sample was attributed to the amino acid with a cn2h3 ( i.e. , omiu ) bound to the -amino group . the free lys contents in ileal digesta collected via a cannula or at slaughter of growing pigs fed soybean meal or rapeseed meal were plotted against the apparent ileal digestible crude protein ( cp ) content of the diets . correlations between the free and total lys content in ileal digesta of growing pigs fed protein - containing diets and between the apparent ileal digestible cp content and the free lys as percentage of total lys were statistically analyzed using the proc corr procedure in sas 9.3 ( sas inst . as expected , the recovery of unreacted lys ( i.e. , having free - and -amino groups ) when crystalline l - lys was incubated with omiu was low . however , the recovery of homoarginine was low as well , resulting in a significant amount of lys ( i.e. , 96% ) being unaccounted for after guanidination ( figure 1 ) . the latter was also observed for the other six amino acids ( figure 1 ) . the unrecovered amino acids after incubation with omiu were likely to have reacted with omiu via their -amino groups and the subsequent inability of the compound to be retained on the ion - exchange column or to be derivatized by o - phthalaldehyde after chromatographic separation . the difference between amino acids in terms of recovery of the unreacted amino acid suggests that there may be a different reaction equilibrium for each amino acid , possibly related to differences between side - chains ( i.e. , charged vs uncharged and polar vs nonpolar ) and the pka of -amino groups of the different amino acids . binding of omiu both to the - and -amino groups of crystalline l - lys and to the -amino group of crystalline l - tyr was confirmed by ms . after incubation of crystalline l - lys with omiu , protonated lys ( 1.38 min , m / z 147.11 ) , protonated monoderivatized lys / homoarginine ( 1.8 min , m / z 189.13 ) , and protonated double derivatized lys ( 2.9 min , m / z 231.16 ) were identified ( figure 2a ) . furthermore , the ratio of these three compounds was dependent on the omiu to lys ratio used for incubation . the m / z values for the peaks at 1.79 min ( figure 2b ) and 2.94 min ( figure 2c ) are consistent with those of nonprotonated homoarginine ( 188.23 g / mol ) and nonprotonated double derivatized lys ( 230.27 g / mol ) . in addition to the m / z value for the intact molecules , several m / z values corresponding to fragment ions were also visible , such as m / z 172.11 ( protonated homoarginine without nh3 ) and m / z 213.14 ( protonated double derivatized lys without 2 h and 1 o ) . after incubation of crystalline l - tyr with omiu , protonated tyr ( 5.5 min , m / z 182.08 ) and protonated monoderivatized tyr ( 6.7 min , m / z 224.10 ) were identified ( figure 2d ) . as was the case with lys , the ratio of these two compounds was dependent on the omiu to tyr ratio . the m / z for the peak at 6.70 min ( figure 2e ) is consistent with those of nonprotonated monoderivatized tyr ( 223.22 g / mol ) . in this case , omiu reacted only with the -amino group of tyr because there is no binding site on the aromatic ring . in peptides , omiu has been reported to bind to the -amino group of gly and partially to the -amino group of met , ser , val , leu , phe , glu , and ala when reaction time was extended to several hours . however , since maldi ms was used , only qualitative results were provided and the extent of the binding of omiu to -amino groups could not be determined . the latter along with the fact that different reactions times were used between the study of beardsley and reilly ( 5 or 10 min ) and the study reported here ( 3 or 7 days ) make comparison of results difficult . nonetheless , under the reaction conditions that were employed in the present study , omiu was found to bind extensively to the -amino group of several crystalline amino acids . moreover , the omiu to amino acid ratio used during incubation appeared to have a major influence on the specificity of omiu for the -amino group of lys . the effects of omiu to amino acid ratio , ph of the omiu solution , and reaction time were subsequently studied to investigate the specificity of omiu to react with the -amino group of crystalline l - lys . recovery of crystalline amino acids after the guanidination reaction using an o - methylisourea ( omiu ) to crystalline amino acid ratio of 1000:1 , ph of the omiu solution of 10.6 , and a reaction time of 7 days . black bars indicate unreacted amino acids , white bar indicates homoarginine , and gray bars indicate nonrecovered amino acids . lc - ms results of o - methylisourea ( omiu ) incubated samples : crystalline l - lys ( a ) and crystalline l - tyr ( d ) incubated at an omiu to crystalline l - lys or crystalline l - tyr ratio of 10:1 , 100:1 , and 1000:1 , and ms spectra of the lc peaks at 1.79 min ( b ) and 2.94 min for crystalline l - lys ( c ) and at 6.70 min for crystalline l - tyr ( e ) . regardless of the omiu to lys ratio , reaction time had little effect on the recovery of unreacted lys and homoarginine ( figure 3 ) and on the quantity of nonrecovered lys ( considered to be double derivatized lys ) . the recovery of unreacted lys and homoarginine decreased from 9 to 1% and from 51 to 1% , respectively , when the omiu to lys ratio increased from 1.5:1 to 1000:1 . these results were consistent with the findings obtained using ms analysis ( figure 2a ) . the impact of the reaction mixture ph on the binding of omiu to the - and -amino group of crystalline l - lys was also examined ( figure 4 ) . when the omiu to lys ratio was 10:1 , the recovery of homoarginine increased from 13 to 75% and the recovery of unreacted lys decreased from 79 to 9% as ph increased from 8.6 to 11.0 . when the omiu to lys ratio was 1000:1 , the recovery of unreacted lys was highest at ph 8.6 and close to 0% for the other ph values while the recovery of homoarginine was highest for ph values between 8.6 and 9.4 and low for ph values between 9.8 and 11.0 . overall , none of the tested combinations of omiu to lys ratio , reaction time , and omiu ph resulted in the complete recovery of crystalline l - lys as homoarginine . moreover , in all cases , between 4 and 99% of the crystalline l - lys was not recovered either as unreacted lys or as homoarginine after incubation with omiu , suggesting that omiu had bound to the -amino group of lys to differing extents . increasing the amount of omiu appeared to drive the equilibrium of the chemical reaction toward double derivatization of the crystalline l - lys . typically , a higher omiu to lys ratio is preferred for the guanidination of protein - bound lys present in food / feed ingredients and diets , having only a free -amino group , in order to completely convert protein - bound lys to homoarginine . however , if lys with a free -amino group , i.e. crystalline l - lys , free lys , or n - terminal lys , is present in those protein sources or diets , then as the omiu to lys ratio is increased , the double derivatization of this lys also appears to increase . lowering the omiu to lys ratio to 1.5:1 , however , resulted in a 51% recovery of homoarginine , indicating that , even at low omiu to lys ratios , it is still possible for omiu to bind to the -amino group of lys . these results appear to be in contrast to those of zhang et al . , who reported a conversion of lys to homoarginine of 99.5% for an omiu to lys ratio of 1.5:1 . conversion of lys to homoarginine in the latter study , however , was calculated as the molar amount of homoarginine divided by the sum of the molar amounts of homoarginine and unreacted lys . this manner of expressing conversions is often used but does not take into account the conversion of lys to double derivatized lys or other lys derivatives . when applying this equation to the data of the current study , conversions of > 90% were found ( data not shown ) . this is in contrast with the low recovery of homoarginine that was actually observed in the present study . thus , the conversion of lys to homoarginine can appear to be high while actually a large proportion of lys is in the double derivatized form . the low recovery of homoarginine could result in an underestimation of the reactive lys content and subsequently an overestimation of lys damage . when considering protein - bound lys to be fully converted to homoarginine , the underestimation of the reactive lys content in food / feed ingredients and diets depends on the amount of lys with a free -amino group ( free + n - terminal lys ) . recovery of crystalline l - lys after the guanidination reaction using four o - methylisourea ( omiu ) to free lys ratios ( 1.5:1 , 10:1 , 100:1 , or 1000:1 ) , three reaction times ( 1 , 3 , or 7 days ) , and a ph of the omiu solution of 10.6 . black bars indicate unreacted lys , white bars indicate homoarginine , and gray bars indicate nonrecovered lys . recovery of crystalline l - lys after the guanidination reaction using two o - methylisourea ( omiu ) to free lys ratios ( 10:1 or 1000:1 ) , seven ph values ( 8.611.0 with 0.4 increments ) of the omiu solution , and a reaction time of 3 days . black bars indicate unreacted lys , white bars indicate homoarginine , and gray bars indicate nonrecovered lys . this reaction depends on the amino group being deprotonated ( i.e. , ph > pka ) for the reaction with omiu to occur . the pka of the -amino group of lys is 10.6 and the recovery of homoarginine should be highest when the ph of the omiu solution is greater than 10.6 . the latter was also found in the current study ( homoarginine recovery of 75% ; figure 4 ) . the pka of the -amino group of lys is 9.0 , and the recovery of unreacted lys ( i.e. , no binding of omiu to either amino group ) should be highest when the ph of the omiu solution is smaller than 9.0 . again , this was found in the current study ( average unreacted lys recovery of 70% ; figure 4 ) . the results are , however , not conclusive with regard to the ph of the omiu solution , since homoarginine is also recovered at ph values smaller than 10.6 . the effect of the ph of the omiu solution was clearly seen for an omiu to lys ratio of 10:1 . for an omiu to lys ratio of 1000:1 , the omiu ph of 8.6 resulted in a high recovery of unreacted lys ( 53% ) and an omiu ph of 9.0 in a high recovery of homoarginine ( 61% ) . the excess of omiu for an omiu ph greater than 9.0 apparently drove the reaction toward both amino groups , irrespective of protonation / deprotonation . several authors have reported different optimal ph values of the omiu solution for different protein sources . the optimal ph for free lys is approximately 10.6 ( figure 4 ) , but none of the ph values resulted in a 100% recovery of homoarginine . to confirm the specificity of omiu for the -amino group of crystalline l - lys , the homoarginine content after incubation with omiu should be equal to the level of lys added to the reaction mixture ( i.e. , complete recovery of lys as homoarginine ) . unfortunately , none of the combinations of reaction time with omiu to lys ratio and ph of the omiu solution with omiu to lys ratio used in the present study resulted in specific binding of omiu to the -amino group of crystalline l - lys . the best reaction conditions ( i.e. , maximal conversion of crystalline l - lys to homoarginine and minimal conversion of crystalline l - lys to double derivatized lys ) were reaction at ph 10.6 for 3 days with an omiu to lys ratio of 10:1 , which resulted in a homoarginine recovery of 75% . it seems unlikely that the guanidination reaction for free and n - terminal lys can be optimized to obtain complete conversion of lys to homoarginine . moreover , it is also unlikely that both protein - bound and free + n - terminal lys can be measured using one set of reaction conditions . the optimized guanidination conditions ( ph 10.6 , omiu to amino acid ratio of 10:1 and reaction time of 3 days ) were applied to crystalline l - lys hcl and a mixture of six amino acids ( arg , phe , val , ile , thr , and gly ) in order to test the reactivity of the -amino groups of crystalline l - lys hcl ( a commercially available form of crystalline l - lys often used as a supplement for pig and poultry diets ) and six other amino acids under these guanidination conditions . an omiu to amino acid ratio of 1000:1 was used , since these conditions have been used previously to determine reactive lys in food / feed ingredients . incubating crystalline l - lys hcl with omiu resulted in a homoarginine recovery of 19.5 and 1.1% whereas the nonrecoverable lys was 79 and 98% , respectively , when the omiu to lys ratio was either 10:1 or 1000:1 , respectively . the recovery of the other six other amino acids ( arg , phe , val , ile , thr , and gly ) was also low ( < 26 and < 38% for an omiu to amino acid ratio of 10:1 and 1000:1 , respectively ) when incubated with omiu as described above . again , these results suggest that omiu can bind to the free -amino groups not only of crystalline l - lys but also of crystalline l - lys hcl and the free -amino groups of crystalline amino acids other than lys , irrespective of the reaction conditions used . the results described above clearly demonstrate that omiu can react with -amino groups of amino acids in addition to the -amino group of lys . furthermore , none of the reaction conditions used in the present study resulted in the complete guanidination of the -amino group of lys without guanidination of the -amino group . thus , it is unlikely that guanidination conditions can be optimized in the future to achieve specificity for lys with a free -amino group ( free + n - terminal lys ) . previously , authors have reported the recovery of all amino acids after guanidination to approximate 100% for lysozyme , soy protein isolate , skim milk powder , lactic casein , whey protein concentrate , soy protein concentrate , blood meal , and cottonseed meal . this suggests that the level of free + n - terminal lys in these ingredients is low . the free lys content in 44 different food / feed ingredients was compiled and found to range from 0 to 5.8% of total lys , with an average of 1.3% ( table 1 ) . consequently , the underestimation of the omiu - reactive lys content for these food / feed ingredients is expected to be low . the estimates of the omiu - reactive lys content are expected to be inaccurate only in those cases where the test material contains a large proportion of free + n - terminal lys . materials for which omiu - reactive lys estimates could be inaccurate are materials that contain crystalline l - lys ( e.g. , practical pig and poultry diets , enteral nutrition formula , and specific pet foods ) , hydrolyzed products ( e.g. , hydrolyzed feather meal , hydrolyzed vegetable protein , infant formula , hypoallergenic diets ) , and potentially digesta obtained from the small intestine . determined after acid hydrolysis in 6 m hcl at 110 c for 24 h. ddgs = distillers dried grain with solubles . in order to determine the potential error involved in the measurement of reactive lys in ileal digesta samples , 23 nonpooled and seven pooled ileal digesta samples from growing pigs and six pooled ileal digesta samples from broilers the free lys as a percentage of total lys for two samples from growing pigs ( one from a protein - free diet and the other from a soybean meal diet ) were considered outliers ( < mean 2 sd ) and were , therefore , excluded from the data analysis . the mean ( sd ) free and total lys contents across the remaining 34 ileal digesta samples from growing pigs and broilers fed protein - free and protein - containing diets were 0.74 ( 0.39 ) and 5.74 ( 2.49 ) g / kg as - is , respectively . the free lys , therefore , was on average 12.8% of the total lys present in the ileal digesta . this amount was unexpectedly high considering that trypsin cleaves at the carboxyl terminal of lys . multiple carrier transport systems are involved in the absorption of different amino acids , and absorption rates differ between amino acids . for example , thr and branched - chain amino acids ( leu , ile , and val ) are rapidly absorbed while lys and arg are more slowly absorbed . moreover , peptides use a different carrier transport system from that used by amino acids and are absorbed more rapidly than free amino acids . a slow absorption rate of lys and a preference for the absorption of peptides might explain the relatively large amount of free lys present in the ileal digesta . of the lys in ileal digesta collected from growing pigs fed a protein - free diet or a protein - containing diet , 13.0 or 12.7% was free lys ( figure 5a ) . reported that approximately 20% of proteinaceous material in the soluble fraction of ileal digesta of growing pigs fed a protein - free diet had a molecular weight less than 1000 da ( considered to consist of free amino acids and small peptides ) while for a corn - soybean meal diet the equivalent value was approximately 13% . reported that endogenous proteins are absorbed at a slower rate compared to dietary proteins , resulting in an increased concentration of endogenous proteins at the end of the ileum . the data of the current study , however , indicate that the presence of free lys in ileal digesta is not related to the presence of protein - containing ingredients in the diet . moreover , the free lys as a percentage of total lys in ileal digesta of growing pigs fed sbm or rsm diets was independent ( r = 0.01 , p = 0.71 ) of the apparent ileal digestible cp content in the diet and of collection method ( figure 5b ) . there appeared to be no difference in the free lys as a percentage of total lys between ileal digesta samples collected from growing pigs or broilers ( 12.7 and 14.4% , respectively ; figure 5a ) , suggesting that the free lys content in ileal digesta is not species specific . the amount of free lys at the terminal ileum of growing pigs and broilers fed protein - containing diets , in the current study , was much higher than the 3.1% reported by moughan and schuttert . this may be due to the relatively slower absorption of free lys compared with peptides or spontaneous nonenzymatic breakdown of peptides due to their instability after hydrolysis . the thawing of fresh samples for subsampling might also have affected the level of free lys in ileal digesta , but this effect is expected to be low . separating pig ileal digesta by centrifugation ( 14,500 relative centrifugal force for 30 min at 4 c ) resulted in the separation of porcine and microbial cells ( precipitate ) from soluble proteins , peptides , free amino acids , and mucins ( supernatant ) . approximately half of the protein present in the supernatant was of microbial origin . while the microbial cells are most likely to be present in the precipitate , the supernatant might contain free lys originating from lysed microbial cells . this source of free lys might also have added to the free lys content in pig ileal digesta analyzed in the current study . there was a linear relation between the total and free lys contents in ileal digesta of growing pigs fed protein - containing diets ( r = 0.54 , p < 0.001 ; figure 5c ) . therefore , the methodology of determining free amino acids which involves the use of 0.1 m hcl and coextraction of nitrogenous macromolecules by sulfosalicylic acid may have hydrolyzed lys from peptides or proteins , thereby , overestimating the free lys content relative to that present in digesta at the terminal ileum . the latter may explain the lower value reported by moughan and schuttert , as these authors used a different methodology to determine free amino acids in ileal digesta of pigs fed protein - free diets . free lys as percentage of total lys in ileal digesta samples from growing pigs and broilers fed protein - free or protein - containing diets ( a ; means are indicated by diamonds ) , the apparent ileal digestible crude protein content of the diet ( g / kg as - fed ) in relation to the free lys as percentage of total lys in ileal digesta samples collected using an ileal cannula ( n = 13 ; open squares ) or at slaughter ( n = 6 ; closed diamonds ) from growing pigs ( b ) and the free lys content in relation to the total lys content in ileal digesta of growing pigs fed protein - containing diets ( c ) . the impact of the nonspecificity of omiu and the free lys content in ileal digesta on the standardized ileal digestibility of omiu - reactive lys was assessed using samples from a previous study . the standardized ileal digestibility was calculated considering supplemented dietary crystalline l - lys hcl to be completely absorbed from the small intestine before the terminal ileum in growing pigs . moreover , it was assumed that all free lys in ileal digesta was double derivatized and , therefore , not determined as omiu - reactive lys . for the soybean meal and rapeseed meal ingredients examined , the determined standardized ileal omiu - reactive lys digestibilities were 92.8 and 83.5% , respectively , and the standardized ileal digestible omiu - reactive lys content was 5.6 and 4.2 g/100 g cp , respectively . the equivalent recalculated values for soybean meal and rapeseed meal considering all free lys to be reactive but not analyzed by the guanidination reaction were 91.5 and 80.1% , respectively , and 5.5 and 4.0 g/100 g cp , respectively . overall , the difference in standardized ileal - reactive lys digestibility where the nonspecific guanidination of free lys was taken into account was small . the overestimation will be greater if ileal digesta contains a significant amount of peptides containing a n - terminal lys residue . in conclusion , omiu was found to be not specific for the -amino group of crystalline l - lys ( hcl ) and able to bind to the -amino groups of crystalline amino acids under the reaction conditions of the assay as developed by moughan and rutherfurd . the various guanidination conditions of the omiu - reactive lysine assay investigated did not result in absolute specificity for the -amino group of lys . it is recommended to analyze the reactive lys content of food / feed ingredients , diets and ileal digesta using an omiu ph of 10.6 , an omiu to lys ratio of 1000:1 , and a reaction time of at least 3 days to fully convert protein - bound lys to homoarginine . these samples should subsequently be analyzed for their free lys content to calculate the reactive lys content of the samples ( i.e. , assuming free lys to be 100% reactive ) . the accurate quantification of free and n - terminal amino acids in ileal digesta warrants further investigation as well as the search for a reagent which is specific for the -amino group of lys .

the specificity of o - methylisourea ( omiu ) to bind to the -amino group of lys , an important supposition for the omiu - reactive lys analysis of foods , feeds , ingredients , and digesta , was investigated . crystalline l - lys incubated under standard conditions with omiu resulted in low homoarginine recoveries . the reaction of omiu with the -amino group of lys was confirmed by ms analysis , with double derivatized lys being identified . none of the changes in reaction conditions ( omiu ph , omiu to lys ratio , and reaction time ) with crystalline l - lys resulted in 100% recovery of homoarginine . the average free lys content in ileal digesta of growing pigs and broilers was found to be 13% of total lys , which could result in a significant underestimation of the reactive lys content . the reaction of omiu with -amino groups may necessitate analysis of free lys to accurately quantify reactive lysine in samples containing a large proportion of lys with a free -amino group .

Introduction Materials and Methods Results and Discussion

in 1935 , greenstein reported that the chemical reagent o - methylisourea ( omiu ) was specific for the -amino group of lys in a guanidination reaction , which was corroborated in a number of subsequent studies . the guanidination reaction with omiu results in the conversion of lys to homoarginine , an acid stable amino acid which can be quantified using conventional amino acid analysis , thereby allowing the omiu - reactive lys content to be determined . however , in 1967 kimmel stated that the reaction of omiu is specific for the -amino group if the -amino group is blocked , suggesting that omiu might be able to bind to the -amino group of amino acids under certain conditions . evidence for the nonspecificity of the guanidination reaction has been observed in the binding of omiu to the free -amino group of gly and to a lesser extent of met , ser , val , leu , phe , glu , and ala when omiu is used to enhance maldi mass spectra of peptides . in addition , the omiu - reactive lys content in diets containing crystalline l - lys hcl was recently reported to be underestimated when analyzed using the guanidination reaction . the authors hypothesized that omiu had reacted with the free -amino group of crystalline l - lys hcl under the specific conditions of the assay . nonspecificity of omiu for the -amino group of lys may have implications when determining reactive lys if foods , feeds , ingredients , and ileal digesta contain appreciable quantities of free and n - terminal lys . since it has been hypothesized that omiu also binds to the -amino groups of amino acids in addition to the -amino group of lys , the current study investigated the specificity of omiu for the -amino group of crystalline l - lys and the binding of omiu to -amino groups of selected crystalline amino acids . reaction conditions ( omiu to lys ratio , reaction time , and ph of the omiu solution ) for the specificity of omiu to react with the -amino group of crystalline l - lys were investigated . reactive lys was determined as being equivalent to the molar amount of homoarginine quantified after incubation of the sample with omiu followed by acid hydrolysis with 6 m hcl for 24 h at 110 c . the influence of omiu to lys ratio and reaction time on the guanidination of crystalline l - lys was assessed using a 4 3 factorial arrangement with four omiu to lys ratios and three reaction times . the omiu to lys ratios were 1.5:1 ( optimal to convert crystalline l - lys to homoarginine ) , 10:1 ( reported to be optimal for casein ) , 100:1 , and 1000:1 . the influence of ph of the omiu solution and omiu to lys ratio on guanidination of crystalline l - lys was assessed using a 7 2 factorial arrangement with seven ph levels and two omiu to lys ratios . the contribution of endogenous or dietary free lys to the total free lys content in ileal digesta was determined by comparing ileal digesta from growing pigs fed protein - free or protein - containing diets . the recovery of amino acids after omiu incubation was calculated using eq 1:1for crystalline l - lys , the difference between lys in the non - omiu incubated sample ( 100% recovery ) and the sum of the recovery of unreacted lys ( i.e. , lys with omiu bound to the -amino group ) , was attributed to lys with a cn2h3 ( i.e. for the other crystalline amino acids , the difference between the recovery of the amino acid in the non - omiu incubated sample ( 100% recovery ) and the recovery of the amino acid in the omiu incubated sample was attributed to the amino acid with a cn2h3 ( i.e. correlations between the free and total lys content in ileal digesta of growing pigs fed protein - containing diets and between the apparent ileal digestible cp content and the free lys as percentage of total lys were statistically analyzed using the proc corr procedure in sas 9.3 ( sas inst . binding of omiu both to the - and -amino groups of crystalline l - lys and to the -amino group of crystalline l - tyr was confirmed by ms . after incubation of crystalline l - lys with omiu , protonated lys ( 1.38 min , m / z 147.11 ) , protonated monoderivatized lys / homoarginine ( 1.8 min , m / z 189.13 ) , and protonated double derivatized lys ( 2.9 min , m / z 231.16 ) were identified ( figure 2a ) . in peptides , omiu has been reported to bind to the -amino group of gly and partially to the -amino group of met , ser , val , leu , phe , glu , and ala when reaction time was extended to several hours . nonetheless , under the reaction conditions that were employed in the present study , omiu was found to bind extensively to the -amino group of several crystalline amino acids . moreover , the omiu to amino acid ratio used during incubation appeared to have a major influence on the specificity of omiu for the -amino group of lys . the effects of omiu to amino acid ratio , ph of the omiu solution , and reaction time were subsequently studied to investigate the specificity of omiu to react with the -amino group of crystalline l - lys . recovery of crystalline amino acids after the guanidination reaction using an o - methylisourea ( omiu ) to crystalline amino acid ratio of 1000:1 , ph of the omiu solution of 10.6 , and a reaction time of 7 days . lc - ms results of o - methylisourea ( omiu ) incubated samples : crystalline l - lys ( a ) and crystalline l - tyr ( d ) incubated at an omiu to crystalline l - lys or crystalline l - tyr ratio of 10:1 , 100:1 , and 1000:1 , and ms spectra of the lc peaks at 1.79 min ( b ) and 2.94 min for crystalline l - lys ( c ) and at 6.70 min for crystalline l - tyr ( e ) . regardless of the omiu to lys ratio , reaction time had little effect on the recovery of unreacted lys and homoarginine ( figure 3 ) and on the quantity of nonrecovered lys ( considered to be double derivatized lys ) . the impact of the reaction mixture ph on the binding of omiu to the - and -amino group of crystalline l - lys was also examined ( figure 4 ) . when the omiu to lys ratio was 1000:1 , the recovery of unreacted lys was highest at ph 8.6 and close to 0% for the other ph values while the recovery of homoarginine was highest for ph values between 8.6 and 9.4 and low for ph values between 9.8 and 11.0 . overall , none of the tested combinations of omiu to lys ratio , reaction time , and omiu ph resulted in the complete recovery of crystalline l - lys as homoarginine . moreover , in all cases , between 4 and 99% of the crystalline l - lys was not recovered either as unreacted lys or as homoarginine after incubation with omiu , suggesting that omiu had bound to the -amino group of lys to differing extents . typically , a higher omiu to lys ratio is preferred for the guanidination of protein - bound lys present in food / feed ingredients and diets , having only a free -amino group , in order to completely convert protein - bound lys to homoarginine . crystalline l - lys , free lys , or n - terminal lys , is present in those protein sources or diets , then as the omiu to lys ratio is increased , the double derivatization of this lys also appears to increase . lowering the omiu to lys ratio to 1.5:1 , however , resulted in a 51% recovery of homoarginine , indicating that , even at low omiu to lys ratios , it is still possible for omiu to bind to the -amino group of lys . thus , the conversion of lys to homoarginine can appear to be high while actually a large proportion of lys is in the double derivatized form . the low recovery of homoarginine could result in an underestimation of the reactive lys content and subsequently an overestimation of lys damage . when considering protein - bound lys to be fully converted to homoarginine , the underestimation of the reactive lys content in food / feed ingredients and diets depends on the amount of lys with a free -amino group ( free + n - terminal lys ) . recovery of crystalline l - lys after the guanidination reaction using four o - methylisourea ( omiu ) to free lys ratios ( 1.5:1 , 10:1 , 100:1 , or 1000:1 ) , three reaction times ( 1 , 3 , or 7 days ) , and a ph of the omiu solution of 10.6 . recovery of crystalline l - lys after the guanidination reaction using two o - methylisourea ( omiu ) to free lys ratios ( 10:1 or 1000:1 ) , seven ph values ( 8.611.0 with 0.4 increments ) of the omiu solution , and a reaction time of 3 days . the pka of the -amino group of lys is 10.6 and the recovery of homoarginine should be highest when the ph of the omiu solution is greater than 10.6 . the pka of the -amino group of lys is 9.0 , and the recovery of unreacted lys ( i.e. for an omiu to lys ratio of 1000:1 , the omiu ph of 8.6 resulted in a high recovery of unreacted lys ( 53% ) and an omiu ph of 9.0 in a high recovery of homoarginine ( 61% ) . the optimal ph for free lys is approximately 10.6 ( figure 4 ) , but none of the ph values resulted in a 100% recovery of homoarginine . to confirm the specificity of omiu for the -amino group of crystalline l - lys , the homoarginine content after incubation with omiu should be equal to the level of lys added to the reaction mixture ( i.e. unfortunately , none of the combinations of reaction time with omiu to lys ratio and ph of the omiu solution with omiu to lys ratio used in the present study resulted in specific binding of omiu to the -amino group of crystalline l - lys . , maximal conversion of crystalline l - lys to homoarginine and minimal conversion of crystalline l - lys to double derivatized lys ) were reaction at ph 10.6 for 3 days with an omiu to lys ratio of 10:1 , which resulted in a homoarginine recovery of 75% . the optimized guanidination conditions ( ph 10.6 , omiu to amino acid ratio of 10:1 and reaction time of 3 days ) were applied to crystalline l - lys hcl and a mixture of six amino acids ( arg , phe , val , ile , thr , and gly ) in order to test the reactivity of the -amino groups of crystalline l - lys hcl ( a commercially available form of crystalline l - lys often used as a supplement for pig and poultry diets ) and six other amino acids under these guanidination conditions . incubating crystalline l - lys hcl with omiu resulted in a homoarginine recovery of 19.5 and 1.1% whereas the nonrecoverable lys was 79 and 98% , respectively , when the omiu to lys ratio was either 10:1 or 1000:1 , respectively . again , these results suggest that omiu can bind to the free -amino groups not only of crystalline l - lys but also of crystalline l - lys hcl and the free -amino groups of crystalline amino acids other than lys , irrespective of the reaction conditions used . the results described above clearly demonstrate that omiu can react with -amino groups of amino acids in addition to the -amino group of lys . furthermore , none of the reaction conditions used in the present study resulted in the complete guanidination of the -amino group of lys without guanidination of the -amino group . the free lys content in 44 different food / feed ingredients was compiled and found to range from 0 to 5.8% of total lys , with an average of 1.3% ( table 1 ) . consequently , the underestimation of the omiu - reactive lys content for these food / feed ingredients is expected to be low . the estimates of the omiu - reactive lys content are expected to be inaccurate only in those cases where the test material contains a large proportion of free + n - terminal lys . in order to determine the potential error involved in the measurement of reactive lys in ileal digesta samples , 23 nonpooled and seven pooled ileal digesta samples from growing pigs and six pooled ileal digesta samples from broilers the free lys as a percentage of total lys for two samples from growing pigs ( one from a protein - free diet and the other from a soybean meal diet ) were considered outliers ( < mean 2 sd ) and were , therefore , excluded from the data analysis . moreover , the free lys as a percentage of total lys in ileal digesta of growing pigs fed sbm or rsm diets was independent ( r = 0.01 , p = 0.71 ) of the apparent ileal digestible cp content in the diet and of collection method ( figure 5b ) . there appeared to be no difference in the free lys as a percentage of total lys between ileal digesta samples collected from growing pigs or broilers ( 12.7 and 14.4% , respectively ; figure 5a ) , suggesting that the free lys content in ileal digesta is not species specific . the amount of free lys at the terminal ileum of growing pigs and broilers fed protein - containing diets , in the current study , was much higher than the 3.1% reported by moughan and schuttert . free lys as percentage of total lys in ileal digesta samples from growing pigs and broilers fed protein - free or protein - containing diets ( a ; means are indicated by diamonds ) , the apparent ileal digestible crude protein content of the diet ( g / kg as - fed ) in relation to the free lys as percentage of total lys in ileal digesta samples collected using an ileal cannula ( n = 13 ; open squares ) or at slaughter ( n = 6 ; closed diamonds ) from growing pigs ( b ) and the free lys content in relation to the total lys content in ileal digesta of growing pigs fed protein - containing diets ( c ) . the impact of the nonspecificity of omiu and the free lys content in ileal digesta on the standardized ileal digestibility of omiu - reactive lys was assessed using samples from a previous study . moreover , it was assumed that all free lys in ileal digesta was double derivatized and , therefore , not determined as omiu - reactive lys . in conclusion , omiu was found to be not specific for the -amino group of crystalline l - lys ( hcl ) and able to bind to the -amino groups of crystalline amino acids under the reaction conditions of the assay as developed by moughan and rutherfurd . the various guanidination conditions of the omiu - reactive lysine assay investigated did not result in absolute specificity for the -amino group of lys . it is recommended to analyze the reactive lys content of food / feed ingredients , diets and ileal digesta using an omiu ph of 10.6 , an omiu to lys ratio of 1000:1 , and a reaction time of at least 3 days to fully convert protein - bound lys to homoarginine . the accurate quantification of free and n - terminal amino acids in ileal digesta warrants further investigation as well as the search for a reagent which is specific for the -amino group of lys .

despite more than 30 years of focused scientific efforts worldwide , creating an effective hiv vaccine has proven difficult . hiv is extraordinary variable ( 1 ) and designing a vaccine able to stimulate immunological responses that will broadly cross - react with circulating variants is a challenge . inducing neutralizing antibodies that can block viral infection of a target cell several breakthrough experimental techniques developed in recent years allowed highly efficient interrogation of human memory b cells and plasma cells and consequently the isolation of many broadly neutralizing antibodies ( bnabs ) ( 2 ) . these bnabs are able to neutralize multiple circulating hiv-1 strains , and large panels of pseudotyped viruses are used to assess the neutralization breadth and potency of these antibodies ( 37 ) . neutralization panel data is usually available in the supplemental materials of the published studies and includes ic50 , and for some studies ic80 , neutralization values ( the concentration at which infectivity is reduced by 50% or 80% , respectively ( 7 ) ) for multiple antibodies and hundreds of viruses . the accumulation of these new monoclonal antibodies together with the vast related neutralization panel information requires storage , comparison and analysis tools . several databases and servers to organize and access this data have become available in recent years ( 810 ) . the standalone program antibodydatabase ( 8) provides an integrated platform for examining sequence , structure , and neutralization data in a holistic way ; however this tool is not a web server . the bnaber database ( 9 ) collects neutralization scores and available antibody structures and sequences of the most important bnabs , and has very useful visualization and analysis tools . the neutralization - based epitope prediction ( nep ) server predicts antibody - specific epitopes at the residue level based on neutralization panels of viral strains , using the user 's data ( 10,11 ) . these important web - based resources , however , do not contain the viral sequence data , and so do not readily enable the exploration of how hiv envelope ( env ) sequence variation is correlated with the neutralization sensitivity . the combination of published neutralization scores , antibody sequences , and viral sequences commonly used for the evaluation of neutralizing antibodies , together with initial analysis of bnab associations with viral sequence mutations has first become available on the web through our new tool catnap ( compile , analyze and tally nab panels ) , available at the los alamos hiv database . in addition , many large env panels are published without accession numbers , and with wide discrepancies in sequence names used by different laboratories , making subsequent comparisons between studies and meta - analysis difficult . given that los alamos hiv database project has a mission of bringing together global hiv sequence and immunology data , we worked with the primary investigators to systematically determine the exact viruses used for neutralization studies in different laboratories . this has enabled an integrated view of hiv envelope sequences , neutralizing antibody ic50 and ic80 data , and descriptions of hiv env / antibody contact residues , collected from multiple studies and put into the framework of our database tools and web services . links to antibody / env structures and complete information of antibody sequence data are also provided , and better visualization tools for this data are under development . in this report , we focus on catnap , a web - based portal of neutralizing antibody ic50 and ic80 values in conjunction with viral data , inspired by studies by west et al . catnap allows users to superimpose neutralization results and virus sequences from published sources and their own data , and perform initial analysis to find potential neutralization antibody signatures . this tool is a part of our larger neutralization antibody resources page at the los alamos hiv database , which includes several other useful sources of information , such as a table of best neutralizing antibodies , a list of external tools for germline antibody reconstruction ( 1318 ) , an hiv genome browser , and the neutralizing antibody contexts and features database . the latter is compiled from multiple references and contains coordinates of important neutralizing antibody contact sites ( 3,4,1928 ) , mutations affecting neutralization sensitivity ( 22,29 ) , cd4 contacts ( 4,26 ) cytoplasmic tail interactions ( 30 ) , and antibody sensitivity signature predictions ( 8,10,11 ) . catnap taps into the neutralizing antibody contexts and features database and links to our hiv immunology and sequence databases , allowing the rich data collected in these databases to be directly incorporated into the analysis . the neutralization panel data ( ic50 and ic80 values for specific monoclonal antibodies and pseudotyped viruses ) were collected from 49 published neutralization studies , mostly from tables in pdf format provided in the supplemental materials . the viral data was collected from los alamos hiv database and in some cases personal communication with the authors , and required careful consideration and systematization to resolve sequence name ambiguities between different laboratories . antibody sequences were downloaded from genbank , and links to the structures in protein data bank are provided . specifically , antibody associations with viral mutations are evaluated by fisher 's exact test , counting the number of antibody - resistant or antibody - sensitive viruses ( above or below threshold of detection ) and the presence or absence of specific amino acids or n - glycosylation motif in the viral sequence alignment position ( 12 ) . catnap includes neutralization data from published studies , as well as curated hiv-1 env alignments corresponding to neutralization panels with carefully standardized virus sequence names . the current collection comprises 172 antibodies and 722 hiv-1 viruses ( 529 with sequences , and the remaining 193 viruses were published by companies , and so the sequences are proprietary ) from 49 published studies , with more being added frequently . the input page allows the user to retrieve details about selected hiv-1 env sequences and antibodies used in neutralization panels , retrieve available details about the donor from whom the antibody was isolated , and select viruses and antibodies both individually and by study , for analysis ( figure 1 ) . the antibody details option provides , for the specified antibody(s ) , links to the corresponding immunology database records , notes , references , links to crystal structures in the protein data bank , antibody donor i d and clonal lineage , data from our neutralizing antibody contexts and features database , and heavy and light chain antibody variable region mrna sequences . the virus details option provides hiv subtype , sampling country , disease stage information , accession number , neutralization tier , and los alamos database comments . ( in some cases , the sequence of the virus used in experiments does not exactly correspond to the genbank sequence . we resolved these issues to the extent possible via personal communication with the authors , and the comments associated with each sequence document whether the genbank sequence or the unpublished one from the authors is used . ) importantly , a sequence name stored in catnap corresponds to the most frequently used name we identified in published neutralization studies . in many cases , however , different names are used in studies by different groups , and they frequently do not exactly correspond to the database sequence name . we provide a list of sequence name aliases and hiv database name for an easy one - to - one correspondence between the various short names , database names and accession numbers , thereby facilitating meta - studies . finally , a set of accession numbers of the selected viruses can be automatically uploaded to the sequence search interface of our hiv sequence database , to obtain information from many additional sequence database fields . the assay details option provides a downloadable table of available ic50 and ic80 data for selected viruses and antibodies from each study . virus combination , both the original data and the geometric mean of the repeated data points is shown . green arrows and green text shown on figures 13 represent comments added on the figure , but not present on the web site . ( a ) antibody details include antibody structures in the protein data bank ; clickable donor i d from whom the antibody was isolated , which leads to donor information stored in the hiv database ; env positions related to antibody neutralization ; and antibody variable chain sequences . the inset on the top of the figure shows an example of env positions related to pg9 neutralization ; env positions shown in the inset have mutations that affect pg9 and related bnab sensitivity . inset on the right shows patient details for the donor from whom the antibody was isolated , and provides the link to hiv-1 sequences from that donor . ( b ) virus details include virus name , subtype , country of isolation , patient health and risk status , genbank accession number , neutralization tier , different names and aliases used in the literature , and our comments . the analysis page superimposes virus data , neutralization data , and aligned viral sequences on one page . for each virus - antibody pair , the user can see virus information ( tier , subtype , country , genbank accession number , sequence aliases ) , neutralization values , both per study and the geometric means , and the virus sequences with highlighted n - linked glycosylation motifs ( figure 2 ) . analysis and additional information are provided under the superimposed data ( figures 2 and 3 ) , and include several options . breadth and potency of each antibody / serum over the selected group of viruses are assessed , with potency calculated as a geometric mean of neutralization scores of both antibody - sensitive viruses and all viruses ( figure 2).list of env - antibody contact positions is displayed for the selected antibodies , with the link to another hiv database tool , quickalign , which aligns these positions to a large set of sequences from the hiv sequence database and shows sequence logos ( amino acid compositions and frequencies ) and summaries of how variable these positions are in hiv circulating sequences ( figure 2).antibody signature analysis options highlight a position of interest in the alignment ( figure 2 ) , and for each sequence report the presence or absence of n - glycosylated motif for in this position ( figure 2 ) , extract further information from the hiv databases and perform basic antibody signature analysis on this position ( figure 3 ) . the extracted information includes entropy scores as a measure of amino acid variability in this position in m group , b clade and c clade database alignments , protein features such as functional domains associated with this position , antibody contacts and other nab - associated features of this position , such as mutations affecting antibody sensitivity , signature predictions , and other features ( figure 3c ) . statistical analysis displays the amino acid makeup of that position in the alignment ( figure 3a ) , the count of viruses with and without n - linked glycosylation motif ( figure 3b ) , the count of antibody sensitive and resistant viruses for each amino acid or n - glycosylation motif found in this position of the alignment , together with the corresponding fisher 's exact test results to detect a potential positive or negative amino acid signature for the selected antibody ( 12 ) . in figure 3b , for example , an n - glycosylation motif at env position 160 is enriched in bnab pg9-detected viruses ( p < 2.2e16 ) , indicating , in agreement with published studies ( 8,31 ) , an association of a glycosylation site at position 160 with increased susceptibility to pg9 neutralization . this analysis should be interpreted with caution , as the counts are not phylogenetically corrected , and significant correlations that are not directly related to antibody sensitivity may arise as a consequence of structure in the phylogenetic tree . still such associations can be informative , particularly for hypothesis forming or validation of a pre - existing hypothesis . a signature analysis tool that incorporates a phylogenetic correction ( 12 ) is in progress at the database . breadth and potency of each antibody / serum over the selected group of viruses are assessed , with potency calculated as a geometric mean of neutralization scores of both antibody - sensitive viruses and all viruses ( figure 2 ) . list of env - antibody contact positions is displayed for the selected antibodies , with the link to another hiv database tool , quickalign , which aligns these positions to a large set of sequences from the hiv sequence database and shows sequence logos ( amino acid compositions and frequencies ) and summaries of how variable these positions are in hiv circulating sequences ( figure 2 ) . antibody signature analysis options highlight a position of interest in the alignment ( figure 2 ) , and for each sequence report the presence or absence of n - glycosylated motif for in this position ( figure 2 ) , extract further information from the hiv databases and perform basic antibody signature analysis on this position ( figure 3 ) . the extracted information includes entropy scores as a measure of amino acid variability in this position in m group , b clade and c clade database alignments , protein features such as functional domains associated with this position , antibody contacts and other nab - associated features of this position , such as mutations affecting antibody sensitivity , signature predictions , and other features ( figure 3c ) . statistical analysis displays the amino acid makeup of that position in the alignment ( figure 3a ) , the count of viruses with and without n - linked glycosylation motif ( figure 3b ) , the count of antibody sensitive and resistant viruses for each amino acid or n - glycosylation motif found in this position of the alignment , together with the corresponding fisher 's exact test results to detect a potential positive or negative amino acid signature for the selected antibody ( 12 ) . in figure 3b , for example , an n - glycosylation motif at env position 160 is enriched in bnab pg9-detected viruses ( p < 2.2e16 ) , indicating , in agreement with published studies ( 8,31 ) , an association of a glycosylation site at position 160 with increased susceptibility to pg9 neutralization . this analysis should be interpreted with caution , as the counts are not phylogenetically corrected , and significant correlations that are not directly related to antibody sensitivity may arise as a consequence of structure in the phylogenetic tree . still such associations can be informative , particularly for hypothesis forming or validation of a pre - existing hypothesis . a signature analysis tool that incorporates a phylogenetic correction ( 12 ) is in progress at the database . each window has vertical and horizontal scroll bars , and each window can be expanded vertically and horizontally by clicking and dragging its right corner . all fields in each window are sortable , and all three windows are sorted simultaneously . for example , clicking on mab 10e8 in the center window will sort all 3 windows by 10e8 neutralization scores . the left window lists virus names included in the analysis as well as neutralization tier , subtype , country , accession number , aliases ( expandable data ) , and the presence of potential n - linked glycosylation motifs ( denoted as nxst ) . the center window lists antibodies with their color - coded ic50 and/or ic80 neutralization scores . virus combination , the geometric mean of the data points is shown and marked with an asterisk ( * ) . the expand link shows the data points from multiple studies used to calculate the geometric means . a mouseover on each individual geometric mean will also show data - points together with the study references . the right window shows aligned env sequences , with the hxb2 reference sequence on top . additional information is provided under the three aligned windows above : breadth of neutralization and potency of each antibody over the selected group of viruses ( potency is calculated two ways : for sensitive viruses that are below neutralization threshold of detection and for all tested viruses where resistant viruses are set to 100 g / ml . ) ; antibody contact hiv position(s ) and a link to the sequence logo of these positions showing their variability in hiv-1 circulating sequences ( see the inset ) . the option to analyze an individual hiv protein sequence position is on the bottom of the figure ( this position is highlighted on the env alignment in the right window with a gray vertical line ) . when a position of interest is entered , a new this column shows the amino acid found in each sequence at the position of interest , and the presence of an n - linked glycosylation motif in that position is indicated by + . n - linked glycosylation motifs are also highlighted in the alignment in red . statistical analysis . ( a ) counts of different amino acids at the chosen hxb2 position of env are shown , together with the counts of bnab pg9 sensitive ( detected , that is below threshold of detection ) and resistant ( undetected , that is above threshold of detection ) viruses for each amino acid . ( b ) counts of viruses with and without an n - linked glycosylation motif at the chosen hxb2 position of env are shown . in ( a ) and ( b ) , fisher 's exact test is based on a contingency table of aa / not aa ( or nxst / not nxst ) and virus sensitive / not sensitive. ( c ) information from the hiv database about the position of interest is shown , and includes entropy calculations using hiv database tool entropy and hiv-1 alignments for m group , and b and c subtypes , information about functional domains , and antibody features of this position , such as how mutations in this position affect various bnabs neutralization sensitivity , signature predictions at this position , mutations affecting antibody binding , etc . custom catnap allows users to superimpose and analyze any table of numerical data in conjunction with genetic sequences ( figure 2 ) , and to look for associated genetic signatures ( figure 3 ) . examples include ic50 neutralization values for monoclonal antibodies , like those used in the basic catnap tool , or id50 titers for plasma neutralization data , but the tool can be used for any numerical data ( binding kinetic data , functional data ) in conjunction with sequence data . as neutralization data frequently use cutoffs , with results below or above a threshold being considered undetected , for the statistical analysis the user should choose the type that describes the most potent scores in the data . for example , ic50 scores for the monoclonal antibodies reflect the concentration needed to achieve 50% neutralization , and so values with a id50 scores for the plasma antibodies , on the other hand , reflect the number of dilutions required to reduce the infectivity by 50% , and the high scores are the most potent , so the values with a like in basic catnap , potential genetic signatures are defined by fisher 's exact test ( 12 ) . users can use their own alignments , or can use a premade alignment that contains subsets of env sequences in the catnap tool , including hiv-1 viral strains commonly used in neutralization panels . catnap includes neutralization data from published studies , as well as curated hiv-1 env alignments corresponding to neutralization panels with carefully standardized virus sequence names . the current collection comprises 172 antibodies and 722 hiv-1 viruses ( 529 with sequences , and the remaining 193 viruses were published by companies , and so the sequences are proprietary ) from 49 published studies , with more being added frequently . the input page allows the user to retrieve details about selected hiv-1 env sequences and antibodies used in neutralization panels , retrieve available details about the donor from whom the antibody was isolated , and select viruses and antibodies both individually and by study , for analysis ( figure 1 ) . the antibody details option provides , for the specified antibody(s ) , links to the corresponding immunology database records , notes , references , links to crystal structures in the protein data bank , antibody donor i d and clonal lineage , data from our neutralizing antibody contexts and features database , and heavy and light chain antibody variable region mrna sequences . the virus details option provides hiv subtype , sampling country , disease stage information , accession number , neutralization tier , and los alamos database comments . ( in some cases , the sequence of the virus used in experiments does not exactly correspond to the genbank sequence . we resolved these issues to the extent possible via personal communication with the authors , and the comments associated with each sequence document whether the genbank sequence or the unpublished one from the authors is used . ) importantly , a sequence name stored in catnap corresponds to the most frequently used name we identified in published neutralization studies . in many cases , however , different names are used in studies by different groups , and they frequently do not exactly correspond to the database sequence name . we provide a list of sequence name aliases and hiv database name for an easy one - to - one correspondence between the various short names , database names and accession numbers , thereby facilitating meta - studies . finally , a set of accession numbers of the selected viruses can be automatically uploaded to the sequence search interface of our hiv sequence database , to obtain information from many additional sequence database fields . the assay details option provides a downloadable table of available ic50 and ic80 data for selected viruses and antibodies from each study . virus combination , both the original data and the geometric mean of the repeated data points is shown . green arrows and green text shown on figures 13 represent comments added on the figure , but not present on the web site . ( a ) antibody details include antibody structures in the protein data bank ; clickable donor i d from whom the antibody was isolated , which leads to donor information stored in the hiv database ; env positions related to antibody neutralization ; and antibody variable chain sequences . the inset on the top of the figure shows an example of env positions related to pg9 neutralization ; env positions shown in the inset have mutations that affect pg9 and related bnab sensitivity . inset on the right shows patient details for the donor from whom the antibody was isolated , and provides the link to hiv-1 sequences from that donor . ( b ) virus details include virus name , subtype , country of isolation , patient health and risk status , genbank accession number , neutralization tier , different names and aliases used in the literature , and our comments . the analysis page superimposes virus data , neutralization data , and aligned viral sequences on one page . for each virus - antibody pair , the user can see virus information ( tier , subtype , country , genbank accession number , sequence aliases ) , neutralization values , both per study and the geometric means , and the virus sequences with highlighted n - linked glycosylation motifs ( figure 2 ) . analysis and additional information are provided under the superimposed data ( figures 2 and 3 ) , and include several options . breadth and potency of each antibody / serum over the selected group of viruses are assessed , with potency calculated as a geometric mean of neutralization scores of both antibody - sensitive viruses and all viruses ( figure 2).list of env - antibody contact positions is displayed for the selected antibodies , with the link to another hiv database tool , quickalign , which aligns these positions to a large set of sequences from the hiv sequence database and shows sequence logos ( amino acid compositions and frequencies ) and summaries of how variable these positions are in hiv circulating sequences ( figure 2).antibody signature analysis options highlight a position of interest in the alignment ( figure 2 ) , and for each sequence report the presence or absence of n - glycosylated motif for in this position ( figure 2 ) , extract further information from the hiv databases and perform basic antibody signature analysis on this position ( figure 3 ) . the extracted information includes entropy scores as a measure of amino acid variability in this position in m group , b clade and c clade database alignments , protein features such as functional domains associated with this position , antibody contacts and other nab - associated features of this position , such as mutations affecting antibody sensitivity , signature predictions , and other features ( figure 3c ) . statistical analysis displays the amino acid makeup of that position in the alignment ( figure 3a ) , the count of viruses with and without n - linked glycosylation motif ( figure 3b ) , the count of antibody sensitive and resistant viruses for each amino acid or n - glycosylation motif found in this position of the alignment , together with the corresponding fisher 's exact test results to detect a potential positive or negative amino acid signature for the selected antibody ( 12 ) . in figure 3b , for example , an n - glycosylation motif at env position 160 is enriched in bnab pg9-detected viruses ( p < 2.2e16 ) , indicating , in agreement with published studies ( 8,31 ) , an association of a glycosylation site at position 160 with increased susceptibility to pg9 neutralization . this analysis should be interpreted with caution , as the counts are not phylogenetically corrected , and significant correlations that are not directly related to antibody sensitivity may arise as a consequence of structure in the phylogenetic tree . still such associations can be informative , particularly for hypothesis forming or validation of a pre - existing hypothesis . a signature analysis tool that incorporates a phylogenetic correction ( 12 ) is in progress at the database . breadth and potency of each antibody / serum over the selected group of viruses are assessed , with potency calculated as a geometric mean of neutralization scores of both antibody - sensitive viruses and all viruses ( figure 2 ) . list of env - antibody contact positions is displayed for the selected antibodies , with the link to another hiv database tool , quickalign , which aligns these positions to a large set of sequences from the hiv sequence database and shows sequence logos ( amino acid compositions and frequencies ) and summaries of how variable these positions are in hiv circulating sequences ( figure 2 ) . antibody signature analysis options highlight a position of interest in the alignment ( figure 2 ) , and for each sequence report the presence or absence of n - glycosylated motif for in this position ( figure 2 ) , extract further information from the hiv databases and perform basic antibody signature analysis on this position ( figure 3 ) . the extracted information includes entropy scores as a measure of amino acid variability in this position in m group , b clade and c clade database alignments , protein features such as functional domains associated with this position , antibody contacts and other nab - associated features of this position , such as mutations affecting antibody sensitivity , signature predictions , and other features ( figure 3c ) . statistical analysis displays the amino acid makeup of that position in the alignment ( figure 3a ) , the count of viruses with and without n - linked glycosylation motif ( figure 3b ) , the count of antibody sensitive and resistant viruses for each amino acid or n - glycosylation motif found in this position of the alignment , together with the corresponding fisher 's exact test results to detect a potential positive or negative amino acid signature for the selected antibody ( 12 ) . in figure 3b , for example , an n - glycosylation motif at env position 160 is enriched in bnab pg9-detected viruses ( p < 2.2e16 ) , indicating , in agreement with published studies ( 8,31 ) , an association of a glycosylation site at position 160 with increased susceptibility to pg9 neutralization . this analysis should be interpreted with caution , as the counts are not phylogenetically corrected , and significant correlations that are not directly related to antibody sensitivity may arise as a consequence of structure in the phylogenetic tree . still such associations can be informative , particularly for hypothesis forming or validation of a pre - existing hypothesis . a signature analysis tool that incorporates a phylogenetic correction ( 12 ) is in progress at the database . each window has vertical and horizontal scroll bars , and each window can be expanded vertically and horizontally by clicking and dragging its right corner . all fields in each window are sortable , and all three windows are sorted simultaneously . for example , clicking on mab 10e8 in the center window will sort all 3 windows by 10e8 neutralization scores . the left window lists virus names included in the analysis as well as neutralization tier , subtype , country , accession number , aliases ( expandable data ) , and the presence of potential n - linked glycosylation motifs ( denoted as nxst ) . the center window lists antibodies with their color - coded ic50 and/or ic80 neutralization scores . virus combination , the geometric mean of the data points is shown and marked with an asterisk ( * ) . expand link shows the data points from multiple studies used to calculate the geometric means . a mouseover on each individual geometric mean will also show data - points together with the study references . the right window shows aligned env sequences , with the hxb2 reference sequence on top . additional information is provided under the three aligned windows above : breadth of neutralization and potency of each antibody over the selected group of viruses ( potency is calculated two ways : for sensitive viruses that are below neutralization threshold of detection and for all tested viruses where resistant viruses are set to 100 g / ml . ) ; antibody contact hiv position(s ) and a link to the sequence logo of these positions showing their variability in hiv-1 circulating sequences ( see the inset ) . the option to analyze an individual hiv protein sequence position is on the bottom of the figure ( this position is highlighted on the env alignment in the right window with a gray vertical line ) . this column shows the amino acid found in each sequence at the position of interest , and the presence of an n - linked glycosylation motif in that position is indicated by + . n - linked glycosylation motifs are also highlighted in the alignment in red . statistical analysis . ( a ) counts of different amino acids at the chosen hxb2 position of env are shown , together with the counts of bnab pg9 sensitive ( detected , that is below threshold of detection ) and resistant ( undetected , that is above threshold of detection ) viruses for each amino acid . ( b ) counts of viruses with and without an n - linked glycosylation motif at the chosen hxb2 position of env are shown . in ( a ) and ( b ) , fisher 's exact test is based on a contingency table of aa / not aa ( or nxst / not nxst ) and virus sensitive / not sensitive. ( c ) information from the hiv database about the position of interest is shown , and includes entropy calculations using hiv database tool entropy and hiv-1 alignments for m group , and b and c subtypes , information about functional domains , and antibody features of this position , such as how mutations in this position affect various bnabs neutralization sensitivity , signature predictions at this position , mutations affecting antibody binding , etc . custom catnap allows users to superimpose and analyze any table of numerical data in conjunction with genetic sequences ( figure 2 ) , and to look for associated genetic signatures ( figure 3 ) . examples include ic50 neutralization values for monoclonal antibodies , like those used in the basic catnap tool , or id50 titers for plasma neutralization data , but the tool can be used for any numerical data ( binding kinetic data , functional data ) in conjunction with sequence data . as neutralization data frequently use cutoffs , with results below or above a threshold being considered undetected , for the statistical analysis the user should choose the type that describes the most potent scores in the data . for example , ic50 scores for the monoclonal antibodies reflect the concentration needed to achieve 50% neutralization , and so values with a id50 scores for the plasma antibodies , on the other hand , reflect the number of dilutions required to reduce the infectivity by 50% , and the high scores are the most potent , so the values with a like in basic catnap , potential genetic signatures are defined by fisher 's exact test ( 12 ) . users can use their own alignments , or can use a premade alignment that contains subsets of env sequences in the catnap tool , including hiv-1 viral strains commonly used in neutralization panels . overall , we believe catnap is a useful tool for the hiv research community , particularly good for integrating and superimposing complete hiv sequence data and broadly neutralizing antibody data across many studies . it provides initial signature analysis on the fly , and it can be used to analyze custom data including genetic sequences in conjunction with numerical data . we consider catnap to be a foundation for a new neutralizing antibody relational database , and we plan to expand this resource to contain many more analysis tools and data tables , such as incorporating phylogenetic correction in evaluating antibody signatures in viral sequences ( 12 ) , listing of germline antibody sequences , antibody variable sequence alignments , hiv - antibody 3d structure visualization tools , and within - patient studies with autologous viral and antibody sequence data . national institutes of health ( nih ) [ contract agraai1200700101000 hiv / siv , database and analysis unit ( b.t.k . , h.y . , j.m . , , k.y . ) and grant hivrad p01 ai100148 ( p.j.b . , a.p.w . ) ] ; bill & melinda gates foundation [ collaboration for aids vaccine discovery , grant 1032144 ( b.t.k . , h.y . ) and grant 1040753 ( p.j.b . , a.p.w . ) ] .

catnap ( compile , analyze and tally nab panels ) is a new web server at los alamos hiv database , created to respond to the newest advances in hiv neutralizing antibody research . it is a comprehensive platform focusing on neutralizing antibody potencies in conjunction with viral sequences . catnap integrates neutralization and sequence data from published studies , and allows users to analyze that data for each hiv envelope protein sequence position and each antibody . the tool has multiple data retrieval and analysis options . as input , the user can pick specific antibodies and viruses , choose a panel from a published study , or supply their own data . the output superimposes neutralization panel data , virus epidemiological data , and viral protein sequence alignments on one page , and provides further information and analyses . the user can highlight alignment positions , or select antibody contact residues and view position - specific information from the hiv databases . the tool calculates tallies of amino acids and n - linked glycosylation motifs , counts of antibody - sensitive and -resistant viruses in conjunction with each amino acid or n - glycosylation motif , and performs fisher 's exact test to detect potential positive or negative amino acid associations for the selected antibody . website name : catnap ( compile , analyze and tally nab panels ) . website address : http://hiv.lanl.gov/catnap .

INTRODUCTION MATERIALS AND METHODS RESULTS Data retrieval Analysis page Custom CATNAP DISCUSSION FUNDING

neutralization panel data is usually available in the supplemental materials of the published studies and includes ic50 , and for some studies ic80 , neutralization values ( the concentration at which infectivity is reduced by 50% or 80% , respectively ( 7 ) ) for multiple antibodies and hundreds of viruses . these important web - based resources , however , do not contain the viral sequence data , and so do not readily enable the exploration of how hiv envelope ( env ) sequence variation is correlated with the neutralization sensitivity . the combination of published neutralization scores , antibody sequences , and viral sequences commonly used for the evaluation of neutralizing antibodies , together with initial analysis of bnab associations with viral sequence mutations has first become available on the web through our new tool catnap ( compile , analyze and tally nab panels ) , available at the los alamos hiv database . given that los alamos hiv database project has a mission of bringing together global hiv sequence and immunology data , we worked with the primary investigators to systematically determine the exact viruses used for neutralization studies in different laboratories . this has enabled an integrated view of hiv envelope sequences , neutralizing antibody ic50 and ic80 data , and descriptions of hiv env / antibody contact residues , collected from multiple studies and put into the framework of our database tools and web services . in this report , we focus on catnap , a web - based portal of neutralizing antibody ic50 and ic80 values in conjunction with viral data , inspired by studies by west et al . catnap allows users to superimpose neutralization results and virus sequences from published sources and their own data , and perform initial analysis to find potential neutralization antibody signatures . this tool is a part of our larger neutralization antibody resources page at the los alamos hiv database , which includes several other useful sources of information , such as a table of best neutralizing antibodies , a list of external tools for germline antibody reconstruction ( 1318 ) , an hiv genome browser , and the neutralizing antibody contexts and features database . the latter is compiled from multiple references and contains coordinates of important neutralizing antibody contact sites ( 3,4,1928 ) , mutations affecting neutralization sensitivity ( 22,29 ) , cd4 contacts ( 4,26 ) cytoplasmic tail interactions ( 30 ) , and antibody sensitivity signature predictions ( 8,10,11 ) . the neutralization panel data ( ic50 and ic80 values for specific monoclonal antibodies and pseudotyped viruses ) were collected from 49 published neutralization studies , mostly from tables in pdf format provided in the supplemental materials . the viral data was collected from los alamos hiv database and in some cases personal communication with the authors , and required careful consideration and systematization to resolve sequence name ambiguities between different laboratories . specifically , antibody associations with viral mutations are evaluated by fisher 's exact test , counting the number of antibody - resistant or antibody - sensitive viruses ( above or below threshold of detection ) and the presence or absence of specific amino acids or n - glycosylation motif in the viral sequence alignment position ( 12 ) . catnap includes neutralization data from published studies , as well as curated hiv-1 env alignments corresponding to neutralization panels with carefully standardized virus sequence names . the current collection comprises 172 antibodies and 722 hiv-1 viruses ( 529 with sequences , and the remaining 193 viruses were published by companies , and so the sequences are proprietary ) from 49 published studies , with more being added frequently . the antibody details option provides , for the specified antibody(s ) , links to the corresponding immunology database records , notes , references , links to crystal structures in the protein data bank , antibody donor i d and clonal lineage , data from our neutralizing antibody contexts and features database , and heavy and light chain antibody variable region mrna sequences . we resolved these issues to the extent possible via personal communication with the authors , and the comments associated with each sequence document whether the genbank sequence or the unpublished one from the authors is used . ) finally , a set of accession numbers of the selected viruses can be automatically uploaded to the sequence search interface of our hiv sequence database , to obtain information from many additional sequence database fields . inset on the right shows patient details for the donor from whom the antibody was isolated , and provides the link to hiv-1 sequences from that donor . the analysis page superimposes virus data , neutralization data , and aligned viral sequences on one page . for each virus - antibody pair , the user can see virus information ( tier , subtype , country , genbank accession number , sequence aliases ) , neutralization values , both per study and the geometric means , and the virus sequences with highlighted n - linked glycosylation motifs ( figure 2 ) . breadth and potency of each antibody / serum over the selected group of viruses are assessed , with potency calculated as a geometric mean of neutralization scores of both antibody - sensitive viruses and all viruses ( figure 2).list of env - antibody contact positions is displayed for the selected antibodies , with the link to another hiv database tool , quickalign , which aligns these positions to a large set of sequences from the hiv sequence database and shows sequence logos ( amino acid compositions and frequencies ) and summaries of how variable these positions are in hiv circulating sequences ( figure 2).antibody signature analysis options highlight a position of interest in the alignment ( figure 2 ) , and for each sequence report the presence or absence of n - glycosylated motif for in this position ( figure 2 ) , extract further information from the hiv databases and perform basic antibody signature analysis on this position ( figure 3 ) . the extracted information includes entropy scores as a measure of amino acid variability in this position in m group , b clade and c clade database alignments , protein features such as functional domains associated with this position , antibody contacts and other nab - associated features of this position , such as mutations affecting antibody sensitivity , signature predictions , and other features ( figure 3c ) . statistical analysis displays the amino acid makeup of that position in the alignment ( figure 3a ) , the count of viruses with and without n - linked glycosylation motif ( figure 3b ) , the count of antibody sensitive and resistant viruses for each amino acid or n - glycosylation motif found in this position of the alignment , together with the corresponding fisher 's exact test results to detect a potential positive or negative amino acid signature for the selected antibody ( 12 ) . in figure 3b , for example , an n - glycosylation motif at env position 160 is enriched in bnab pg9-detected viruses ( p < 2.2e16 ) , indicating , in agreement with published studies ( 8,31 ) , an association of a glycosylation site at position 160 with increased susceptibility to pg9 neutralization . breadth and potency of each antibody / serum over the selected group of viruses are assessed , with potency calculated as a geometric mean of neutralization scores of both antibody - sensitive viruses and all viruses ( figure 2 ) . list of env - antibody contact positions is displayed for the selected antibodies , with the link to another hiv database tool , quickalign , which aligns these positions to a large set of sequences from the hiv sequence database and shows sequence logos ( amino acid compositions and frequencies ) and summaries of how variable these positions are in hiv circulating sequences ( figure 2 ) . antibody signature analysis options highlight a position of interest in the alignment ( figure 2 ) , and for each sequence report the presence or absence of n - glycosylated motif for in this position ( figure 2 ) , extract further information from the hiv databases and perform basic antibody signature analysis on this position ( figure 3 ) . statistical analysis displays the amino acid makeup of that position in the alignment ( figure 3a ) , the count of viruses with and without n - linked glycosylation motif ( figure 3b ) , the count of antibody sensitive and resistant viruses for each amino acid or n - glycosylation motif found in this position of the alignment , together with the corresponding fisher 's exact test results to detect a potential positive or negative amino acid signature for the selected antibody ( 12 ) . in figure 3b , for example , an n - glycosylation motif at env position 160 is enriched in bnab pg9-detected viruses ( p < 2.2e16 ) , indicating , in agreement with published studies ( 8,31 ) , an association of a glycosylation site at position 160 with increased susceptibility to pg9 neutralization . the left window lists virus names included in the analysis as well as neutralization tier , subtype , country , accession number , aliases ( expandable data ) , and the presence of potential n - linked glycosylation motifs ( denoted as nxst ) . when a position of interest is entered , a new this column shows the amino acid found in each sequence at the position of interest , and the presence of an n - linked glycosylation motif in that position is indicated by + . n - linked glycosylation motifs are also highlighted in the alignment in red . ( a ) counts of different amino acids at the chosen hxb2 position of env are shown , together with the counts of bnab pg9 sensitive ( detected , that is below threshold of detection ) and resistant ( undetected , that is above threshold of detection ) viruses for each amino acid . ( b ) counts of viruses with and without an n - linked glycosylation motif at the chosen hxb2 position of env are shown . in ( a ) and ( b ) , fisher 's exact test is based on a contingency table of aa / not aa ( or nxst / not nxst ) and virus sensitive / not sensitive. ( c ) information from the hiv database about the position of interest is shown , and includes entropy calculations using hiv database tool entropy and hiv-1 alignments for m group , and b and c subtypes , information about functional domains , and antibody features of this position , such as how mutations in this position affect various bnabs neutralization sensitivity , signature predictions at this position , mutations affecting antibody binding , etc . custom catnap allows users to superimpose and analyze any table of numerical data in conjunction with genetic sequences ( figure 2 ) , and to look for associated genetic signatures ( figure 3 ) . examples include ic50 neutralization values for monoclonal antibodies , like those used in the basic catnap tool , or id50 titers for plasma neutralization data , but the tool can be used for any numerical data ( binding kinetic data , functional data ) in conjunction with sequence data . for example , ic50 scores for the monoclonal antibodies reflect the concentration needed to achieve 50% neutralization , and so values with a id50 scores for the plasma antibodies , on the other hand , reflect the number of dilutions required to reduce the infectivity by 50% , and the high scores are the most potent , so the values with a like in basic catnap , potential genetic signatures are defined by fisher 's exact test ( 12 ) . catnap includes neutralization data from published studies , as well as curated hiv-1 env alignments corresponding to neutralization panels with carefully standardized virus sequence names . the current collection comprises 172 antibodies and 722 hiv-1 viruses ( 529 with sequences , and the remaining 193 viruses were published by companies , and so the sequences are proprietary ) from 49 published studies , with more being added frequently . the antibody details option provides , for the specified antibody(s ) , links to the corresponding immunology database records , notes , references , links to crystal structures in the protein data bank , antibody donor i d and clonal lineage , data from our neutralizing antibody contexts and features database , and heavy and light chain antibody variable region mrna sequences . we resolved these issues to the extent possible via personal communication with the authors , and the comments associated with each sequence document whether the genbank sequence or the unpublished one from the authors is used . ) finally , a set of accession numbers of the selected viruses can be automatically uploaded to the sequence search interface of our hiv sequence database , to obtain information from many additional sequence database fields . the analysis page superimposes virus data , neutralization data , and aligned viral sequences on one page . for each virus - antibody pair , the user can see virus information ( tier , subtype , country , genbank accession number , sequence aliases ) , neutralization values , both per study and the geometric means , and the virus sequences with highlighted n - linked glycosylation motifs ( figure 2 ) . breadth and potency of each antibody / serum over the selected group of viruses are assessed , with potency calculated as a geometric mean of neutralization scores of both antibody - sensitive viruses and all viruses ( figure 2).list of env - antibody contact positions is displayed for the selected antibodies , with the link to another hiv database tool , quickalign , which aligns these positions to a large set of sequences from the hiv sequence database and shows sequence logos ( amino acid compositions and frequencies ) and summaries of how variable these positions are in hiv circulating sequences ( figure 2).antibody signature analysis options highlight a position of interest in the alignment ( figure 2 ) , and for each sequence report the presence or absence of n - glycosylated motif for in this position ( figure 2 ) , extract further information from the hiv databases and perform basic antibody signature analysis on this position ( figure 3 ) . statistical analysis displays the amino acid makeup of that position in the alignment ( figure 3a ) , the count of viruses with and without n - linked glycosylation motif ( figure 3b ) , the count of antibody sensitive and resistant viruses for each amino acid or n - glycosylation motif found in this position of the alignment , together with the corresponding fisher 's exact test results to detect a potential positive or negative amino acid signature for the selected antibody ( 12 ) . in figure 3b , for example , an n - glycosylation motif at env position 160 is enriched in bnab pg9-detected viruses ( p < 2.2e16 ) , indicating , in agreement with published studies ( 8,31 ) , an association of a glycosylation site at position 160 with increased susceptibility to pg9 neutralization . breadth and potency of each antibody / serum over the selected group of viruses are assessed , with potency calculated as a geometric mean of neutralization scores of both antibody - sensitive viruses and all viruses ( figure 2 ) . list of env - antibody contact positions is displayed for the selected antibodies , with the link to another hiv database tool , quickalign , which aligns these positions to a large set of sequences from the hiv sequence database and shows sequence logos ( amino acid compositions and frequencies ) and summaries of how variable these positions are in hiv circulating sequences ( figure 2 ) . antibody signature analysis options highlight a position of interest in the alignment ( figure 2 ) , and for each sequence report the presence or absence of n - glycosylated motif for in this position ( figure 2 ) , extract further information from the hiv databases and perform basic antibody signature analysis on this position ( figure 3 ) . the extracted information includes entropy scores as a measure of amino acid variability in this position in m group , b clade and c clade database alignments , protein features such as functional domains associated with this position , antibody contacts and other nab - associated features of this position , such as mutations affecting antibody sensitivity , signature predictions , and other features ( figure 3c ) . statistical analysis displays the amino acid makeup of that position in the alignment ( figure 3a ) , the count of viruses with and without n - linked glycosylation motif ( figure 3b ) , the count of antibody sensitive and resistant viruses for each amino acid or n - glycosylation motif found in this position of the alignment , together with the corresponding fisher 's exact test results to detect a potential positive or negative amino acid signature for the selected antibody ( 12 ) . in figure 3b , for example , an n - glycosylation motif at env position 160 is enriched in bnab pg9-detected viruses ( p < 2.2e16 ) , indicating , in agreement with published studies ( 8,31 ) , an association of a glycosylation site at position 160 with increased susceptibility to pg9 neutralization . the left window lists virus names included in the analysis as well as neutralization tier , subtype , country , accession number , aliases ( expandable data ) , and the presence of potential n - linked glycosylation motifs ( denoted as nxst ) . the option to analyze an individual hiv protein sequence position is on the bottom of the figure ( this position is highlighted on the env alignment in the right window with a gray vertical line ) . this column shows the amino acid found in each sequence at the position of interest , and the presence of an n - linked glycosylation motif in that position is indicated by + . n - linked glycosylation motifs are also highlighted in the alignment in red . ( a ) counts of different amino acids at the chosen hxb2 position of env are shown , together with the counts of bnab pg9 sensitive ( detected , that is below threshold of detection ) and resistant ( undetected , that is above threshold of detection ) viruses for each amino acid . ( b ) counts of viruses with and without an n - linked glycosylation motif at the chosen hxb2 position of env are shown . ( c ) information from the hiv database about the position of interest is shown , and includes entropy calculations using hiv database tool entropy and hiv-1 alignments for m group , and b and c subtypes , information about functional domains , and antibody features of this position , such as how mutations in this position affect various bnabs neutralization sensitivity , signature predictions at this position , mutations affecting antibody binding , etc . custom catnap allows users to superimpose and analyze any table of numerical data in conjunction with genetic sequences ( figure 2 ) , and to look for associated genetic signatures ( figure 3 ) . examples include ic50 neutralization values for monoclonal antibodies , like those used in the basic catnap tool , or id50 titers for plasma neutralization data , but the tool can be used for any numerical data ( binding kinetic data , functional data ) in conjunction with sequence data . for example , ic50 scores for the monoclonal antibodies reflect the concentration needed to achieve 50% neutralization , and so values with a id50 scores for the plasma antibodies , on the other hand , reflect the number of dilutions required to reduce the infectivity by 50% , and the high scores are the most potent , so the values with a like in basic catnap , potential genetic signatures are defined by fisher 's exact test ( 12 ) . overall , we believe catnap is a useful tool for the hiv research community , particularly good for integrating and superimposing complete hiv sequence data and broadly neutralizing antibody data across many studies . it provides initial signature analysis on the fly , and it can be used to analyze custom data including genetic sequences in conjunction with numerical data . we consider catnap to be a foundation for a new neutralizing antibody relational database , and we plan to expand this resource to contain many more analysis tools and data tables , such as incorporating phylogenetic correction in evaluating antibody signatures in viral sequences ( 12 ) , listing of germline antibody sequences , antibody variable sequence alignments , hiv - antibody 3d structure visualization tools , and within - patient studies with autologous viral and antibody sequence data .

kinetoplastidae are flagellated protozoan parasites , including serious human pathogens that are transmitted by different insect vectors . diseases caused by kinetoplastids include human african trypanosomiasis ( hat , also known as african sleeping sickness ) , which is due to infection with trypanosoma brucei gambiense or t. brucei rhodesiense ; chagas disease , which is caused by t. cruzi ; and various forms of leishmaniasis caused by infection with different species of leishmania . those living in tropical and subtropical areas of the world are at risk of contracting these diseases . sleeping sickness , which is found predominantly across sub - saharan africa , is fatal if not treated , and all drugs used in the treatment of hat have issues relating to efficacy , administration , and side effects . additionally , increasing levels of drug resistance demonstrate the need for new , improved , and affordable drugs . we have previously identified the bifunctional enzyme fold as an interesting target for antibacterial drug discovery . this enzyme produces n - formyl - tetrahydrofolate ( thf ) in a two - step reaction . first , n , n - methylene - thf is converted to n , n - methenyl - thf by the nadp or nad - dependent n , n - methylene - thf dehydrogenase ( dh ) ; then , n - formyl - thf is produced by the action of n , n - methenyl - thf cyclohydrolase ( ch , figure 1 ) . proposed reaction mechanism of tbfold . fold crystal structures have been reported from different organisms , including leishmania major(8 ) but not from t. brucei . we initiated an inhibitor discovery program targeting the t. brucei enzyme ( tbfold ) and selected as a lead compound ly374571 ( compound 1 ) developed by eli lilly and company because it inhibits bacterial and human fold . however , following the protocol reported for the synthesis of 1 , we obtained a new compound whose structure has been unambiguously determined as 2 ( figure 2 ) . we judge it likely that the original report refers to compound 2 not 1 . to confirm 2 as a suitable lead compound for the development of tbfold inhibitors , we were able to obtain the first x - ray structure of tbfold in the presence of nadp and the inhibitor . the x - ray crystal structure of the complex tbfold-2-nadp revealed molecular details that were relevant to the binding and inhibition of the enzyme , and allowed , with the help of molecular modeling , a rational design of several analogues ( compounds 1620 ) . multiple sequence alignment of fold from several different organisms ( see figure s1 ) and superimposition of the corresponding three - dimensional structures provided insight into the major determinants of ligand binding and selectivity for the different fold forms ( see figure s2 ) . antiparasitic activity against the bloodstream form of t. brucei and cytotoxicity against human leukemia macrophages , differentiated from thp1 monocytes , were evaluated for all compounds under study . ( a ) structures of 1 and 2 ; ( b ) molecular formula of one symmetry - independent molecule of intermediate 3 and of the dmso unit showing the atom numbering scheme . compound 2 was prepared following a procedure originally reported to afford 1 ( figure 2 ) . however , in contrast to what was reported by schmidt et al . reaction of 4-hydroxy-2,5,6-triaminopyrimidine sulfate with ethyl 4-isocyanatobenzoate ( scheme 1 ) afforded a derivative with the urea moiety linked at position 5 ( compound 3 ) instead of position 6 . our structural assignment is based on the x - ray analysis of 3 , which was crystallized as a thin plate by slow evaporation from dimethyl sulfoxide ( dmso ) . figure 2b shows the molecular formula of one symmetry - independent molecule of 3 with dmso and the atom numbering scheme ( see supporting information for details on crystal determination : figure s3 , s4 , and s5 ) . the latter can be obtained free of charge from the cambridge crystallographic data centre via www.ccdc.cam.ac.uk/data_request/cif . a series of new analogues of compound 2 were designed on the basis of the crystallographic data and molecular modeling studies , as discussed in detail later . as shown in scheme 1 intermediate 4 was reacted with a series of different -amino acids 69 or with -amino butyric acid 10 , all as methyl esters , using n - methylmorpholine and 2-chloro-4,6-dimethoxy-1,3,5-triazine as coupling reagents , affording derivatives 1115 , which were finally converted into the desired compounds 1620 by alkaline hydrolysis with 1 n naoh ( scheme 2 ) . the molecular structure of 3 , as obtained from x - ray diffraction analysis , is shown in figure s3 . the asymmetric unit consists of four independent molecules ( labeled a , b , c , and d ) , each of them being strictly associated with a dmso host molecule ( called e , f , g , and h ) ; see figure s4 . the four unique molecule 3 entities mainly differ by the torsion angles between the pyrimidine ring and the ureic moieties ( table s1 ) . in general , the pyrimidine ring is almost perpendicular to the phenyl mean plane , whereas the ureic group roughly lies in the same plane as the phenyl system . for example , the (o3-c10-n1-c7 ) angle is as low as 6(2 ) , 0(2 ) , 2(2 ) and 7(2 ) degrees in molecules a , b , c , and d , respectively . as for the crystal packing , a / b and c / d alternating pairs of closely associated independent molecules are arranged along the [ 110 ] axis ( figure s5a ) . within each pair , a cyclic network of n3h3o4 hydrogen - bonded contacts is set up between almost coplanar facing pyrimidine rings ( figure s5b ) . the other pyrimidinic nitrogen ( n4 ) acts as an acceptor of an intermolecular hydrogen bond donated from the n6h2 group ( figure s5c ) , determining the formation of two symmetry - independent infinite hydrogen - bonded zigzag ribbons in the ( 110 ) plane ( figure s5a ) . moreover , the ureic system is involved in hydrogen - bonded contacts with the oxygen of the cocrystallized solvent , whereas the o3 oxygen is a hydrogen - bond acceptor interacting with a neighboring n5h2 group . table s2 summarizes all the relevant intermolecular hydrogen - bonded contacts in crystalline 3 . the preparation of an efficient recombinant escherichia coli protein production system as well as the purification , crystallization , and structure determination of tbfold in a ternary complex with nadp and the inhibitor ( s)-2-(4-(3-(2,4-diamino-6-oxo-1,6-dihydro pyrimidin-5-yl ) ureido)benzamido ) pentanedioic acid ( 2 ) is detailed in the supporting information . the inhibitor and adp component of the cofactor were clearly defined by the high - resolution electron density , but the density was less well ordered for the nicotinate moiety . the tbfold subunit , which is a polypeptide of 297 amino acids , displays a distinctive tertiary structure typical of this enzyme family . this subunit consists of 11 -helices and 11 -strands forming a two - domain structure.tbfold forms a dimer in solution , as shown by size exclusion chromatography , and a dimer constitutes the asymmetric unit of the crystal structure . approximately 10% of a subunit surface is occluded from the solvent by dimer formation . the c - terminal domain displays the rossmann fold and binds nadp in a deep cleft . the adenine occupies a hydrophobic pocket near the surface of the enzyme , and the nicotinate is then located between the c- and n - terminal domains . the amino acids that form the cofactor - binding site and interact with the cofactor are highly conserved . we previously noted in different crystal structures of bacterial fold that the conformation of a loop adjacent to the active site , the 8-10 loop , was variable . the loop occludes the active site in the structure of the pseudomonas aeruginosa fold ( pafold ) . in the case of the tbfold , the loop adopts an open configuration , lining and helping to create the active site ( figure s6 ) . the folate - binding catalytic center is a deep , solvent - filled cavity on the n - terminal domain . here , 2 binds with an extended conformation ( figure 3 ) . the pyrimidine headgroup is wedged between k56 and q100 on one side and i174 ( not shown ) on the other . direct hydrogen bonding interactions between the inhibitor headgroup and the enzyme involve the side chain of d123 , the main chain carbonyls of l101 and v99 , and the main chain amide of l101 . additionally , a number of solvent - mediated interactions link 2 o4 to the side chains of k56 and d123 . the k56 side chain also binds to the carbonyl group of the linking amide . the 2 benzyl forms -stacking interactions on one side with y52 ; on the other side , there are van der waals interactions with g276 , p277 , and t279 . the -carboxylate group of the ligand glutamate moiety participates in a direct charge - reinforced hydrogen bond with the g273 main chain nitrogen and a long - range ionic interaction with the side chain of r10 . in similar fashion , the -carboxylate interacts with r54 . in addition , there are several solvent - mediated interactions that link these acidic groups to g274 , g276 , and y250 . the side chains of t51 , l55 , and l252 together with p272 form a hydrophobic clamp to position the tail of the inhibitor . the polypeptide is depicted as off - white ribbon , the interacting residues , and the nicotinate moiety of the cofactor , which was modeled , are represented with c atoms as orange sticks , the ligand as cyan sticks . all o , n , and h atom positions are red , blue , and white , respectively . compound 2 was tested for the inhibition of fold dehydrogenase activity using an established assay . to enlarge the sar study , we decided to include in the panel of compounds under evaluation also the synthetic intermediates 35 . we selected compound ly354899 ( 21 ) , a competitive inhibitor of the human ( ki 29 nm ) , pseudomonas aeruginosa ( 30 nm ) and l. major ( 105 nm ) fold enzymes , as a reference compound , whose structure represents a rigid cyclized analogue of compound 2 . superimposition of the available a. baumanii fold/21 structure ( pdb code 4v4v ) on the tbfold/2 complex , herein described , shows that the two ligands adopt the same binding conformation ( figure s7 in supporting information ) . in accordance , compound 21 displayed tbfold inhibitory activity with a ki comparable to that of compound 2 ( ki = 8.5 m and 1.1 m , respectively ) . in vitro growth inhibition expressed as ic50 ( m ) of all compounds against the t. brucei bloodstream form and human thp1 differentiated macrophages . compound structures , enzyme inhibition , antiparasitic activity , and human macrophage growth inhibition are reported . in stark contrast , the inactivity or very low level of inhibition observed with compounds 35 confirms that the aforementioned interactions displayed by the glutamate tail are essential for the inhibitory activity . indeed , both the elimination of the glutamate portion ( compounds 34 ) and the esterification of the -carboxylate group ( compound 5 ) led to a significant drop in the inhibitory properties of these compounds . on the basis of the tbfold/2/nadp ternary complex presented here , new compounds 1620 were designed , synthesized , and tested in an enzyme assay . specifically , compounds 18 and 19 were designed to fill the hydrophobic cleft formed by the side chains of y52 , y250 , l252 , and t51 . indeed , derivative 18 ( ki = 0.54 m ) displayed a 2-fold increase of the inhibitory potency , compared to that of compound 2 . in contrast , the lower activity of 19 suggests that the hydroxyl group on the terminal phenyl ring is not well accepted , probably due to its electron - donating properties that unfavorably affect the interaction with y52 and y250 . we considered that a direct interaction with r54 would be beneficial for inhibition , and compound 16 ( ki = 0.48 m ) was designed for that purpose . the 2-fold increase in the inhibitory potency of this compound , compared to that of compound 2 , seems to confirm our hypothesis . however , compounds 17 and 20 were synthesized as proof - of - concept for the importance of the c and c carboxylate groups , respectively . both derivatives showed a decreased inhibitory potency , confirming , once again , that at least two strong interactions should be established by the amino acidic tail in order to achieve submicromolar inhibition , either one charge - reinforced h - bond and one ionic interaction , as for compounds 2 and 16 , or one charge - reinforced h - bond and one hydrophobic interaction , as in the case of compound 18 . potential binding poses of 16 and 18 , the most potent inhibitors identified in this series , were calculated by means of glide 5.5 software in extra precision ( xp ) mode , using glidescore for ligand ranking ( see below ) . as shown in figure 4 , the elongation of the -carboxylate chain ( 16 vs 2 ) allows for a salt bridge interaction with the side chains of r54 or r10 . specifically , in the tbfold-2-nadp x - ray structure the shortest distance between the 2 carboxylate oxygen and an r54 guanidine hydrogen was around 5 , while in the docking - derived pose the same distance is reduced to 2.6 . this direct and by implication stronger association is likely the reason for the 2-fold increase in inhibitory potency of 16 with respect to 2 . ( a ) two possible binding modes of 16 within tbfold as resulted from docking studies . compound 18 was originally designed to fill the hydrophobic pocket shaped by the three residues y52 , y250 , and l252 residues . indeed , two favored binding poses have been found , one in which the phenyl ring occupies the above - mentioned hydrophobic cleft forming a t - shaping assembly with the two tyrosine residue side chains ( see figure 4 ) and another in which the ligand phenyl group is directed toward r10 establishing a cation- interaction ( data not shown ) . however , the lower activity of 19 would perhaps suggest that the latter pose may not be relevant but would support the interaction of the terminal phenyl ring with y52 and y25 . these latter interactions could be , in turn , the reason for the increased inhibitory potency of 18 with respect to 2 . superimposition of the three - dimensional x - ray structures of human and tbfolds demonstrates that the enzyme presents a similar structure in these two diverse species and that the amino acids lining the active sites are overall well conserved ( figure s2 , overall sequence identity of 40% ) . nevertheless , a number of residues differ between the two enzymes , and a close inspection of the different residues lining the active sites reveals structural differences that could be exploited for the discovery of new trypanocidal agents endowed with low activity against the human fold . specifically , in human fold , residues k10 , l51 , n54 , v55 , k175 , c236 , and i238 are replaced by r10 , t51 , r54 , l55 , d173 , v236 , and t238 , respectively , in tbfold ( figure 5 ) . the lysine arginine asparagine arginine differences at positions 10 and 54 might be targeted to achieve selectivity in inhibitor both tbfold r10 and r54 are in close proximity to the glutamate tail of 2 and may be reached via appropriate modification of the ligand s glutamate residue . thus , the crystal structure of the tbfold-2-nadp ternary complex and comparison with the human fold structure may help address the challenging problem of how to achieve both potency and selectivity toward the parasite and not the human enzyme . superimposition of the human fold ( pdb code 1dig ) and tbfold/2 ( pdb 4lrr ) complexes , represented as green and orange ribbons and sticks , respectively . all compounds were screened against the bloodstream form of t. brucei and showed in vitro dose - dependent killing , as determined from the reduction of the resazurin marker for cell viability ( table 1 ) . despite the fact that several compounds within this series showed a micromolar or even submicromolar activity against the target enzyme tbfold , they did not display any remarkable antiparasitic activity . only compounds 25 showed a moderate antiparasitic activity , and 2 was the most active , with an ic50 value of 49 3.2 m . the reference compound , analogue 21 , exhibited a similar activity . all compounds were less active than suramin , a standard drug used in hat therapy . a counter - screen testing all compounds with human macrophages differentiated from thp1 monocytes showed more effective toxic activity on the parasite than in mammalian cells . compounds 2 , 5 , and 21 were 4- , 2.2- , and 1.8-fold more effective , respectively , against the t. brucei bloodstream form than against that from human macrophages . we have reported the first crystal structure of tbfold , a potential therapeutic target , with the bonus of obtaining information on the molecular basis of inhibition by compound 2 . this molecule displays a micromolar enzyme inhibitory activity against tbfold and modest antiparasitic properties . a key part of the analysis was the clear assignment of the molecular structure of 2 using single crystal x - ray analysis of a synthetic intermediate , 3 . the crystal structure of the enzyme ligand complex provides an accurate template to support structure - based approaches in early stage drug discovery . in addition , we have suggested that , although fold is a highly conserved enzyme , as indicated by the primary sequence alignment and structural comparisons , some critical differences in the active sites could be exploited to reduce the activity on the human form of the enzyme , and this possibility will be pursued in future development of this class of compounds . moreover , because molecular recognition is a dynamic process , differences in structural flexibility between the human and t. brucei enzymes might provide additional information to support this effort . despite the fact that almost all compounds showed low micromolar or submicromolar inhibition of tbfold , the antiparasitic activity on the bloodstream form parasite was modest . considering the polar nature of these molecules , an explanation for the modest level of antiparasitic activity is that structural modification of the amino acidic tail , while facilitating a better interaction with the target enzyme , has compromised the ability to cross the parasite membrane , likely due to a reduced affinity for membrane transporters . indeed , very little modification of the glu chain , such as the one carbon homologation of compound 2 leading to 16 , has produced a complete loss of antiparasitic activity . this suggests that , to obtain more efficacious analogues , also the interaction with membrane transporters should be considered at the onset of the design process . h nmr and c nmr spectra were recorded with a varian mercury 300 ( 300 mhz ) spectrometer . chemical shifts ( ) are expressed in ppm , and coupling constants ( j ) are expressed in hz . tlc analyses were performed on commercial silica gel 60 f254 aluminum sheets ; spots were further evidenced by spraying with a dilute alkaline potassium permanganate solution or ninhydrin . melting points were determined on a model b 540 bchi apparatus and are uncorrected . mass spectrometry was carried out on a triple quadrupole spectrometer type varian 320-ms coupled with esi source . elemental analyses were performed on a perkinelmer pe 2400 elemental analyzer , and the data for c , h , and n were within 0.4% of the theoretical values . all target compounds possessed a purity of 95% as verified by elemental analyses by comparison with the theoretical values . 4-hydroxy-2,5,6-triaminopyrimidine sulfate ( 0.91 g , 3.8 mmol ) was suspended in water ( 2 ml ) and mixed with a 1 n naoh solution ( 11.4 ml ) . a solution of ethyl 4-isocyanatobenzoate ( 0.73 mg , 3.8 mmol ) in 6 ml of acetonitrile was added dropwise . after stirring at room temperature for 2.5 h , 1 n hcl ( 11.4 ml ) was added , and the mixture was stirred for 5 min . the solid was filtered off and washed with water ( 50 ml ) , etoh ( 30 ml ) , and diethyl ether ( 30 ml ) . after drying , the product was obtained as a pale orange solid ( 0.95 g , 2.86 mmol , 75% yield ) . m.p . h nmr ( 300 mhz , dmso - d6 ) ( ppm ) : 10.0 ( bs , 1h , -nhconh- ) , 8.98 ( bs , 1h , -nhconh ) , 7.84 ( d , 2h , j = 8.4 , ar h ) , 6.76 ( s , 1h , -nh ) , 6.16 ( bs , 2h , -nh2 ) , 5.96 ( bs , 2h , -nh2 ) , 4.26 ( q , 2h , j = 7.0 , ch2ch3 ) , 1.31 ( t , 3h , j = 7.0 , ch2ch3 ) . c nmr ( 75 mhz , dmso - d6 ) ( ppm ) : 166.22 , 162.38 , 160.97 , 155.26 , 154.02 , 145.89 , 130.89 , 122.60 , 117.61 , 89.94 , 60.85 , 14.94 . calcd for c14h16n6o4 : c 50.60 , h 4.85 , n 25.29 ; found , c 50.62 , h 4.98 , n 25.03 . compound 3 ( 0.95 g , 2.86 mmol ) was suspended in water ( 25 ml ) , and 1 n naoh ( 29 ml ) was added . the mixture was stirred at room temperature for 3 h , and it slowly turned into a clear solution . 1 n hcl ( 29 ml ) was added , and the precipitate was collected by centrifugation ( 5000 rpm for 3 min ) . the solid was sequentially washed and centrifuged ( 5000 rpm for 3 min ) with water ( 20 ml ) , etoh ( 20 ml ) , and diethyl ether ( 20 ml ) . the product was collected as a pale orange solid ( 0.80 g , 2.63 mmol , 92% yield ) . m.p . : h nmr ( 300 mhz , dmso - d6 ) ( ppm ) : 12.50 ( bs , 1h , cooh ) , 9.98 ( bs , 1h , -nhconh- ) , 8.90 ( bs , 1h , -nhconh ) , 7.80 ( d , 2h , j = 7.6 , ar h ) , 6.71 ( s , 1h , -nh ) , 6.18 ( bs , 2h , -nh2 ) , 5.88 ( bs , 2h , -nh2 ) . c nmr ( 75 mhz , dmso - d6 ) ( ppm ) : 167.81 , 162.20 , 160.88 , 155.26 , 153.98 , 145.58 , 131.07 , 123.50 , 117.49 , 90.05 . calcd for c12h12n6o4 : c 47.37 , h 3.98 , n 27.62 ; found , c 47.41 , h 4.06 , n 27.40 . a suspension of compound 4 ( 0.1 g , 0.33 mmol ) in dry dmf ( 7 ml ) was placed under a nitrogen atmosphere and sonicated for 5 min . n - methylmorpholine ( 0.146 ml , 1.32 mmol ) was added to the mixture , followed by 2-chloro-4,6-dimethoxy-1,3,5-triazine ( 232 mg , 1.32 mmol ) , and the mixture was stirred at 35 c under nitrogen for 5 h. the suspension slowly turned into an orange - red solution . the desired amino acid ( 610 ) as methyl or ethyl ester hydrochloride ( 1.32 mmol ) was added to the solution followed by n - methylmorpholine ( 0.146 ml , 1.32 mmol ) , and the mixture was stirred at 30 c overnight . the solvent was removed under vacuum , keeping the temperature below 45 c , and the crude mixture was resuspended in etoh ( 10 ml ) and stirred for 5 min . the solid was recovered by vacuum filtration and washed with etoh ( 10 ml ) and diethyl ether ( 10 ml ) and finally dried under vacuum overnight . h nmr ( 300 mhz , dmso - d6 ) ( ppm ) : 9.95 ( bs , 1h , -nhconh- ) , 8.82 ( bs , 1h , -nhconh ) , 8.48 ( d , 1h , j = 7.0 , arco - nh- ) , 7.80 ( d , 2h , j = 8.8 , ar h ) , 6.70 ( s , 1h , -nh ) , 6.18 ( bs , 2h , -nh2 ) , 5.88 ( bs , 2h , -nh2 ) , 4.404.37 ( m , 1h , h ) , 4.07 ( q , 2h , j = 7.3 , ch2ch3 ) , 4.00 ( q , 2h , j = 7.3 , ch2ch3 ) , 2.452.38 ( m , 2h , ch2ch2cooet ) , 2.171.95 ( m , 2h , chch2ch2cooet ) , 1.17 ( t , 3h , j = 7.3 , ch2ch3 ) , 1.13 ( t , 3h , j = 7.3 , ch2ch3 ) . c nmr ( 75 mhz , dmso - d6 ) ( ppm ) : 172.92 , 172.66 , 166.98 , 162.17 , 160.88 , 155.37 , 153.95 , 144.36 , 129.06 , 126.59 , 117.37 , 90.10 , 61.17 , 60.60 , 52.65 , 30.91 , 26.46 , 14.76 ; ms 490.2 [ m + h ] . calcd for c21h27n7o7 : c 51.53 , h 5.56 , n 20.03 ; found , c 51.61 , h 5.60 , n 19.91 . h nmr ( 300 mhz , dmso - d6 ) 9.96 ( bs , 1h , conhar ) , 8.82 ( bs , 1h , pyrim - nhco ) , 8.50 ( d , 1h , j = 7.4 , conhc ) , 7.77 ( d , 2h , j = 8.8 , ar h ) , 7.50 ( d , 2h , j = 8.8 , ar h ) , 6.69 ( s , 1h , oh ) , 6.14 ( bs , 2h , -nh2 ) , 5.88 ( bs , 2h , -nh2 ) , 4.38 ( dt , 1h , j = 6.9 , 7.4 , h ) , 3.62 ( s , 3h , och3 ) , 3.56 ( s , 3h , och3 ) , 2.32 ( t , 2h , j = 7.3 , ch2ch2ch2coome ) , 1.891.72 ( m , 2h , ch2ch2ch2coome ) , 1.681.53 ( m , 2h , ch2ch2ch2cooch3 ) . c nmr ( 75 mhz , dmso - d6 ) 173.76 , 173.45 , 166.88 , 162.29 , 160.88 , 155.37 , 153.97 , 144.37 , 129.07 , 126.53 , 117.34 , 90.05 , 53.00 , 52.51 , 51.94 , 33.46 , 30.51 , 22.00 . calcd for c20h25n7o7 : c 50.52 ; h 5.30 ; n 20.62 ; found , c 50.62 , h 5.58 , n 20.43 . h nmr ( 300 mhz , dmso - d6 ) 9.98 ( bs , 1h , conhar ) , 8.82 ( bs , 1h , pyrim - nhco ) , 8.55 ( d , 1h , j = 6.9 , conhc ) , 7.77 ( d , 2h , j = 8.7 , ar h ) , 7.50 ( d , 2h , j = 8.7 , ar h ) , 6.70 ( s , 1h , oh ) , 6.17 ( bs , 2h , -nh2 ) , 5.89 ( bs , 2h , -nh2 ) , 4.43 ( dq , 1h , j = 6.9 , 7.1 , h ) , 3.62 ( s , 3h , och3 ) , 1.37 ( d , 3h , j = 7.1 , chch3 ) . c nmr ( 75 mhz , dmso - d6 ) 174.08 , 166.52 , 162.20 , 160.88 , 155.37 , 153.95 , 144.33 , 129.03 , 126.52 , 117.31 , 90.03 , 52.50 , 48.86 , 17.49 . calcd for c16h19n7o5 : c 49.35 ; h 4.92 ; n 25.18 ; found , c 49.38 , h 4.95 , n 24.98 . dmso - d6 ) 9.98 ( bs , 1h , conhar ) , 8.81 ( bs , 1h , pyrim - nhco ) , 8.61 ( d , 1h , j = 7.0 , conhc ) , 7.70 ( d , 2h , j = 8.8 , ar h ) , 7.48 ( d , 2h , j = 8.8 , ar h ) , 7.307.21 ( m , 4h , ch2c6h5 ) , 7.207.13 ( m , 1h , ch2c6h5 ) , 6.70 ( s , 1h , oh ) , 6.16 ( bs , 2h , -nh2 ) , 5.89 ( bs , 2h , -nh2 ) , 4.60 ( ddd , 1h , j = 6.0 , 8.0 , 9.1 , h ) , 3.61 ( s , 3h , och3 ) 3.173.04 ( m , 2h , j = 7.3 , ch2-c6h5 ) . c nmr ( 75 mhz , dmso - d6 ) 173.11 , 166.74 , 162.27 , 160.87 , 155.35 , 153.96 , 144.36 , 138.49 , 129.74 , 128.99 , 128.93 , 127.15 , 126.48 , 117.34 , 90.05 , 54.71 , 52.57 , 36.97 . calcd for c22h23n7o5 : c 56.77 ; h 4.98 ; n 21.06 ; found , c 56.96 , h 5.18 , n 20.82 . h nmr ( 300 mhz , dmso - d6 ) 9.97 ( bs , 1h , conhar ) , 9.19 ( s , 1h , ch2c6h4oh ) , 8.83 ( bs , 1h , pyrim - nhco ) , 8.55 ( d , 1h , j = 7.8 , conhc ) , 7.70 ( d , 2h , j = 8.7 , ar h ) , 7.48 ( d , 2h , j = 8.7 , ar h ) , 7.06 ( d , 2h , j = 8.4 , ch2c6h4oh ) , 6.71 ( s , 1h , oh ) , 6.63 ( d , 2h , j = 8.4 , ch2c6h4oh ) , 6.15 ( bs , 2h , -nh2 ) , 5.89 ( bs , 2h , -nh2 ) , 4.564.45 ( m , 1h , h ) , 3.60 ( s , 3h , och3 ) , 3.052.87 ( m , 2h , ch2c6h4oh ) . c nmr ( 75 mhz , dmso - d6 ) 173.26 , 166.73 , 162.31 , 160.89 , 156.58 , 155.36 , 153.97 , 144.34 , 130.67 , 129.00 , 128.43 , 126.53 , 117.30 , 115.71 , 90.03 , 55.37 , 52.49 , 36.26 . calcd for c22h23n7o6 : c 54.88 ; h 4.82 ; n 20.36 ; found , c , 54.97 ; h , 4.97 ; n , 20.01 . , dec t > 210 c ; h nmr ( 300 mhz , dmso - d6 ) 9.98 ( bs , 1h , conhar ) , 8.77 ( bs , 1h , pyrim - nhco ) , 8.26 ( t , 1h , j = 5.5 , conhch2 ) , 7.71 ( d , 2h , j = 8.5 , ar h ) , 7.47 ( d , 2h , j = 8.5 , ar h ) , 6.69 ( s , 1h , oh ) , 6.16 ( bs , 2h , -nh2 ) , 5.88 ( bs , 2h , -nh2 ) , 3.56 ( s , 3h , och3 ) , 3.23 ( dt , 2h , j = 5.5 , 6.6 , ch2ch2ch2coome ) , 2.34 ( t , 2h , j = 7.1 , ch2ch2ch2coome ) , 1.75 ( tt , 2h , j = 6.6 , 7.1 , ch2ch2ch2coome ) . c nmr ( 75 mhz , dmso - d6 ) 173.88 , 166.57 , 162.28 , 160.88 , 155.39 , 153.96 , 143.94 , 128.65 , 127.49 , 117.36 , 90.11 , 51.92 , 39.09 , 31.53 , 25.28 . calcd for c17h21n7o5 : c 50.62 ; h 5.25 ; n 24.31 ; found , c 50.52 , h 5.38 , n 24.33 . compound 5 ( or analogues 1115 ) ( 0.2 mmol ) was dissolved in 1 n naoh ( 0.8 mmol ) , and the solution was stirred at room temperature for 5 h. the solution was neutralized with 1 n hcl , and the precipitate was recovered by vacuum filtration and washed with water ( 10 ml ) , etoh ( 20 ml ) , and diethyl ether ( 20 ml ) and finally dried under vacuum overnight . h nmr ( 300 mhz , dmso - d6 ) ( ppm ) : 12.35 ( bs , 1h , cooh ) , 9.98 ( bs , 1h , -nhconh- ) , 8.82 ( bs , 1h , -nhconh ) , 8.38 ( d , 1h , j = 6.6 , arco - nh- ) , 7.79 ( d , 2h , j = 8.0 , ar h ) , 6.68 ( s , 1h , -nh ) , 6.18 ( bs , 2h , -nh2 ) , 5.90 ( bs , 2h , -nh2 ) , 4.404.30 ( m , 1h , h ) , 2.382.30 ( m , 2h , ch2ch2cooh ) , 2.102.00 ( m , 1h , chch2ch2cooh ) , 2.001.90 ( m , 1h , chch2ch2cooh ) . c nmr ( 75 mhz , dmso - d6 ) ( ppm ) : 174.58 , 174.28 , 166.85 , 162.30 , 160.90 , 155.39 , 153.98 , 144.26 , 129.01 , 126.82 , 117.36 , 90.10 , 52.55 , 31.15 , 26.68 . ms : 434.1 [ m + h ] . anal . calcd for c17h19n7o7 : c 47.11 , h 4.42 , n 22.62 ; found , c 47.20 , h 4.50 , n 22.46 . h nmr ( 300 mhz , d6-dmso ) 12.26 ( bs , 2h , cooh ) , 9.97 ( bs , 1h , conhar ) , 8.80 ( bs , 1h , pyrim - nhco ) , 8.35 ( d , 1h , j = 7.7 , conhc ) , 7.78 ( d , 2h , j = 8.7 , ar h ) , 7.50 ( d , 2h , j = 8.7 , ar h ) , 6.69 ( s , 1h , oh ) , 6.14 ( bs , 2h , -nh2 ) , 5.87 ( bs , 2h , -nh2 ) , 4.31 ( dt , 1h , j = 5.1 , 7.7 , h ) , 2.22 ( t , 2h , j = 7.4 , ch2ch2ch2cooh ) , 1.891.68 ( m , 2h , ch2ch2ch2cooh ) , 1.681.49 ( m , 2h , ch2ch2ch2cooch3 ) . c nmr ( 75 mhz , d6-dmso ) 174.96 , 174.54 , 166.69 , 162.27 , 160.90 , 155.38 , 153.99 , 144.21 , 128.97 , 126.95 , 117.35 , 90.14 , 53.09 , 33.97 , 30.90 , 22.15 . ms 448.1 m / z [ m + h ] . calcd for c18h21n7o7 : c 48.32 ; h 4.73 ; n 21.91 ; found , c 48.42 , h 4.90 , n 21.63 . h nmr ( 300 mhz , dmso - d6 ) 12.44 ( bs , 1h , cooh ) , 9.98 ( bs , 1h , conhar ) , 8.82 ( bs , 1h , pyrim - nhco ) , 8.40 ( d , 1h , j = 7.1 , conhc ) , 7.77 ( d , 2h , j = 8.8 , ar h ) , 7.49 ( d , 2h , j = 8.8 , ar h ) , 6.69 ( s , 1h , oh ) , 6.13 ( bs , 2h , -nh2 ) , 5.87 ( bs , 2h , -nh2 ) , 5.74 ( s , 1h , oh ) , 4.37 ( dq , 1h , j = 7.1 , 7.4 , h ) , 1.36 ( d , 3h , j = 7.4 , chch3 ) . c nmr ( 75 mhz , dmso - d6 ) 175.15 , 166.33 , 162.32 , 160.90 , 155.40 , 153.99 , 144.19 , 128.94 , 126.88 , 117.33 , 90.07 , 48.85 , 17.8 . calcd for c15h17n7o5 : c 48.00 ; h 4.57 ; n 26.12 ; found , c 47.84 , h 4.78 , n 25.93 . h nmr ( 300 mhz , dmso - d6 ) 12.76 ( bs , 1h , cooh ) , 9.97 ( bs , 1h , conhar ) , 8.79 ( bs , 1h , pyrim - nhco ) , 8.46 ( d , 1h , j = 8.2 , conhc ) , 7.69 ( d , 2h , j = 8.8 , ar h ) , 7.48 ( d , 2h , j = 8.8 , ar h ) , 7.337.21 ( m , 4h , ch2c6h5 ) , 7.197.12 ( m , 1h , ch2c6h5 ) , 6.69 ( s , 1h , oh ) , 6.14 ( bs , 2h , -nh2 ) , 5.87 ( bs , 2h , -nh2 ) , 4.56 ( ddd , 1h , j = 4.6 , 8.2 , 10.5 , h ) , 3.15 ( dd , 1h , j = 4.6 , 13.8 , ch2-c6h5 ) , 3.04 ( dd , 1h , j = 10.5 , 13.8 , ch2-c6h5 ) . c nmr ( 75 mhz , dmso - d6 ) 174.09 , 166.64 , 162.27 , 160.88 , 155.35 , 153.97 , 144.22 , 138.99 , 129.74 , 128.91 , 128.85 , 126.99 , 126.81 , 117.33 , 90.08 , 54.88 , 37.01 . calcd for c21h21n7o5 : c 55.87 ; h 4.69 ; n 21.72 ; found , c 55.57 , h 4.94 , n 21.52 . dec t > 198 c ; h nmr ( 300 mhz , dmso - d6 ) 12.61 ( bs , 1h , cooh ) , 9.96 ( bs , 1h , conhar ) , 9.16 ( s , 1h , ch2c6h4oh ) , 8.81 ( bs , 1h , pyrim - nhco ) , 8.39 ( d , 1h , j = 8.2 , conhc ) , 7.69 ( d , 2h , j = 8.8 , ar h ) , 7.47 ( d , 2h , j = 8.8 , ar h ) , 7.07 ( d , 2h , j = 8.4 , ch2c6h4oh ) , 6.69 ( s , 1h , oh ) , 6.62 ( d , 2h , j = 8.4 , ch2c6h4oh ) , 6.15 ( bs , 2h , -nh2 ) , 5.89 ( bs , 2h , -nh2 ) , 4.534.41 ( m , 1h , h ) , 3.02 ( dd , 1h , j = 4.5 , 13.8 , ch2-c6h4oh ) , 2.91 ( dd , 1h , j = 10.1 , 13.8 , ch2-c6h4oh ) ; c nmr ( 75 mhz , dmso - d6 ) 174.24 , 166.64 , 162.27 , 160.88 , 156.48 , 155.37 , 153.96 , 144.21 , 130.66 , 128.94 , 126.86 , 126.81 , 117.32 , 115.65 , 90.03 , 55.24 , 36.25 . ms 468.2 m / z [ m + h ] . calcd for c21h21n7o6 : c 53.96 ; h 4.53 ; n 20.98 ; found , c 54.12 , h 4.78 , n 20.64 . h nmr ( 300 mhz , dmso - d6 ) 12.02 ( bs , 1h , cooh ) , 9.97 ( bs , 1h , conhar ) , 8.78 ( bs , 1h , pyrim - nhco ) , 8.25 ( t , 1h , j = 5.8 , conhch2 ) , 7.72 ( d , 2h , j = 8.8 , ar h ) , 7.47 ( d , 2h , j = 8.8 , ar h ) , 6.68 ( s , 1h , oh ) , 6.13 ( bs , 2h , -nh2 ) , 5.86 ( bs , 2h , -nh2 ) , 3.23 ( dt , 2h , j = 5.8 , 6.6 , ch2ch2ch2cooh ) , 2.25 ( t , 2h , j = 7.4 , ch2ch2ch2cooh ) , 1.72 ( tt , 2h , j = 5.8 , 6.6 , ch2ch2ch2cooh ) . c nmr ( 75 mhz , dmso - d6 ) 174.99 , 166.56 , 162.25 , 160.88 , 155.39 , 153.96 , 143.92 , 128.64 , 127.57 , 117.37 , 90.16 , 39.24 , 31.94 , 25.36 . calcd for c16h19n7o5 : c 49.35 ; h 4.92 ; n 25.18 ; found , c 49.32 , h 4.78 , n 25.02 . with the aim to test the glide 5.5 program for its ability to reproduce the crystallized binding geometry of 2 , the latter ligand was subjected to automated docking calculations using extra precision ( xp ) mode and glidescore for ligand ranking . before docking , the program was successful in reproducing the experimentally found binding mode of 2 , as it corresponds to the best ranked solution with an rmsd of only 0.87 . the herein reported x - ray structure of tbfold was prepared through the protein preparation wizard within the maestro 9.0.2112 package using the opls-2001 force field . compound 3 is poorly soluble in most organic solvents at room temperature , with the exception of dmso . we therefore tried to grow crystals from dmso ( in different conditions ) and from mixtures of dmso / ch3cn . eventually , crystallization by slow evaporation ( 16 days ) of a solution of 3 in reagent - grade dmso ( sigma - aldrich ) at room temperature produced small plates suitable for the determination of the molecular connectivity . after testing several samples , a specimen ( 0.25 0.20 0.05 mm ) grown at the liquor / air interface was selected for the crystallographic analysis . the specimen manifested a significant pleochroism ( colorless to violet ) under polarized light . it was mounted on the top of a glass capillary fiber in dense perfluorinated oil . the x - ray data collection was performed at room temperature on a 3-circle bruker smart apex goniometer equipped with a ccd apex - ii detector , using graphite - monochromated mo k radiation ( = 0.71073 ) . a total of 26180 reflections ( 8144 unique ) were recorded up to ( sin /)max = 0.5017 . a high exposure time ( 120 s / frame ) was employed due to the small dimensions of the sample and its very low scattering power . the compound was found to crystallize in the triclinic centrosymmetric p1 space group ( no . 2 ) , with unit cell parameters a = 8.0260(16 ) , b = 10.054(2 ) , c = 48.956(10 ) , = 84.44(3) , = 89.13(3) , = 80.14(3) , and v = 3873.8(14 ) . careful visual inspection of the reciprocal lattice showed some weak off - lattice spots attributable to a minor non- merohedral epitaxial twin component of the same 3 phase . experimental structure factor amplitudes were extracted by integration of the diffraction frames through the saint+ program suite , taking into account the presence of the additional lattice . corrections for sample absorption and x - ray beam anisotropy were applied by the program twinabs . eventually , the molecular structure was solved and refined with the shelx program package . in the final least - squares refined model , the nonmerohedral twinning was explicitly accounted for , and the fractional contribution of the minority component was estimated to be 0.161(3 ) . all of the hydrogen atoms in the asymmetric unit were idealized , and their coordinates were indirectly determined through a riding motion moreover , all the bonds involving non - h atoms were subjected to a rigid - bond restraint , i.e. , the component of the atomic anisotropic displacement parameters along the bond direction were restrained to be equal within a tolerance of 0.01 . this strategy was motivated by ( i ) the rather poor quality of the sample and , consequently , the relatively low maximum resolution available for the data ; ( ii ) the quite low data - to - parameter ratio ( 8) , due to the high number of symmetry - unrelated molecules in a large unit cell . however , these drawbacks did not hamper us from reliably determining the molecular connectivity of 3 , even though our estimates for bond distances and angles have , on average , a rather low precision . the latter can be obtained free of charge from the cambridge crystallographic data centre via www.ccdc.cam.ac.uk/data_request/cif . the gene encoding 5,10-methylene tetrahydrofolate dehydrogenase/5,10 methenyl tetrahydrofolate cyclohydrolase ( dhch1 ) was identified in genedb ( http://www.genedb.org , accession number tb927.7.1600 ) . genomic dna from t. brucei ( lister 927 strain ) was used as template for pcr with the following primers designed to amplify the dhch1 open reading frame using ndei and xho1 restriction sites ( bold ) , respectively : 5-cat - atg - cct - gag - gcg - gtt - g-3 and 5-ctc - gag - tca - caa - ggc - acg - aa-3. the pcr product was inserted into pcr - bluntii - topo vector using the zero blunt topo pcr cloning kit ( invitrogen ) prior to excision and ligation into a modified pet15b vector ( novagen ) containing a tobacco etch virus ( tev ) protease recognition sequence ( pet15btev ) . this results in recombinant expression of a product carrying an n - terminal hexa - histidine tag ( his - tag ) , which is cleavable with tev protease . the recombinant plasmid was amplified in xl-1 blue e. coli , and the gene sequence verified by dna sequencing services ( dundee university ) , before being transformed into e. coli bl21 ( de3 ) for protein production . protein was produced in cells cultured at 37 c in 1 l lb media containing 50 mg / ml ampicillin . when an od of 0.8 was attained , expression was induced with iptg ( 1 mm ) for 16 h at 21 c . cells were collected by centrifugation at 4000 rpm for 30 min at 4 c and frozen at 80 c until required . after thawing , the cells were resuspended in 50 mm bicine and 50 mm nacl , ph 8.0 ( using 2 ml / g pellet ) , with the addition of complete ( roche edta - free protease inhibitor ) then lysed by sonication ( 7 10 s bursts ) , with cooling to < 4 c between pulses . the lysate was clarified with centrifugation ( 40000 g , 30 min , 4 c ) . the supernatant was loaded , at 2 ml min , onto a histrap hp ml column ( prepacked with ni sepharose high performance - ge healthcare ) equilibrated with buffer 50 mm bicine and 50 mm nacl , ph 8.0 . retained proteins were eluted with a linear gradient at 1 ml min of 0100% of buffer 50 mm bicine , 50 mm nacl , and 1 m imidazole , ph 8.0 . the sample of tbfold was passed through a hitrap desalting ( ge healthcare ) column to exchange the buffer with 50 mm bicine , 250 mm nacl , 0.5 mm dtt , and 10% glycerol , ph 8.0 . the addition of 10% glycerol in all buffers was required to prevent the loss of activity when the samples were flash - frozen . crystallization trials were carried out with a phoenix liquid handling system ( art robbins instruments / rigaku ) using commercially available screens ( hampton research ) with a 1:1 ratio of 100 nl of protein solution and an equivalent volume of reservoir equilibrated against a 70 l reservoir at 20 c . crystals , small orthorhombic blocks with approximate dimensions of 0.1 0.1 0.1 mm , were observed after 3 days in conditions with 20% peg 6000 and 0.1 m citric acid , ph 5.0 . we were unable to increase the size of the crystals using hanging drop or sitting drop vapor diffusion methods . crystals were transferred into a cryo - solution containing the reservoir supplemented with 20% glycerol prior to flash freezing at 173 c . crystals were characterized in - house with a micromax-007 rotating anode generator and r - axis iv dual image plate detector ( rigaku ) , prior to storage in liquid nitrogen . x - ray diffraction data were then collected on the microfocus beamline of id23 - 2 at the european synchrotron radiation facility ( esrf ) . the crystals belonged to the orthorhombic space group p212121 with unit cell parameters a = 58.22 , b = 77.33 , c = 128.91 , = = = 90. the molecular weight of a subunit is 31.9 kda , and the asymmetric unit consists of two subunits with a vm value of 2.79 da and solvent content of approximately 55% . the structure of tbfold was solved by molecular replacement and refined to a resolution of 2.05 . the search model was a monomer of the l. major fold structure , which shares a sequence identity of 85% ( pdb code a26 ) , with side chains removed . the rotation and translation functions were determined with phaser with a log likelihood gain of 84 . inspection of the solution in the graphics program coot showed that a dimer , consistent with gel filtration data , was indeed present . side chains were built into electron and difference density maps , followed by iterative rounds of restrained refinement , model manipulation , and addition of solvent molecules using coot and refmac5 . geometric restrained parameters were manually adjusted and applied in addition to translation / libration / screw analysis ( tls ) during the latter stages of refinement . structure superpositions were calculated using lsqkab , and figures were prepared using pymol ( schrdinger llc ) . the dehydrogenase activity of fold was measured spectrophotometrically , using a beckman du-640 spectrophotometer , following the formation of 5,10-ch = thf from 5,10-ch2-thf . 5,10-ch2-thf dehydrogenase assays were carried out in a 0.5 ml volume at 27 c and contained 25 mm mops , ph 7.3 , 30 mm 2-mercaptoethanol , 35 m mthf , and 1 mm nadp . the km values of substrate and cofactor having been previously elucidated as approximately 35 and 70 m . the reaction was initiated by the addition of nadp and incubated for 5 min then stopped by the addition of an equal volume of 1 m hcl and the 5,10-ch = thf produced quantified at 350 nm , using an extinction coefficient of 24.9 mm cm . the enzyme activity was expressed as moles 5,10-ch = thf produced per minute . enzyme inhibition was measured using the dehydrogenase assay , with stocks of compounds dissolved in dmso ( 10 mm ) . data from inhibition assays were fitted to a competitive model by linear regression using origin ( originlab corporation ) . the bloodstream - form t. brucei brucei strain 427 was grown in hmi-9 medium 17.66 mg / ml imdm ( gibco ) , 3.020 mg / ml sodium bicarbonate ( sigma - aldrich ) , 0.136 mg / ml hypoxanthine ( sigma - aldrich ) , 0.039 mg / ml thymidine ( sigma - aldrich ) , and 0.028 mg / ml bathocuproine sulfonic acid ( sigma - aldrich ) supplemented with 10% heat - inactivated fetal bovine serum ( fbs ) ( biowhittaker ) , 1.5 mm l - cysteine hydrochloride monohydrate ( merck ) , 0.2 mm -mercaptoethanol ( sigma - aldrich ) , 100 u / ml penicillin , and 100 u / ml streptomycin ( biowhittaker ) at 37 c under a humidified 5% co2 atmosphere . cultures were grown in t25 or t75 vented cap culture flasks ( sarstedt ) and subcultured every 23 days by 100- to 1000-fold dilution , respectively . parasites were counted directly using a neubauer chamber ( marienfeld ) and diluted appropriately in complete hmi-9 medium . the compounds under study and 21 were tested in a serial drug dilution assay in order to determine the ic50 values ( concentration of drug causing 50% growth inhibition ) by using the alamar blue assay . serial drug dilutions were prepared in 96-well microtiter plates containing culture medium as described above , and wells were inoculated with approximately 2,000 bloodstream form t. b. brucei cells . cultures were incubated for 72 h at 37 c under a humidified 5% co2 atmosphere . after this time the plates were incubated for an additional 4 h , and then the fluorescence read in a microplate reader ( sinergy 2 , biotek ) using an excitation wavelength of 528 nm and an emission wavelength of 590 nm . the cytotoxicity of the compounds under study was assessed by a colorimetric mtt assay . thp1 differentiated macrophages were seeded at a density of 10 cells / well in 96-well plates and allowed to adhere overnight . cells were incubated with the compound concentration range for 72 h at 37 c . at the end of the incubation period , 200 l of 0.5 mg / ml mtt reagent ( thiazolyl blue tetrazolium bromide , sigma ) solution was added to each well . the plates were further incubated for 4 h at 37 c in the dark . the culture medium was subsequently discarded , and 200 l of isopropanol was added to dissolve the dark - blue formazan crystals . cell viability was quantified spectrophotometrically by measuring the absorbance of the formazan product at wavelength 570 nm and the background at 660 nm with a microplate reader ( synergy 2 , biotek , usa ) . the data are expressed as the percentages of viable cells compared to the survival of a control group ( untreated cells ) . the ic50 value , i.e. , the concentration of compounds necessary to decrease cell viability to 50% of the untreated control was determined by linear regression analysis . differences between in vitro anti - trypanosoma and thp1 cell cytotoxicity were examined using student s t test . the data are presented as the means sd , and all experiments were independently repeated at least three times . p - values < 0.05 ( two - sided ) were considered to be statistically significant .

the bifunctional enzyme n5,n10-methylenetetrahydrofolate dehydrogenase / cyclo hydrolase ( fold ) is essential for growth in trypanosomatidae . we sought to develop inhibitors of trypanosoma brucei fold ( tbfold ) as potential antiparasitic agents . compound 2 was synthesized , and the molecular structure was unequivocally assigned through x - ray crystallography of the intermediate compound 3 . compound 2 showed an ic50 of 2.2 m , against tbfold and displayed antiparasitic activity against t. brucei ( ic50 49 m ) . using compound 2 , we were able to obtain the first x - ray structure of tbfold in the presence of nadp+ and the inhibitor , which then guided the rational design of a new series of potent tbfold inhibitors .

Introduction Results Conclusions Experimental Section

diseases caused by kinetoplastids include human african trypanosomiasis ( hat , also known as african sleeping sickness ) , which is due to infection with trypanosoma brucei gambiense or t. brucei rhodesiense ; chagas disease , which is caused by t. cruzi ; and various forms of leishmaniasis caused by infection with different species of leishmania . sleeping sickness , which is found predominantly across sub - saharan africa , is fatal if not treated , and all drugs used in the treatment of hat have issues relating to efficacy , administration , and side effects . we have previously identified the bifunctional enzyme fold as an interesting target for antibacterial drug discovery . we initiated an inhibitor discovery program targeting the t. brucei enzyme ( tbfold ) and selected as a lead compound ly374571 ( compound 1 ) developed by eli lilly and company because it inhibits bacterial and human fold . however , following the protocol reported for the synthesis of 1 , we obtained a new compound whose structure has been unambiguously determined as 2 ( figure 2 ) . to confirm 2 as a suitable lead compound for the development of tbfold inhibitors , we were able to obtain the first x - ray structure of tbfold in the presence of nadp and the inhibitor . the x - ray crystal structure of the complex tbfold-2-nadp revealed molecular details that were relevant to the binding and inhibition of the enzyme , and allowed , with the help of molecular modeling , a rational design of several analogues ( compounds 1620 ) . antiparasitic activity against the bloodstream form of t. brucei and cytotoxicity against human leukemia macrophages , differentiated from thp1 monocytes , were evaluated for all compounds under study . compound 2 was prepared following a procedure originally reported to afford 1 ( figure 2 ) . our structural assignment is based on the x - ray analysis of 3 , which was crystallized as a thin plate by slow evaporation from dimethyl sulfoxide ( dmso ) . figure 2b shows the molecular formula of one symmetry - independent molecule of 3 with dmso and the atom numbering scheme ( see supporting information for details on crystal determination : figure s3 , s4 , and s5 ) . a series of new analogues of compound 2 were designed on the basis of the crystallographic data and molecular modeling studies , as discussed in detail later . the molecular structure of 3 , as obtained from x - ray diffraction analysis , is shown in figure s3 . the preparation of an efficient recombinant escherichia coli protein production system as well as the purification , crystallization , and structure determination of tbfold in a ternary complex with nadp and the inhibitor ( s)-2-(4-(3-(2,4-diamino-6-oxo-1,6-dihydro pyrimidin-5-yl ) ureido)benzamido ) pentanedioic acid ( 2 ) is detailed in the supporting information . the inhibitor and adp component of the cofactor were clearly defined by the high - resolution electron density , but the density was less well ordered for the nicotinate moiety . this subunit consists of 11 -helices and 11 -strands forming a two - domain structure.tbfold forms a dimer in solution , as shown by size exclusion chromatography , and a dimer constitutes the asymmetric unit of the crystal structure . the adenine occupies a hydrophobic pocket near the surface of the enzyme , and the nicotinate is then located between the c- and n - terminal domains . the loop occludes the active site in the structure of the pseudomonas aeruginosa fold ( pafold ) . direct hydrogen bonding interactions between the inhibitor headgroup and the enzyme involve the side chain of d123 , the main chain carbonyls of l101 and v99 , and the main chain amide of l101 . the side chains of t51 , l55 , and l252 together with p272 form a hydrophobic clamp to position the tail of the inhibitor . the polypeptide is depicted as off - white ribbon , the interacting residues , and the nicotinate moiety of the cofactor , which was modeled , are represented with c atoms as orange sticks , the ligand as cyan sticks . in vitro growth inhibition expressed as ic50 ( m ) of all compounds against the t. brucei bloodstream form and human thp1 differentiated macrophages . indeed , both the elimination of the glutamate portion ( compounds 34 ) and the esterification of the -carboxylate group ( compound 5 ) led to a significant drop in the inhibitory properties of these compounds . on the basis of the tbfold/2/nadp ternary complex presented here , new compounds 1620 were designed , synthesized , and tested in an enzyme assay . indeed , derivative 18 ( ki = 0.54 m ) displayed a 2-fold increase of the inhibitory potency , compared to that of compound 2 . the 2-fold increase in the inhibitory potency of this compound , compared to that of compound 2 , seems to confirm our hypothesis . specifically , in the tbfold-2-nadp x - ray structure the shortest distance between the 2 carboxylate oxygen and an r54 guanidine hydrogen was around 5 , while in the docking - derived pose the same distance is reduced to 2.6 . superimposition of the three - dimensional x - ray structures of human and tbfolds demonstrates that the enzyme presents a similar structure in these two diverse species and that the amino acids lining the active sites are overall well conserved ( figure s2 , overall sequence identity of 40% ) . nevertheless , a number of residues differ between the two enzymes , and a close inspection of the different residues lining the active sites reveals structural differences that could be exploited for the discovery of new trypanocidal agents endowed with low activity against the human fold . the lysine arginine asparagine arginine differences at positions 10 and 54 might be targeted to achieve selectivity in inhibitor both tbfold r10 and r54 are in close proximity to the glutamate tail of 2 and may be reached via appropriate modification of the ligand s glutamate residue . all compounds were screened against the bloodstream form of t. brucei and showed in vitro dose - dependent killing , as determined from the reduction of the resazurin marker for cell viability ( table 1 ) . only compounds 25 showed a moderate antiparasitic activity , and 2 was the most active , with an ic50 value of 49 3.2 m . compounds 2 , 5 , and 21 were 4- , 2.2- , and 1.8-fold more effective , respectively , against the t. brucei bloodstream form than against that from human macrophages . we have reported the first crystal structure of tbfold , a potential therapeutic target , with the bonus of obtaining information on the molecular basis of inhibition by compound 2 . this molecule displays a micromolar enzyme inhibitory activity against tbfold and modest antiparasitic properties . a key part of the analysis was the clear assignment of the molecular structure of 2 using single crystal x - ray analysis of a synthetic intermediate , 3 . in addition , we have suggested that , although fold is a highly conserved enzyme , as indicated by the primary sequence alignment and structural comparisons , some critical differences in the active sites could be exploited to reduce the activity on the human form of the enzyme , and this possibility will be pursued in future development of this class of compounds . despite the fact that almost all compounds showed low micromolar or submicromolar inhibition of tbfold , the antiparasitic activity on the bloodstream form parasite was modest . considering the polar nature of these molecules , an explanation for the modest level of antiparasitic activity is that structural modification of the amino acidic tail , while facilitating a better interaction with the target enzyme , has compromised the ability to cross the parasite membrane , likely due to a reduced affinity for membrane transporters . indeed , very little modification of the glu chain , such as the one carbon homologation of compound 2 leading to 16 , has produced a complete loss of antiparasitic activity . elemental analyses were performed on a perkinelmer pe 2400 elemental analyzer , and the data for c , h , and n were within 0.4% of the theoretical values . 1 n hcl ( 29 ml ) was added , and the precipitate was collected by centrifugation ( 5000 rpm for 3 min ) . n - methylmorpholine ( 0.146 ml , 1.32 mmol ) was added to the mixture , followed by 2-chloro-4,6-dimethoxy-1,3,5-triazine ( 232 mg , 1.32 mmol ) , and the mixture was stirred at 35 c under nitrogen for 5 h. the suspension slowly turned into an orange - red solution . the desired amino acid ( 610 ) as methyl or ethyl ester hydrochloride ( 1.32 mmol ) was added to the solution followed by n - methylmorpholine ( 0.146 ml , 1.32 mmol ) , and the mixture was stirred at 30 c overnight . the solvent was removed under vacuum , keeping the temperature below 45 c , and the crude mixture was resuspended in etoh ( 10 ml ) and stirred for 5 min . compound 5 ( or analogues 1115 ) ( 0.2 mmol ) was dissolved in 1 n naoh ( 0.8 mmol ) , and the solution was stirred at room temperature for 5 h. the solution was neutralized with 1 n hcl , and the precipitate was recovered by vacuum filtration and washed with water ( 10 ml ) , etoh ( 20 ml ) , and diethyl ether ( 20 ml ) and finally dried under vacuum overnight . h nmr ( 300 mhz , dmso - d6 ) 12.76 ( bs , 1h , cooh ) , 9.97 ( bs , 1h , conhar ) , 8.79 ( bs , 1h , pyrim - nhco ) , 8.46 ( d , 1h , j = 8.2 , conhc ) , 7.69 ( d , 2h , j = 8.8 , ar h ) , 7.48 ( d , 2h , j = 8.8 , ar h ) , 7.337.21 ( m , 4h , ch2c6h5 ) , 7.197.12 ( m , 1h , ch2c6h5 ) , 6.69 ( s , 1h , oh ) , 6.14 ( bs , 2h , -nh2 ) , 5.87 ( bs , 2h , -nh2 ) , 4.56 ( ddd , 1h , j = 4.6 , 8.2 , 10.5 , h ) , 3.15 ( dd , 1h , j = 4.6 , 13.8 , ch2-c6h5 ) , 3.04 ( dd , 1h , j = 10.5 , 13.8 , ch2-c6h5 ) . dec t > 198 c ; h nmr ( 300 mhz , dmso - d6 ) 12.61 ( bs , 1h , cooh ) , 9.96 ( bs , 1h , conhar ) , 9.16 ( s , 1h , ch2c6h4oh ) , 8.81 ( bs , 1h , pyrim - nhco ) , 8.39 ( d , 1h , j = 8.2 , conhc ) , 7.69 ( d , 2h , j = 8.8 , ar h ) , 7.47 ( d , 2h , j = 8.8 , ar h ) , 7.07 ( d , 2h , j = 8.4 , ch2c6h4oh ) , 6.69 ( s , 1h , oh ) , 6.62 ( d , 2h , j = 8.4 , ch2c6h4oh ) , 6.15 ( bs , 2h , -nh2 ) , 5.89 ( bs , 2h , -nh2 ) , 4.534.41 ( m , 1h , h ) , 3.02 ( dd , 1h , j = 4.5 , 13.8 , ch2-c6h4oh ) , 2.91 ( dd , 1h , j = 10.1 , 13.8 , ch2-c6h4oh ) ; c nmr ( 75 mhz , dmso - d6 ) 174.24 , 166.64 , 162.27 , 160.88 , 156.48 , 155.37 , 153.96 , 144.21 , 130.66 , 128.94 , 126.86 , 126.81 , 117.32 , 115.65 , 90.03 , 55.24 , 36.25 . with the aim to test the glide 5.5 program for its ability to reproduce the crystallized binding geometry of 2 , the latter ligand was subjected to automated docking calculations using extra precision ( xp ) mode and glidescore for ligand ranking . before docking , the program was successful in reproducing the experimentally found binding mode of 2 , as it corresponds to the best ranked solution with an rmsd of only 0.87 . the herein reported x - ray structure of tbfold was prepared through the protein preparation wizard within the maestro 9.0.2112 package using the opls-2001 force field . eventually , crystallization by slow evaporation ( 16 days ) of a solution of 3 in reagent - grade dmso ( sigma - aldrich ) at room temperature produced small plates suitable for the determination of the molecular connectivity . experimental structure factor amplitudes were extracted by integration of the diffraction frames through the saint+ program suite , taking into account the presence of the additional lattice . corrections for sample absorption and x - ray beam anisotropy were applied by the program twinabs . eventually , the molecular structure was solved and refined with the shelx program package . in the final least - squares refined model , the nonmerohedral twinning was explicitly accounted for , and the fractional contribution of the minority component was estimated to be 0.161(3 ) . all of the hydrogen atoms in the asymmetric unit were idealized , and their coordinates were indirectly determined through a riding motion moreover , all the bonds involving non - h atoms were subjected to a rigid - bond restraint , i.e. genomic dna from t. brucei ( lister 927 strain ) was used as template for pcr with the following primers designed to amplify the dhch1 open reading frame using ndei and xho1 restriction sites ( bold ) , respectively : 5-cat - atg - cct - gag - gcg - gtt - g-3 and 5-ctc - gag - tca - caa - ggc - acg - aa-3. we were unable to increase the size of the crystals using hanging drop or sitting drop vapor diffusion methods . x - ray diffraction data were then collected on the microfocus beamline of id23 - 2 at the european synchrotron radiation facility ( esrf ) . the crystals belonged to the orthorhombic space group p212121 with unit cell parameters a = 58.22 , b = 77.33 , c = 128.91 , = = = 90. the molecular weight of a subunit is 31.9 kda , and the asymmetric unit consists of two subunits with a vm value of 2.79 da and solvent content of approximately 55% . the structure of tbfold was solved by molecular replacement and refined to a resolution of 2.05 . the search model was a monomer of the l. major fold structure , which shares a sequence identity of 85% ( pdb code a26 ) , with side chains removed . inspection of the solution in the graphics program coot showed that a dimer , consistent with gel filtration data , was indeed present . the bloodstream - form t. brucei brucei strain 427 was grown in hmi-9 medium 17.66 mg / ml imdm ( gibco ) , 3.020 mg / ml sodium bicarbonate ( sigma - aldrich ) , 0.136 mg / ml hypoxanthine ( sigma - aldrich ) , 0.039 mg / ml thymidine ( sigma - aldrich ) , and 0.028 mg / ml bathocuproine sulfonic acid ( sigma - aldrich ) supplemented with 10% heat - inactivated fetal bovine serum ( fbs ) ( biowhittaker ) , 1.5 mm l - cysteine hydrochloride monohydrate ( merck ) , 0.2 mm -mercaptoethanol ( sigma - aldrich ) , 100 u / ml penicillin , and 100 u / ml streptomycin ( biowhittaker ) at 37 c under a humidified 5% co2 atmosphere . after this time the plates were incubated for an additional 4 h , and then the fluorescence read in a microplate reader ( sinergy 2 , biotek ) using an excitation wavelength of 528 nm and an emission wavelength of 590 nm . cell viability was quantified spectrophotometrically by measuring the absorbance of the formazan product at wavelength 570 nm and the background at 660 nm with a microplate reader ( synergy 2 , biotek , usa ) .

the tnm staging system was initially devised more than 50 years ago by pierre denoix in france . from then on , many revisions were made in advent of changes in breast cancer management and treatment . in 1987 , the international union against cancer ( uicc ) and the ajcc staging systems were unified into a single tnm staging system to be able to communicate clinical information without ambiguity . t is for the size or direct extent of the primary tumor , n is for the spread of regional lymph nodes and m is for the presence of distant metastasis . since the first publication in 1977 , currently ajcc / uicc system revised and published the 7th edition . it is arguable that n part of the staging changed most significantly since the sln , biopsy first described by giuliano et al . in 1994 in breast cancer as a potential altering the role of alnd by using intraoperative lymphatic mapping . along with sln biopsy advancement by surgical oncologists , pathology assessment of sln biopsy sample led to significant changes in n - stage by the ajcc staging manual . starting from the 5th edition in 1997 when the first sln was reported , the 6th edition in 2003 and the 7th edition in 2010 had some changes regarding sln reporting . as sln biopsy became more a standard treatment in the management of breast cancer , the ajcc staging manual needed revision to reflect the continual development and knowledge in this field . the 6th edition of the ajcc cancer staging manual when compared to the previous one contains some of the most extensive and significant revisions pertaining to sln related to the growing knowledge of new technology such as ihc staining and reverse transcriptase - polymerase chain reaction ( rt - pcr ) . the principal changes are related to the size , number , location , and method of detection of regional metastases to the lymph nodes . a breast task force was constituted to serve in an advisory role to the ajcc in the fall of 2001 and the 6th edition reflected the significant changes . the breast task force used the guideline to recommend changes and additions based on evidence - based findings in published clinical outcome data , reflecting a wide - spread clinical consensus and changes in nomenclature and coding system which support the uniform accrual of outcome information in national databanks . in the ajcc 5th edition , micrometastases were defined as metastatic lesions no larger than 2 mm in greatest dimension and classified as pn1 . the terminology occult metastases was interchangeably used to describe a small metastatic carcinoma in the lymph node in the literature . the upper limit of 2 mm cut - off for separating micro- and macrometastases was determined by two studies 30 years ago . the major change in the ajcc 6th edition was to define the lower limit for micrometastasis defined as a metastatic lesion larger than 0.2 mm in diameter and the upper limit was kept in 2 mm and itc ( single cells or cell deposits ) no larger than 0.2 mm and classified as pn0 . this lower limit of > 0.2 mm ( 10 times smaller than the upper limit ) had been tested in only one retrospective study . with the advent of more sensitive technique , such as ihc stains , it was easier to detect itc that were not readily visible from the h&e stain slides which was the gold standard for detection of metastatic carcinoma in the axillary lymph nodes . for example , when h&e stain shows no metastatic carcinoma but ihc stain with itc , then the classifier became pn0(i+ ) for positive ihc , no cluster > 0.2 mm . the designation of pn1mi ( i+ ) was used when h&e stain negative but ihc stain detected micrometastasis . separating the upper and lower limits of cutoff for itc and micrometastatic lymph node is not an easy task since there is no pure study that has evaluated differences in overall survival compounding factors such as with or without tumor size , chemotherapy and radiation therapy . the pn0(mol- ) and pn0(mol+ ) classifiers were also added in the 6th edition of ajcc based on the molecular findings using rt - pcr . the additional designation of ( sn ) for sentinel node was used in the classification of tnm staging system when only the sln was excised without the full alnd . , knowing sentinel nodes are more likely to contain metastases than non - slns , pathologists began to perform much more thorough and comprehensive examinations which included grossing by submitting the entire node by sectioning 2.0 mm thickness in a longest axis , serial sections for h&e stains instead of just one section , and adding ihc stains which deviated from the reference population assay in which lymph node examination was much simpler- previously , grossly negative large lymph nodes were not necessarily entirely submitted for histologic examination . introduction of itc was to prevent overtreatment of low - volume nodal involvement since some of the itc is caused by passive tumor cell transport to the sln after pre - operative fine needle aspiration ( fna ) and/or core needle biopsy procedures and iatrogenic displacement due to breast massage causing benign epithelial elements in lymph node resulting false positive especially in papillary lesions . as we all know , finding metastatic carcinoma in lymph nodes is a statistical exercise of probability . with more h&e and ihc stain sections we get from the block , the probability of having positive node increases . we have the capability to detect even one cell in the sln if this is clinically meaningful but first , we need to figure out whether this effort is paid off by clinical significance . also , there are differences in terminology in literature . prior to ajcc 2003 and 2006 , the term isolated tumor cells and micrometastasis were not clearly defined . some studies referred to these as occult metastasis . later on the ajcc definition of sln tumor deposits were measured using a micrometer and placed in one of three categories : macrometastasis , micrometastasis , or itc . a macrometastasis was classified as one or more tumor deposits greater than 2 mm . itc were defined as single cells or small cluster of cells not greater than 0.2 mm in largest dimension . a cluster was defined as a confluent focus of tumor cells touching other tumor cells . single dispersed cells throughout the lymph node were regarded as itc if the largest cluster measured less than or equal to 0.2 mm . the presence of metastatic disease is measured by the size of the largest contiguous metastasis for multiple foci in sln . the main differences between the ajcc 6th edition to the ajcc 7th edition in regards to sln is to count the number of metastatic cells ; if it is less than 200 cells versus over 200 cells for itc and micrometastasis respectively . i stands for isolated tumor cells and not ihc stain for the 7th edition ajcc . the main reason for a 200 cell cutoff was to have better reproducibility between pathologists . the arbitrary cutoff of the exact size and number was listed for improvement of agreement between pathologists without any clinical trial of the significance in prognosis and clinical validation . in fact , this was incorporated into the 7th edition for large dishesive , non - confluent tumor cells throughout the lymph node typically seen in metastatic lobular carcinoma . in addition , the 7th edition of ajcc states these thresholds are meant to be guidelines , and not absolute cutoffs , to help pathologists determine if the tumor burden in a given lymph nodes is likely to be clinically important or not . the pathologist should use judgment and not an absolute cut - off of 0.2 mm or exact 200 cells , in determining the likelihood of whether the cluster of cells is an itc or a true micrometastasis . see table 2 . according to the 7th edition ajcc , a positive node is defined as a metastasis measuring at least 0.2 mm , or > 200 tumor cells ( pn1mi ) . whenever there is a positive lymph node , the pathologist also needs to describe the following : the size of metastasis ( or number of tumor cells ) , the anatomical node involved such as axilla , internal mammary , intramammary , infraclavicular or supraclavicular lymph node , the total number of positive nodes , and the method of detection such as clinical , fna or tissue biopsy . positive internal mammary sln affects pn stage depending on status of other nodes ( metastasis in clinically apparent ipsilateral internal mammary lymph nodes in the presence of 1 or more positive axillary lymph nodes ; or in more than 3 axillary lymph nodes and in internal mammary nodes with microscopic disease detected by sln dissection but not clinically apparent ) . positive infraclavicular node is staged as pn3a and supraclavicular node is staged as pn3c . in addition , there is a troubling statement in the ajcc 2010 data elements with asterisks ( small clusters of cells not greater than 0.2 mm or single tumor cells , or a cluster of fewer than 200 cells in a single histologic cross - section ) are not required . however , these elements may be clinically important but are not yet validated or regularly used in patient management . further disclaimer statements in the 7th edition of ajcc are as follows : approximately 1,000 tumor cells are contained in a 3-dimensional 0.2-mm cluster . thus , if more than 200 individual tumor cells are identified as single dispersed tumor cells or as a nearly confluent elliptical or spherical focus in a single histologic section of a lymph node , there is a high probability that more than 1,000 cells are present in the node . in these situations , cells in different lymph node cross sections or longitudinal sections or levels of the block are not added together ; the 200 cells must be in a single node profile even if the node has been thinly sectioned into multiple slices . it is recognized that there is substantial overlap between the upper limit of the itc and the lower limit of the micrometastasis categories because of inherent limitations in pathologic nodal evaluation and detection of minimal tumor burden in lymph nodes . thus , the threshold of 200 cells in a single cross - section is a guideline to help pathologists distinguish between these 2 categories . and final statement is as follows : the pathologist should use judgment regarding whether it is likely that the cluster of cells represents a true micrometastasis or is simply a small group of isolated tumor cells . in 2010 , both the uicc and tnm joined in the provision in guidelines for sln classification , however , studies have shown poor reproducibility in the application of in both invasive ductal and invasive lobular carcinomas . furthermore , previous studies have shown that application of ihc stain on h&e negative lymph node can detect small volume metastatic carcinoma in 12%29% of cases . however , all the previous data of prognosis based on lymph node status has been from one h&e section evaluation . therefore due to more thorough examination of sln , more cases of itc have been reported [ 1 - 6 ] . for this reason , the college of american pathologists ( cap ) published a consensus statement in july 2000 recommending a single h&e slide section from each lymph node block without gross evidence of metastasis without routine cytokeratin ihc stain or routine serial sectioning based on the preliminary study from john wayne cancer center which found no differences in five - year disease - free survival rate between node negative by h&e negative , ihc negative versus h&e negative , ihc positive groups [ 18 - 20 ] . although cap guidelines and recommendations for sln biopsy for pathologists did not include serial sectioning with multiple h&e slides and ihc stains , many pathologists reported and continued to perform comprehensive evaluation on sln including our institution to this date . with the advent of the comprehensive sln evaluation , probability of finding small metastasis it is reported that among patients with a positive sln by h&e stain were found to have additional nodal metastasis in alnd in 48.3% of cases . in addition , metastatic carcinoma is found in non - sentinel nodes during alnd in 9%15% and 15%35% of patients with itc and micrometastasis in the sln respectively [ 22 - 25 ] . traditionally , complete alnd was done for sln with pn1mi and macrometastasis but not for pn0(i+ ) . therefore , it would be important to distinguish an accurate size of metastatic tumor deposits in the sln . most pathologists submit clinically negative sln entirely for microscopic examination by slicing lymph node in a longest axis into 0.2 cm thickness based on cap guideline . cap states that a single h&e section from each block is sufficient and multiple level sections and ihc stain for keratin are not necessary . however , there are many institutions performing multiple levels and ihc stains for each block for sln method is still a matter of debate . in our institution , we get 3 multiple levels for h&e stain and 2 intervening keratin ihc stains for entirely submitted each sln blocks . the reason for this is that ihc stain highlights small metastatic cells easier than h&e stain , especially for the metastatic lobular carcinoma cases . while the 7th edition ajcc criteria and definition are fairly straightforward , its application may be difficult in reality for pathologists to uniformly classify n - stage correctly connolly described some of the most practical challenges in the classification in the assessment of sln biopsy separating itc , micrometastases , and macrometastases . reported that there is 24% discordance in their review of 512 cases of low - volume nodal metastasis and a plea for better formulated supplemented tnm definition . literature reported the kappa values for pathologist s agreement is at best moderate to substantial reproducibility [ 28 - 32 ] . the kappa value for interobserver agreement was reported moderate ( 0.55 and 0.56 ) for pn0(i+ ) and substantial ( 0.62 ) for pn1mi . this is an improvement from the previous interobserver agreement amongst expert breast pathologists ( kappa : 0.49 ) and community hospital - based pathologists ( kappa : 0.47 ) based on european working group for breast screening pathology ( ewgbsp ) . despite the intent for the better interpretation of metastatic tumor cells in lymph node in the 7th edition ajcc , many issues have not been resolved . in 2012 , netherlands group published the discrepancy rate between central to local pathologists in which changed pn status in 24% with kappa value of 0.69 . this is stating that one of four cases in breast cancer , pathologists do not agree with n - stage even though the descriptor for n - stage is easily understandable in the ajcc staging manual . there are still some circumstances pathologists have difficulties knowing how to accurately assign n- stage based on the 7th edition ajcc . here the entire area of metastatic carcinoma is > 2 mm but the largest contiguous metastatic carcinoma measures < 0.2 mm . the ajcc staging criteria states that we should not add these noncontiguous metastatic foci but should nt there be an upper limit to number of foci of micrometastases to upgrade to macrometastasis based on the sheer volume of tumor deposits ? does this actually make a common sense to classify as pn1mi ? using the best pathologist s judgment , macrometastasis may be best for this case although by strict criteria , it is pn1mi . the entire area will be classified as micrometastasis but one of the longest contiguous areas will be classified as itc . metastatic carcinoma seen in axillary adipose or fibrous tissue without any residual and apparent lymph node structure . should this be counted as positive lymph node or a carcinoma arising in axillary breast tissue ? based on the 6th edition , cancerous nodules in the axillary fat without evidence of residual lymph node tissue should be classified as positive axillary lymph nodes . but what if these cancerous nodules in the axilla are cancer cells from the axillary breast tissue ? should it be classified as extensive extracapsular extension ? many pathologists do not classify cancerous nodules in axilla without apparent lymph node structure as lymph node metastasis ( unpublished personal observation ) . the dimension of the tumor from this scenario may end up in t - stage rather than in n - stage . 4 . when there is pericapsular lymph - vascular invasion ( lvi ) in which tumor cells are not in the parenchyma or subcapsular part of lymph node , is that considered metastatic or lvi ? deeper sections still did not show intracapsular or intraparenchymal involvement . anecdotally , this kind of the case was sent out to other breast pathology experts and came back with different opinions ; some considered metastatic carcinoma pn0(i+ ) or pn1mi depending on the size of the largest contiguous dimension of metastatic focus and some just lvi . based on cap guidelines , capsular lvi is considered metastatic carcinoma and the largest dimension is measured as the size of metastasis . 5 . when most of the metastatic carcinoma is seen outside the lymph node capsule ( in this case by metastatic lobular carcinoma ) with only one itc within the lymph node parenchyma , is it considered extracapsular invasion with itc or is it micrometastasis based on the total number of cells of > 200 or > 0.2 mm ? invasive lobular carcinoma often has loss of e - cadherin which results in individual cell pattern in both breast and lymph node . this scenario was written by turner et al . who reported inconsistence in nodal metastases by breast pathologists with the initial kappa before the training program was 0.575 and post - test was 0.947 . we reported that strict adherence of the 6th edition ajcc criteria will result in down staging n in many lobular carcinoma cases . the 7th edition ajcc was bit more helpful in this situation because one needs to count the number of metastatic cells in sln ; if it is less than 200 cells versus over 200 cells for isolated tumor cells and micrometastasis respectively . however , the number 200 cells is arbitrarily chosen without any background published research data with clinical follow - up . although some metastatic lobular carcinoma cases have obvious macrometastasis , diffuse single or few cell clusters of infiltrating pattern is typical for invasive lobular carcinoma metastatic to lymph node ( fig . all cases such as this will be classified as pn1mi based on > 200 cells but why ca nt it be macrometastasis ? is there an upper limit such as 500 cells or 1,000 cells to qualify as macrometastasis ? 8 . for n - stage , if there is stromal desmoplasia or stromal proliferation around small volume metastasis , the 6th edition ajcc states that the size of the tumor deposit should include the stromal reaction . in general , after neoadjuvant chemotherapy , the classification of lymph nodes should be the same as the patients who did not have neoadjuvant chemotherapy . lymph nodes after neoadjuvant chemotherapy more frequently show stromal fibrosis without typical desmoplasia , foamy histiocytes with multinucleated giant cell reaction , elastosis and small clusters of non - confluent residual and viable metastatic tumor cells . after all , even small residual metastatic deposits after neoadjuvant chemotherapy is probably not true itc but a remnant of macrometastasis prior to therapy and hence , it should be classified as pn1 . currently , the ajcc staging manual is not clear about this scenario and hence , pathologists are stating in their report how they measured the size of metastatic deposit with a long explanation . location of metastatic tumor cells whether it is subcapsular or intraparenchymal is not specified in the 7th edition ajcc , however , many european pathologists based on previous uicc related publications , tumor cells located within the parenchyma measuring even less than 0.2 mm is considered micrometastasis and not considered as itc . regardless of the location , most of american pathologists would consider itc purely depending on the size of metastasis . the above stated scenarios are difficult to resolve and ultimately depends on the pathologists to make appropriate judgments resulting high subjectivity and inconsistence in n - stage . finally , we need to ask ourselves , is this distinction between pn0 ( i+ ) and pn1mi useful for patient s prognosis , treatment plans and overall survival ? for that matter , is macrometastasis different in prognosis and overall survival when compared to pn0(i+ ) and pn1mi ? our goal is to determine the significance of these findings , albeit that there is difficulty in interobserver reproducibility among the pathologists . also we should keep in mind that both retrospective and prospective studies in literature composed of variability in interpretation of n - stages ; pn0(i+ ) , pn1mi and pn1a even with the best intention of adhering to ajcc criterion . most pathologists submit clinically negative sln entirely for microscopic examination by slicing lymph node in a longest axis into 0.2 cm thickness based on cap guideline . cap states that a single h&e section from each block is sufficient and multiple level sections and ihc stain for keratin are not necessary . however , there are many institutions performing multiple levels and ihc stains for each block for sln method is still a matter of debate . in our institution , we get 3 multiple levels for h&e stain and 2 intervening keratin ihc stains for entirely submitted each sln blocks . the reason for this is that ihc stain highlights small metastatic cells easier than h&e stain , especially for the metastatic lobular carcinoma cases . while the 7th edition ajcc criteria and definition are fairly straightforward , its application may be difficult in reality for pathologists to uniformly classify n - stage correctly . connolly described some of the most practical challenges in the classification in the assessment of sln biopsy separating itc , micrometastases , and macrometastases . reported that there is 24% discordance in their review of 512 cases of low - volume nodal metastasis and a plea for better formulated supplemented tnm definition . literature reported the kappa values for pathologist s agreement is at best moderate to substantial reproducibility [ 28 - 32 ] . the kappa value for interobserver agreement was reported moderate ( 0.55 and 0.56 ) for pn0(i+ ) and substantial ( 0.62 ) for pn1mi . this is an improvement from the previous interobserver agreement amongst expert breast pathologists ( kappa : 0.49 ) and community hospital - based pathologists ( kappa : 0.47 ) based on european working group for breast screening pathology ( ewgbsp ) . despite the intent for the better interpretation of metastatic tumor cells in lymph node in the 7th edition ajcc , many issues have not been resolved . in 2012 , netherlands group published the discrepancy rate between central to local pathologists in which changed pn status in 24% with kappa value of 0.69 . this is stating that one of four cases in breast cancer , pathologists do not agree with n - stage even though the descriptor for n - stage is easily understandable in the ajcc staging manual . there are still some circumstances pathologists have difficulties knowing how to accurately assign n- stage based on the 7th edition ajcc . here the entire area of metastatic carcinoma is > 2 mm but the largest contiguous metastatic carcinoma measures < 0.2 mm . is this macrometastasis or micrometastasis ? the ajcc staging criteria states that we should not add these noncontiguous metastatic foci but should nt there be an upper limit to number of foci of micrometastases to upgrade to macrometastasis based on the sheer volume of tumor deposits ? using the best pathologist s judgment , macrometastasis may be best for this case although by strict criteria , it is pn1mi . 3 . the entire area will be classified as micrometastasis but one of the longest contiguous areas will be classified as itc . metastatic carcinoma seen in axillary adipose or fibrous tissue without any residual and apparent lymph node structure . should this be counted as positive lymph node or a carcinoma arising in axillary breast tissue ? based on the 6th edition , cancerous nodules in the axillary fat without evidence of residual lymph node tissue should be classified as positive axillary lymph nodes . but what if these cancerous nodules in the axilla are cancer cells from the axillary breast tissue ? many pathologists do not classify cancerous nodules in axilla without apparent lymph node structure as lymph node metastasis ( unpublished personal observation ) . the dimension of the tumor from this scenario may end up in t - stage rather than in n - stage . 4 . when there is pericapsular lymph - vascular invasion ( lvi ) in which tumor cells are not in the parenchyma or subcapsular part of lymph node , is that considered metastatic or lvi ? anecdotally , this kind of the case was sent out to other breast pathology experts and came back with different opinions ; some considered metastatic carcinoma pn0(i+ ) or pn1mi depending on the size of the largest contiguous dimension of metastatic focus and some just lvi . capsular lvi is considered metastatic carcinoma and the largest dimension is measured as the size of metastasis . when most of the metastatic carcinoma is seen outside the lymph node capsule ( in this case by metastatic lobular carcinoma ) with only one itc within the lymph node parenchyma , is it considered extracapsular invasion with itc or is it micrometastasis based on the total number of cells of > 200 or > 0.2 mm ? 7 and 8 . should the extracapsular extension be included as a maximum linear dimension of metastasis ? 6 invasive lobular carcinoma often has loss of e - cadherin which results in individual cell pattern in both breast and lymph node . this scenario was written by turner et al . who reported inconsistence in nodal metastases by breast pathologists with the initial kappa before the training program was 0.575 and post - test was 0.947 . we reported that strict adherence of the 6th edition ajcc criteria will result in down staging n in many lobular carcinoma cases . the 7th edition ajcc was bit more helpful in this situation because one needs to count the number of metastatic cells in sln ; if it is less than 200 cells versus over 200 cells for isolated tumor cells and micrometastasis respectively . however , the number 200 cells is arbitrarily chosen without any background published research data with clinical follow - up . although some metastatic lobular carcinoma cases have obvious macrometastasis , diffuse single or few cell clusters of infiltrating pattern is typical for invasive lobular carcinoma metastatic to lymph node ( fig . all cases such as this will be classified as pn1mi based on > 200 cells but why ca nt it be macrometastasis ? is there an upper limit such as 500 cells or 1,000 cells to qualify as macrometastasis ? 8 . for n - stage , if there is stromal desmoplasia or stromal proliferation around small volume metastasis , the 6th edition ajcc states that the size of the tumor deposit should include the stromal reaction . in general , after neoadjuvant chemotherapy , the classification of lymph nodes should be the same as the patients who did not have neoadjuvant chemotherapy . lymph nodes after neoadjuvant chemotherapy more frequently show stromal fibrosis without typical desmoplasia , foamy histiocytes with multinucleated giant cell reaction , elastosis and small clusters of non - confluent residual and viable metastatic tumor cells . after all , even small residual metastatic deposits after neoadjuvant chemotherapy is probably not true itc but a remnant of macrometastasis prior to therapy and hence , it should be classified as pn1 . currently , the ajcc staging manual is not clear about this scenario and hence , pathologists are stating in their report how they measured the size of metastatic deposit with a long explanation . 9 . location of metastatic tumor cells whether it is subcapsular or intraparenchymal is not specified in the 7th edition ajcc , however , many european pathologists based on previous uicc related publications , tumor cells located within the parenchyma measuring even less than 0.2 mm is considered micrometastasis and not considered as itc . regardless of the location , most of american pathologists would consider itc purely depending on the size of metastasis . the above stated scenarios are difficult to resolve and ultimately depends on the pathologists to make appropriate judgments resulting high subjectivity and inconsistence in n - stage . finally , we need to ask ourselves , is this distinction between pn0 ( i+ ) and pn1mi useful for patient s prognosis , treatment plans and overall survival ? for that matter , is macrometastasis different in prognosis and overall survival when compared to pn0(i+ ) and pn1mi ? our goal is to determine the significance of these findings , albeit that there is difficulty in interobserver reproducibility among the pathologists . also we should keep in mind that both retrospective and prospective studies in literature composed of variability in interpretation of n - stages ; pn0(i+ ) , pn1mi and pn1a even with the best intention of adhering to ajcc criterion . in 2005 , an american society of clinical oncology ( asco ) guideline recommendation for sln biopsy in early - breast cancer was published . the recommendation and guideline were based on the literature review from one prospective randomized controlled trial , four limited meta - analysis , 69 published single - institution and multicenter trials . sln biopsy alone without complete alnd is considered standard of care for sln negative patients . completion alnd was standard treatment for patients who had macro- and micrometastasis in sln for t1 and t2 tumors . for large or locally advanced inflammatory breast cancer ( t3 and t4 ) , ductal carcinoma in situ , when breast - conserving surgery is to be done , pregnancy , in the setting of prior non - oncologic breast surgery or axillary surgery , and in the presence of clinically suspicious axillary lymph node , sln biopsy was not recommended . survival data on pn0(i+ ) , pn1mi and pn1 showed mixed results . survival benefit of preforming alnd after itc and/or micrometastasis in sln was not clear cut . again , the mixed results may have been due to the variable definitions of micrometastases and itc in literature and different mode of detection . the surveillance epidemiology and end results ( seer ) database reported that micrometastasis represents a significant prognostic marker . their data consists of 209,720 patient numbers between 1992 and 2003 and the 10 year survival among pn1 , pn1mi and pn0(i+ ) is 73% , 77% , and 78% , respectively . also , n1mi diagnoses increased from 2.3% to 7% . de boer et al . published mirror ( micrometastasis and isolated tumor cells : relevant and robust or rubbish ? ) study in netherlands which included three cohorts with the median follow - up of 5.1 years : 865 patient with negative regional lymph nodes who did not receive adjuvant chemotherapy , 865 patients with either itc or micrometastasis in lymph nodes who did not receive adjuvant chemotherapy and 994 with either itc or micrometastasis in lymph nodes who did receive adjuvant chemotherapy . the authors found itc or micrometastases in regional lymph nodes were associated with a reduced disease - free survival who did not receive adjuvant chemotherapy and those who received adjuvant chemotherapy had improved disease - free survival rate . pugliese et al . studied a total of 954 sln biopsies consist of 491 n0(i- ) , 86 n0(i+ ) , 73 n1mi , 146 n1a , 29 n2a , and 11 n3a patients with a median follow - up of 45.4 months and found no differences in overall survival ( os ) and relapse - free survival ( rfs ) between pn0(i- ) and pn0(i+ ) or pn1mi but worse prognosis was noted when the size of the metastases reached the pn1a . reported no difference in disease - free survival and os with a median follow up of 8 years between pn0 , pn0 ( i+ ) and pn1mi in their study cohort of 790 with patients . only patients with macrometastasis had a statistical significant decrease in disease - free survival and os . bilimoria et al . reported a retrospective study from the national cancer data base on 97,314 sln positive patients from19982006 subdivided into those who had alnd and those who did not have alnd had a similar axillary local recurrence and 5 year relative survival ; 23% macrometastases ( pn1 ) and 55% with sln micrometastases ( pn1mi ) . the seer database by yi et al . reported a total of 26,986 composed of 11% pn1 and 33% pn1mi from 19982004 had no difference in os at a median follow - up of 50 months . in addition , there are nine smaller studies comprising a total of 1,035 patients with positive sln without alnd and reported low axillary local recurrence rate , in the range of 0% to 2% at 2882 months follow - up . our institution also supported that sln biopsy alone as adequate intervention in 302 patients with an average clinical follow - up of 24 months . even for the mastectomy cases with pn1 disease in sln did not affect os and rfs between the radiation therapy without alnd and complete alnd surgery . there are three significant prospective randomized studies relevant to sln metastasis : the national surgical adjuvant breast and bowel project ( nsabp ) trial-32 , the american college of surgeons oncology group ( acosog ) trial z0011 , and the international breast cancer study group ( ibcsg 23 - 01 ) [ 45 - 47 ] . the nsabp b-32 was a randomized controlled phase 3 trial composed of 5,611 patient sample divided into group 1 ( sln and alnd ) and group 2 ( sln alone if no metastasis sln with alnd if metastatic carcinoma was present in sln ) . the adjuvant chemotherapy and radiation therapy was relatively equally divided in between these groups : 85% in group 1 and 84.1% in group 2 . the authors found no differences in os , disease - free survival and regional control between two groups . comparing pn0 , pn0(i+ ) and pn1mi groups and found no difference in overall survival among these groups during 5-year follow - up period . the authors concluded that additional evaluation including ihc stains and multiple levels of sln are not indicated because a clinical benefit was not significant . the acosog z0011 trial 2 is a landmark article that changed paradigm shifts in 2011 . acosog z0011 is a randomized trial , non - inferiority study which suggested that completion alnd is not necessary in t1 and t2 breast cancer patients who received whole - breast radiation since disease - free survival and recurrence rates did not appear to differ between the patients who had completion alnd versus those who had sln biopsy alone and had up to two positive slns . the study consisted of 813 positive sln patients randomized to 388 patients who had alnd and 425 patients without alnd . criticisms of acosog z0011 study have been that the case selection was biased toward older women , predominantly estrogen receptor ( er ) positive tumors ( younger women with er negative tumors were underrepresented ) , lost to follow - up was significant ( 21% in the complete alnd and 17% in the sln alone groups ) and short clinical follow - up of 6.3 years . the ibcsg 23 - 01 was a multicenter randomized phase 3 trial in breast cancer size of less or equal to 5 cm , separating pn0(i+ ) and pn1mi without extracapsular invasion of sln based on h&e slide to 464 patients who had alnd and 467 patients who did not have alnd with a median follow - up of 5 years . the result was similar to the acosog z0011 trial stating that alnd was not necessary since there is no adverse effect on survival . additionally , the european organization for research and treatment of cancer ( eortc ) 10981 - 22023 , the after mapping of the axilla : radiotherapy or surgery ( amaros ) which is a randomized , multicentric , phase 3 non - inferiority trial also supported acosog z0011 findings that there are no differences when complete alnd verses axillary radiation therapy from positive sln patients in early breast cancer when the usage of chemotherapy , or hormone therapy was factored in . traditionally alnd has been a guide to adjuvant chemotherapy and based on this study , the information provided by complete alnd is no longer useful . another prospective clinical trial study , the posnoc ( positive sentinel node : adjuvant therapy alone versus adjuvant therapy plus clearance or axillary radiotherapy ) trial is still open and recruiting patients till 3/31/18 in the united kingdom which is a pragmatic , randomized , multicentric and non - inferiority trial for early breast cancer with one or two sln macrometastases . the surveillance epidemiology and end results ( seer ) database reported that micrometastasis represents a significant prognostic marker . their data consists of 209,720 patient numbers between 1992 and 2003 and the 10 year survival among pn1 , pn1mi and pn0(i+ ) is 73% , 77% , and 78% , respectively . also , n1mi diagnoses increased from 2.3% to 7% . de boer et al . published mirror ( micrometastasis and isolated tumor cells : relevant and robust or rubbish ? ) study in netherlands which included three cohorts with the median follow - up of 5.1 years : 865 patient with negative regional lymph nodes who did not receive adjuvant chemotherapy , 865 patients with either itc or micrometastasis in lymph nodes who did not receive adjuvant chemotherapy and 994 with either itc or micrometastasis in lymph nodes who did receive adjuvant chemotherapy . the authors found itc or micrometastases in regional lymph nodes were associated with a reduced disease - free survival who did not receive adjuvant chemotherapy and those who received adjuvant chemotherapy had improved disease - free survival rate . pugliese et al . studied a total of 954 sln biopsies consist of 491 n0(i- ) , 86 n0(i+ ) , 73 n1mi , 146 n1a , 29 n2a , and 11 n3a patients with a median follow - up of 45.4 months and found no differences in overall survival ( os ) and relapse - free survival ( rfs ) between pn0(i- ) and pn0(i+ ) or pn1mi but worse prognosis was noted when the size of the metastases reached the pn1a . reported no difference in disease - free survival and os with a median follow up of 8 years between pn0 , pn0 ( i+ ) and pn1mi in their study cohort of 790 with patients . only patients with macrometastasis had a statistical significant decrease in disease - free survival and os . bilimoria et al . reported a retrospective study from the national cancer data base on 97,314 sln positive patients from19982006 subdivided into those who had alnd and those who did not have alnd had a similar axillary local recurrence and 5 year relative survival ; 23% macrometastases ( pn1 ) and 55% with sln micrometastases ( pn1mi ) . the seer database by yi et al . reported a total of 26,986 composed of 11% pn1 and 33% pn1mi from 19982004 had no difference in os at a median follow - up of 50 months . in addition , there are nine smaller studies comprising a total of 1,035 patients with positive sln without alnd and reported low axillary local recurrence rate , in the range of 0% to 2% at 2882 months follow - up . our institution also supported that sln biopsy alone as adequate intervention in 302 patients with an average clinical follow - up of 24 months . even for the mastectomy cases with pn1 disease in sln did not affect os and rfs between the radiation therapy without alnd and complete alnd surgery . there are three significant prospective randomized studies relevant to sln metastasis : the national surgical adjuvant breast and bowel project ( nsabp ) trial-32 , the american college of surgeons oncology group ( acosog ) trial z0011 , and the international breast cancer study group ( ibcsg 23 - 01 ) [ 45 - 47 ] . the nsabp b-32 was a randomized controlled phase 3 trial composed of 5,611 patient sample divided into group 1 ( sln and alnd ) and group 2 ( sln alone if no metastasis sln with alnd if metastatic carcinoma was present in sln ) . the adjuvant chemotherapy and radiation therapy was relatively equally divided in between these groups : 85% in group 1 and 84.1% in group 2 . the authors found no differences in os , disease - free survival and regional control between two groups . comparing pn0 , pn0(i+ ) and pn1mi groups and found no difference in overall survival among these groups during 5-year follow - up period . the authors concluded that additional evaluation including ihc stains and multiple levels of sln are not indicated because a clinical benefit was not significant . the acosog z0011 trial 2 is a landmark article that changed paradigm shifts in 2011 . acosog z0011 is a randomized trial , non - inferiority study which suggested that completion alnd is not necessary in t1 and t2 breast cancer patients who received whole - breast radiation since disease - free survival and recurrence rates did not appear to differ between the patients who had completion alnd versus those who had sln biopsy alone and had up to two positive slns . the study consisted of 813 positive sln patients randomized to 388 patients who had alnd and 425 patients without alnd . criticisms of acosog z0011 study have been that the case selection was biased toward older women , predominantly estrogen receptor ( er ) positive tumors ( younger women with er negative tumors were underrepresented ) , lost to follow - up was significant ( 21% in the complete alnd and 17% in the sln alone groups ) and short clinical follow - up of 6.3 years . the ibcsg 23 - 01 was a multicenter randomized phase 3 trial in breast cancer size of less or equal to 5 cm , separating pn0(i+ ) and pn1mi without extracapsular invasion of sln based on h&e slide to 464 patients who had alnd and 467 patients who did not have alnd with a median follow - up of 5 years . the result was similar to the acosog z0011 trial stating that alnd was not necessary since there is no adverse effect on survival . additionally , the european organization for research and treatment of cancer ( eortc ) 10981 - 22023 , the after mapping of the axilla : radiotherapy or surgery ( amaros ) which is a randomized , multicentric , phase 3 non - inferiority trial also supported acosog z0011 findings that there are no differences when complete alnd verses axillary radiation therapy from positive sln patients in early breast cancer when the usage of chemotherapy , or hormone therapy was factored in . traditionally alnd has been a guide to adjuvant chemotherapy and based on this study , the information provided by complete alnd is no longer useful . another prospective clinical trial study , the posnoc ( positive sentinel node : adjuvant therapy alone versus adjuvant therapy plus clearance or axillary radiotherapy ) trial is still open and recruiting patients till 3/31/18 in the united kingdom which is a pragmatic , randomized , multicentric and non - inferiority trial for early breast cancer with one or two sln macrometastases . completion alnd is not providing benefit of os and disease - free survival in microscopic metastatic sln [ pn0(i+ ) and pn1mi ] . even macrometastasis in one or two sln(s ) in acosog z0011 did not affect os . there is mounting evidence to support that sln biopsy alone can be a standard practice demonstrating its efficacy , accuracy in staging and equivalent survival outcome when compared to complete alnd and slnb alone in t1t2 breast cancer [ 45,53 - 57 ] . recently , danish breast cancer cooperative group ( dbcg ) looked into 2,074 patients who had wither itc or micrometastasis in sln and found no significant difference in os and axillary recurrence rate between patients who had completion alnd or no alnd . the study add growing literature that it is safe to omit alnd in minimal metastatic disease in the sln . from a pathologists point of view , we need to ask the following questions : are we reporting small volume metastasis in vain ? is separating pn0(i+ ) or pn1mi really necessary ? while most of the cases n - staging are straightforward , we do still face ambiguous cases . when we perform tedious work by counting metastatic tumor cells , we want to make sure this work has value in clinical contribution . the evidence shows that separating small volume n - stage or even macrometastasis is not affecting clinical management to perform completion alnd or os in early stage breast cancers . also all other organ systems in the same ajcc staging manual for colon cancer , gynecologic cancer , even melanoma which the technique of sln was first described do not practice as in breast n - staging ; there is no pn0 ( i+ ) or pn1mi . metastatic carcinoma when seen by either h&e or ihc stain slide is called metastatic carcinoma . ihc stain is used discretionally by pathologists to confirm the presence of metastasis as needed if there is suspicious area by h&e slide . why should breast cancer n - stage be any different from other organs ? as we understand that alnd dissection is no longer needed or necessary in many early breast cancers , the necessity of performing multiple levels , ihc stain , frozen section and molecular studies on sln needs to be revisited . recently the ajcc executive committee announced a mid-2016 publication date for the 8th edition ajcc cancer staging system during its september 2013 annual meeting held in chicago . the 8th edition will be effective for all cancer cases recorded on or after january 1 , 2017 . collaboration with the cancer care and surveillance community to set the standard to anticipate , communicate , and help incorporate staging system changes . breast expert panel leaders are gabriel n. hortobagyi , md , breast medical oncologist and armando giuliano , md , breast surgical oncologist . as a pathologist , i hope to see the 8th edition ajcc cancer staging system for n - stage to be clinically relevant with simple and practical application without further arbitrary cutoffs to reduce subjective interpretation and to enhance agreement between both academic and community based pathologists all over the world .

breast cancer staging , in particular n - stage changed most significantly due to the advanced technique of sentinel lymph node biopsy two decades ago . pathologists have more thoroughly examined and scrutinized sentinel lymph node and found increased number of small volume metastases . while pathologists use the strict criteria from the tumor lymph node metastasis ( tnm ) classification , studies have shown poor reproducibility in the application of american joint committee on cancer and international union against cancer / tnm guidelines for sentinel lymph node classification in breast cancer . in this review article , a brief history of tnm with a focus on n - stage is described , followed by innate problems with the guidelines , and why pathologists may have difficulties in assessing lymph node metastases uniformly . finally , clinical significance of isolated tumor cells , micrometastasis , and macrometastasis is described by reviewing historical retrospective data and significant prospective clinical trials .

BRIEF HISTORY OF SENTINEL LYMPH NODE STAGING PROBLEMS IN DETERMINING METASTATIC SENTINEL LYMPH NODE SIZE IN PATHOLOGY Grossing axillary SLN Microscopic examination of SLN CLINICAL SIGNIFICANCE OF SENTINEL LYMPH NODE Retrospective clinical data Prospective clinical data CONCLUSION AND COMMENTARY

from then on , many revisions were made in advent of changes in breast cancer management and treatment . in 1987 , the international union against cancer ( uicc ) and the ajcc staging systems were unified into a single tnm staging system to be able to communicate clinical information without ambiguity . it is arguable that n part of the staging changed most significantly since the sln , biopsy first described by giuliano et al . in 1994 in breast cancer as a potential altering the role of alnd by using intraoperative lymphatic mapping . along with sln biopsy advancement by surgical oncologists , pathology assessment of sln biopsy sample led to significant changes in n - stage by the ajcc staging manual . as sln biopsy became more a standard treatment in the management of breast cancer , the ajcc staging manual needed revision to reflect the continual development and knowledge in this field . the 6th edition of the ajcc cancer staging manual when compared to the previous one contains some of the most extensive and significant revisions pertaining to sln related to the growing knowledge of new technology such as ihc staining and reverse transcriptase - polymerase chain reaction ( rt - pcr ) . the principal changes are related to the size , number , location , and method of detection of regional metastases to the lymph nodes . a breast task force was constituted to serve in an advisory role to the ajcc in the fall of 2001 and the 6th edition reflected the significant changes . the terminology occult metastases was interchangeably used to describe a small metastatic carcinoma in the lymph node in the literature . with the advent of more sensitive technique , such as ihc stains , it was easier to detect itc that were not readily visible from the h&e stain slides which was the gold standard for detection of metastatic carcinoma in the axillary lymph nodes . the additional designation of ( sn ) for sentinel node was used in the classification of tnm staging system when only the sln was excised without the full alnd . , knowing sentinel nodes are more likely to contain metastases than non - slns , pathologists began to perform much more thorough and comprehensive examinations which included grossing by submitting the entire node by sectioning 2.0 mm thickness in a longest axis , serial sections for h&e stains instead of just one section , and adding ihc stains which deviated from the reference population assay in which lymph node examination was much simpler- previously , grossly negative large lymph nodes were not necessarily entirely submitted for histologic examination . introduction of itc was to prevent overtreatment of low - volume nodal involvement since some of the itc is caused by passive tumor cell transport to the sln after pre - operative fine needle aspiration ( fna ) and/or core needle biopsy procedures and iatrogenic displacement due to breast massage causing benign epithelial elements in lymph node resulting false positive especially in papillary lesions . we have the capability to detect even one cell in the sln if this is clinically meaningful but first , we need to figure out whether this effort is paid off by clinical significance . prior to ajcc 2003 and 2006 , the term isolated tumor cells and micrometastasis were not clearly defined . later on the ajcc definition of sln tumor deposits were measured using a micrometer and placed in one of three categories : macrometastasis , micrometastasis , or itc . the main differences between the ajcc 6th edition to the ajcc 7th edition in regards to sln is to count the number of metastatic cells ; if it is less than 200 cells versus over 200 cells for itc and micrometastasis respectively . i stands for isolated tumor cells and not ihc stain for the 7th edition ajcc . in fact , this was incorporated into the 7th edition for large dishesive , non - confluent tumor cells throughout the lymph node typically seen in metastatic lobular carcinoma . in addition , the 7th edition of ajcc states these thresholds are meant to be guidelines , and not absolute cutoffs , to help pathologists determine if the tumor burden in a given lymph nodes is likely to be clinically important or not . the pathologist should use judgment and not an absolute cut - off of 0.2 mm or exact 200 cells , in determining the likelihood of whether the cluster of cells is an itc or a true micrometastasis . according to the 7th edition ajcc , a positive node is defined as a metastasis measuring at least 0.2 mm , or > 200 tumor cells ( pn1mi ) . whenever there is a positive lymph node , the pathologist also needs to describe the following : the size of metastasis ( or number of tumor cells ) , the anatomical node involved such as axilla , internal mammary , intramammary , infraclavicular or supraclavicular lymph node , the total number of positive nodes , and the method of detection such as clinical , fna or tissue biopsy . in addition , there is a troubling statement in the ajcc 2010 data elements with asterisks ( small clusters of cells not greater than 0.2 mm or single tumor cells , or a cluster of fewer than 200 cells in a single histologic cross - section ) are not required . further disclaimer statements in the 7th edition of ajcc are as follows : approximately 1,000 tumor cells are contained in a 3-dimensional 0.2-mm cluster . thus , if more than 200 individual tumor cells are identified as single dispersed tumor cells or as a nearly confluent elliptical or spherical focus in a single histologic section of a lymph node , there is a high probability that more than 1,000 cells are present in the node . and final statement is as follows : the pathologist should use judgment regarding whether it is likely that the cluster of cells represents a true micrometastasis or is simply a small group of isolated tumor cells . in 2010 , both the uicc and tnm joined in the provision in guidelines for sln classification , however , studies have shown poor reproducibility in the application of in both invasive ductal and invasive lobular carcinomas . furthermore , previous studies have shown that application of ihc stain on h&e negative lymph node can detect small volume metastatic carcinoma in 12%29% of cases . for this reason , the college of american pathologists ( cap ) published a consensus statement in july 2000 recommending a single h&e slide section from each lymph node block without gross evidence of metastasis without routine cytokeratin ihc stain or routine serial sectioning based on the preliminary study from john wayne cancer center which found no differences in five - year disease - free survival rate between node negative by h&e negative , ihc negative versus h&e negative , ihc positive groups [ 18 - 20 ] . with the advent of the comprehensive sln evaluation , probability of finding small metastasis it is reported that among patients with a positive sln by h&e stain were found to have additional nodal metastasis in alnd in 48.3% of cases . while the 7th edition ajcc criteria and definition are fairly straightforward , its application may be difficult in reality for pathologists to uniformly classify n - stage correctly connolly described some of the most practical challenges in the classification in the assessment of sln biopsy separating itc , micrometastases , and macrometastases . despite the intent for the better interpretation of metastatic tumor cells in lymph node in the 7th edition ajcc , many issues have not been resolved . this is stating that one of four cases in breast cancer , pathologists do not agree with n - stage even though the descriptor for n - stage is easily understandable in the ajcc staging manual . there are still some circumstances pathologists have difficulties knowing how to accurately assign n- stage based on the 7th edition ajcc . based on the 6th edition , cancerous nodules in the axillary fat without evidence of residual lymph node tissue should be classified as positive axillary lymph nodes . but what if these cancerous nodules in the axilla are cancer cells from the axillary breast tissue ? many pathologists do not classify cancerous nodules in axilla without apparent lymph node structure as lymph node metastasis ( unpublished personal observation ) . the dimension of the tumor from this scenario may end up in t - stage rather than in n - stage . when there is pericapsular lymph - vascular invasion ( lvi ) in which tumor cells are not in the parenchyma or subcapsular part of lymph node , is that considered metastatic or lvi ? when most of the metastatic carcinoma is seen outside the lymph node capsule ( in this case by metastatic lobular carcinoma ) with only one itc within the lymph node parenchyma , is it considered extracapsular invasion with itc or is it micrometastasis based on the total number of cells of > 200 or > 0.2 mm ? the 7th edition ajcc was bit more helpful in this situation because one needs to count the number of metastatic cells in sln ; if it is less than 200 cells versus over 200 cells for isolated tumor cells and micrometastasis respectively . for n - stage , if there is stromal desmoplasia or stromal proliferation around small volume metastasis , the 6th edition ajcc states that the size of the tumor deposit should include the stromal reaction . location of metastatic tumor cells whether it is subcapsular or intraparenchymal is not specified in the 7th edition ajcc , however , many european pathologists based on previous uicc related publications , tumor cells located within the parenchyma measuring even less than 0.2 mm is considered micrometastasis and not considered as itc . the above stated scenarios are difficult to resolve and ultimately depends on the pathologists to make appropriate judgments resulting high subjectivity and inconsistence in n - stage . also we should keep in mind that both retrospective and prospective studies in literature composed of variability in interpretation of n - stages ; pn0(i+ ) , pn1mi and pn1a even with the best intention of adhering to ajcc criterion . most pathologists submit clinically negative sln entirely for microscopic examination by slicing lymph node in a longest axis into 0.2 cm thickness based on cap guideline . while the 7th edition ajcc criteria and definition are fairly straightforward , its application may be difficult in reality for pathologists to uniformly classify n - stage correctly . connolly described some of the most practical challenges in the classification in the assessment of sln biopsy separating itc , micrometastases , and macrometastases . despite the intent for the better interpretation of metastatic tumor cells in lymph node in the 7th edition ajcc , many issues have not been resolved . this is stating that one of four cases in breast cancer , pathologists do not agree with n - stage even though the descriptor for n - stage is easily understandable in the ajcc staging manual . there are still some circumstances pathologists have difficulties knowing how to accurately assign n- stage based on the 7th edition ajcc . based on the 6th edition , cancerous nodules in the axillary fat without evidence of residual lymph node tissue should be classified as positive axillary lymph nodes . but what if these cancerous nodules in the axilla are cancer cells from the axillary breast tissue ? many pathologists do not classify cancerous nodules in axilla without apparent lymph node structure as lymph node metastasis ( unpublished personal observation ) . the dimension of the tumor from this scenario may end up in t - stage rather than in n - stage . when there is pericapsular lymph - vascular invasion ( lvi ) in which tumor cells are not in the parenchyma or subcapsular part of lymph node , is that considered metastatic or lvi ? when most of the metastatic carcinoma is seen outside the lymph node capsule ( in this case by metastatic lobular carcinoma ) with only one itc within the lymph node parenchyma , is it considered extracapsular invasion with itc or is it micrometastasis based on the total number of cells of > 200 or > 0.2 mm ? the 7th edition ajcc was bit more helpful in this situation because one needs to count the number of metastatic cells in sln ; if it is less than 200 cells versus over 200 cells for isolated tumor cells and micrometastasis respectively . for n - stage , if there is stromal desmoplasia or stromal proliferation around small volume metastasis , the 6th edition ajcc states that the size of the tumor deposit should include the stromal reaction . location of metastatic tumor cells whether it is subcapsular or intraparenchymal is not specified in the 7th edition ajcc , however , many european pathologists based on previous uicc related publications , tumor cells located within the parenchyma measuring even less than 0.2 mm is considered micrometastasis and not considered as itc . the above stated scenarios are difficult to resolve and ultimately depends on the pathologists to make appropriate judgments resulting high subjectivity and inconsistence in n - stage . also we should keep in mind that both retrospective and prospective studies in literature composed of variability in interpretation of n - stages ; pn0(i+ ) , pn1mi and pn1a even with the best intention of adhering to ajcc criterion . for large or locally advanced inflammatory breast cancer ( t3 and t4 ) , ductal carcinoma in situ , when breast - conserving surgery is to be done , pregnancy , in the setting of prior non - oncologic breast surgery or axillary surgery , and in the presence of clinically suspicious axillary lymph node , sln biopsy was not recommended . again , the mixed results may have been due to the variable definitions of micrometastases and itc in literature and different mode of detection . published mirror ( micrometastasis and isolated tumor cells : relevant and robust or rubbish ? ) studied a total of 954 sln biopsies consist of 491 n0(i- ) , 86 n0(i+ ) , 73 n1mi , 146 n1a , 29 n2a , and 11 n3a patients with a median follow - up of 45.4 months and found no differences in overall survival ( os ) and relapse - free survival ( rfs ) between pn0(i- ) and pn0(i+ ) or pn1mi but worse prognosis was noted when the size of the metastases reached the pn1a . in addition , there are nine smaller studies comprising a total of 1,035 patients with positive sln without alnd and reported low axillary local recurrence rate , in the range of 0% to 2% at 2882 months follow - up . there are three significant prospective randomized studies relevant to sln metastasis : the national surgical adjuvant breast and bowel project ( nsabp ) trial-32 , the american college of surgeons oncology group ( acosog ) trial z0011 , and the international breast cancer study group ( ibcsg 23 - 01 ) [ 45 - 47 ] . the ibcsg 23 - 01 was a multicenter randomized phase 3 trial in breast cancer size of less or equal to 5 cm , separating pn0(i+ ) and pn1mi without extracapsular invasion of sln based on h&e slide to 464 patients who had alnd and 467 patients who did not have alnd with a median follow - up of 5 years . another prospective clinical trial study , the posnoc ( positive sentinel node : adjuvant therapy alone versus adjuvant therapy plus clearance or axillary radiotherapy ) trial is still open and recruiting patients till 3/31/18 in the united kingdom which is a pragmatic , randomized , multicentric and non - inferiority trial for early breast cancer with one or two sln macrometastases . published mirror ( micrometastasis and isolated tumor cells : relevant and robust or rubbish ? ) studied a total of 954 sln biopsies consist of 491 n0(i- ) , 86 n0(i+ ) , 73 n1mi , 146 n1a , 29 n2a , and 11 n3a patients with a median follow - up of 45.4 months and found no differences in overall survival ( os ) and relapse - free survival ( rfs ) between pn0(i- ) and pn0(i+ ) or pn1mi but worse prognosis was noted when the size of the metastases reached the pn1a . in addition , there are nine smaller studies comprising a total of 1,035 patients with positive sln without alnd and reported low axillary local recurrence rate , in the range of 0% to 2% at 2882 months follow - up . there are three significant prospective randomized studies relevant to sln metastasis : the national surgical adjuvant breast and bowel project ( nsabp ) trial-32 , the american college of surgeons oncology group ( acosog ) trial z0011 , and the international breast cancer study group ( ibcsg 23 - 01 ) [ 45 - 47 ] . the ibcsg 23 - 01 was a multicenter randomized phase 3 trial in breast cancer size of less or equal to 5 cm , separating pn0(i+ ) and pn1mi without extracapsular invasion of sln based on h&e slide to 464 patients who had alnd and 467 patients who did not have alnd with a median follow - up of 5 years . another prospective clinical trial study , the posnoc ( positive sentinel node : adjuvant therapy alone versus adjuvant therapy plus clearance or axillary radiotherapy ) trial is still open and recruiting patients till 3/31/18 in the united kingdom which is a pragmatic , randomized , multicentric and non - inferiority trial for early breast cancer with one or two sln macrometastases . recently , danish breast cancer cooperative group ( dbcg ) looked into 2,074 patients who had wither itc or micrometastasis in sln and found no significant difference in os and axillary recurrence rate between patients who had completion alnd or no alnd . when we perform tedious work by counting metastatic tumor cells , we want to make sure this work has value in clinical contribution . the evidence shows that separating small volume n - stage or even macrometastasis is not affecting clinical management to perform completion alnd or os in early stage breast cancers . also all other organ systems in the same ajcc staging manual for colon cancer , gynecologic cancer , even melanoma which the technique of sln was first described do not practice as in breast n - staging ; there is no pn0 ( i+ ) or pn1mi . why should breast cancer n - stage be any different from other organs ? collaboration with the cancer care and surveillance community to set the standard to anticipate , communicate , and help incorporate staging system changes . as a pathologist , i hope to see the 8th edition ajcc cancer staging system for n - stage to be clinically relevant with simple and practical application without further arbitrary cutoffs to reduce subjective interpretation and to enhance agreement between both academic and community based pathologists all over the world .

decays of b mesons to two - body final states containing a charmonium resonance such as a j/ or (2s ) offer a powerful way of studying electroweak transitions . such decays probe charmonium properties and play a role in the study of cp violation and mixing in the neutral b system . the relative branching fractions of b , b and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s}$\end{document } mesons into j/ and (2s ) mesons have previously been studied by both the cdf and d0 collaborations [ 24 ] . since the current experimental results for the study of cp violation in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } mixing using the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow j/\psi \phi$\end{document } decay [ 57 ] are statistically limited , it is important to establish other channels where this analysis can be done . one such channel is the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow \psi ( 2s)\phi$\end{document } decay . in this paper , measurements of the ratios of the branching fractions of b mesons decaying to (2s)x and j/x are reported , where b denotes a b , b or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s}$\end{document } meson ( charge conjugate decays are implicitly included ) and x denotes a k , k or meson . the data were collected by the lhcb experiment in pp collisions at the centre - of - mass energy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt{s } = 7~\mathrm{tev}$\end{document } during 2011 and correspond to an integrated luminosity of 0.37 fb . the lhcb detector is a single - arm forward spectrometer covering the pseudorapidity range 2<<5 , designed for the study of b- and c - hadrons . the detector includes a high precision tracking system consisting of a silicon - strip vertex detector surrounding the pp interaction region , a large - area silicon - strip detector located upstream of a dipole magnet with a bending power of about 4 tm , and three stations of silicon - strip detectors and straw drift - tubes placed downstream . the combined tracking system has a momentum resolution p / p that varies from 0.4 % at 5 gev / c to 0.6 % at 100 gev / c , and an impact parameter resolution of 20 m for tracks with high transverse momentum . data were taken with both magnet polarities to reduce systematic effects due to detector asymmetries . photon , electron and hadron candidates are identified by a calorimeter system consisting of scintillating - pad and pre - shower detectors , and electromagnetic and hadronic calorimeters . muons are identified by a muon system composed of alternating layers of iron and multiwire proportional chambers . the trigger consists of a hardware stage based on information from the calorimeter and muon systems , followed by a software stage which applies a full event reconstruction . events with a j/ final state are triggered using two hardware trigger decisions : the single - muon decision , which requires one muon candidate with a transverse momentum pt larger than 1.5 gev / c , and the di - muon decision , which requires two muon candidates with transverse momenta \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_{\mathrm{t}_{1}}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_{\mathrm{t}_{2}}$\end{document } satisfying the relation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt{p_{\mathrm{t}_{1 } } \cdot p_{\mathrm{t}_{2 } } } > 1.3~\mathrm{gev}/c$\end{document}. the di - muon trigger decision in the software trigger requires muon pairs of opposite charge with pt>500 mev / c , forming a common vertex and with an invariant mass in excess of 2.9 gev / c . in this analysis , the decays \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{+}\rightarrow \psi k^{+}(b^{0}\rightarrow \psi k^{*0},\allowbreak b^{0}_{s}\rightarrow \psi\phi)$\end{document } are reconstructed , where represents (2s ) or j/ , reconstructed in the decay modes . a k(k, ) candidate is added to the di - muon pair to form a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{+}(b^{0 } , b^{0}_{s})$\end{document } candidate . the starting point of the analysis is the reconstruction of either a j/ or (2s ) meson decaying into a di - muon pair . candidates are formed from pairs of opposite sign tracks that both have a transverse momentum larger than 500 mev / c . good reconstruction quality is assured by requiring the per degree of freedom of the track fit to satisfy /ndf<5 . this is achieved by requiring the muon identification variable , the difference in logarithm of the likelihood of the muon and hadron hypotheses provided by the muon detection system , to satisfy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta\log\mathcal{l}^{\mu - h } > -5$\end{document}. the muons are required to form a common vertex of good quality ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\chi^{2}_{\mathrm{vtx}}<20$\end{document } ) . the resulting di - muon candidate is required to have decay length significance from its associated primary vertex greater than 5 and have an invariant mass between 3020 and 3135 mev / c in the case of a j/ candidate or between 3597 and 3730 mev / c for a (2s ) candidate . the selected j/ and (2s ) candidates are then combined with a k , k or to create b meson candidates . pion - kaon separation is provided by the ring - imaging cherenkov detectors . to identify kaons the difference in logarithm of the likelihood of the kaon and pion hypotheses is required to satisfy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta\log\mathcal{l}^ { k- \pi } > -5$\end{document}. in the case of pions the difference in logarithm of the likelihood of the pion and kaon hypotheses is required to satisfy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta\log\mathcal{l}^ { \pi - k } > -5$\end{document}. as in the case of muons , a cut is applied on the track /ndf provided by the track fit at 5 . the kaons and pions are required to have a transverse momentum larger than 250 mev / c and to have an impact parameter significance with respect to any primary vertex larger than 2 . in the b channel , the mass of the kaon and pion system is required to be \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$842<\mathrm{m } _ { k^{+ } \pi^{-}}<942~\mathrm{mev}/c^{2}$\end{document } and in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s}$\end{document } channel the mass of the kaon pair is required to be \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1010<\mathrm{m } _ { k^{+ } k^{- } } < 1030~\mathrm{mev}/c^{2}$\end{document}. in addition , we require the decay time of the b candidate ( c ) to be larger than 100 m to reduce the large combinatorial background from particles produced in the primary pp interaction . a global refit of the three - prong ( four - prong ) combination is performed with a primary vertex constraint and with the di - muon pair mass constrained to the nominal value using the decay tree fit ( dtf ) procedure . the reduced of this fit ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\chi^{2}_{\mathrm{dtf}}/\mathrm{ndf}$\end{document } ) is required to be less than 5 , where the dtf algorithm takes into account the number of decay products to determine the number of degrees of freedom . the b candidates , where a muon from the (2s) decay is reconstructed as both muon and kaon , are removed by requiring the angle between the same sign muon and kaon to be greater than 3 mrad . the number of the bk candidates is estimated by performing an unbinned maximum likelihood fit . the same procedure is used to determine the number of the bk candidates in a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$842<\mathrm{m } _ { k^{+ } \pi^{-}}<942~\mathrm{mev}/c^{2}$\end{document } mass window and the number of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow \psi k^{+ } k^{-}$\end{document } candidates in a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1010<\mathrm{m } _ { k^{+ } k^{-}}<1030~\mathrm{mev}/c^{2}$\end{document } mass window . the number of signal candidates is determined by fitting a double - sided crystal ball function [ 12 , 13 ] for signal together with an exponential function to model the background . the tail parameters of the crystal ball function are fixed to values determined from simulation . 1mass distributions of ( a ) b j/k , ( b ) b (2s)k , ( c ) b j/k , ( d ) b (2s)k , ( e ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow j/\psi k^{+ } k^{-}$\end{document } and ( f ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow \psi(2s ) k^{+ } k^{-}$\end{document}. the total fitted function ( solid ) and the combinatorial background ( dashed ) are shown . the variation in resolution of the different modes is fully consistent with the energy released in the decays and in agreement with simulation mass distributions of ( a ) b j/k , ( b ) b (2s)k , ( c ) b j/k , ( d ) b (2s)k , ( e ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow j/\psi k^{+ } k^{-}$\end{document } and ( f ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow \psi(2s ) k^{+ } k^{-}$\end{document}. the total fitted function ( solid ) and the combinatorial background ( dashed ) are shown . the variation in resolution of the different modes is fully consistent with the energy released in the decays and in agreement with simulation to estimate the contribution from non - resonant decays bk and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow \psi k^{+ } k^{-}$\end{document } , the k and kk invariant mass distributions have been studied after relaxing requirements on the k and kk invariant masses , see fig . 2 . the k and kk invariant mass distributions are then fitted with the sum of a relativistic breit - wigner function convolved with a gaussian , to describe the resonant contribution from the k or , two - body phase space function multiplied by a second order polynomial , to describe the non - resonant k or kk contribution . the splot technique is used to unfold the k or kk invariant mass of the non - resonant ( in k and kk ) candidates . this unfolded distribution contains a mixture of combinatorial background and non - resonant bk or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow \psi k^{+ } k^{-}$\end{document } decays . is then used to estimate the contribution from the non - resonant b decays , which is subtracted from the total yield to estimate the contribution from resonant bk or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow \psi\phi$\end{document } decays . the contribution of the resonant decays can also be extracted by unfolding the contribution of resonant k or kk decays to the k or kk invariant mass distribution . 2mass distributions of k or k k in ( a ) b j/k , ( b ) b (2s)k , ( c ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow j/\psi k^{+ } k^{-}$\end{document } and ( d ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow \psi(2s ) k^{+ } k^{-}$\end{document } decays . the total fitted function ( solid ) and the combination of the non - resonant component and the combinatorial background ( dashed ) 3mass distributions of a ( a ) b j/k , ( b ) b (2s)k , ( c ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow j/\psi k^{+ } k^{-}$\end{document } and ( d ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow \psi(2s ) k^{+ } k^{-}$\end{document } for resonant component ( full circles ) and non - resonant component ( open circles ) . the total fitted function ( solid ) and the combinatorial background ( dashed ) are shown . the fit is described in the texttable 1summary of the signal yields for the six b modes considered and the ratios of the number of j/ and (2s ) decays : n is the summed signal yield for resonant and non - resonant modes , n is the signal yield for non - resonant modes only and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$n^{\mathrm{res}}_{\psi x}$\end{document } is the signal yield for resonant decays ( through k or ) . the uncertainties are statistical only b decay modes n n n \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$n^{\mathrm{res}}_{\psi(2s)x}/n^{\mathrm{res}}_{j/\psi x}$\end{document } b j/k 141,769410141,7694100.08570.0009 b (2s)k 12,15413012,154130 b j/k 35,7702071,2533034,5172090.06120.0018 b (2s)k 2,22360112122,11161 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow j/\psi k^{+ } k^{-}$\end{document } 7,6549266137,588930.06520.0034 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow \psi(2s ) k^{+ } k^{-}$\end{document } 49525 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$0^{+1}_{-0}$\end{document } 49525 mass distributions of k or k k in ( a ) b j/k , ( b ) b (2s)k , ( c ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow j/\psi k^{+ } k^{-}$\end{document } and ( d ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow \psi(2s ) k^{+ } k^{-}$\end{document } decays . the total fitted function ( solid ) and the combination of the non - resonant component and the combinatorial background ( dashed ) the fit is described in the text mass distributions of a ( a ) b j/k , ( b ) b (2s)k , ( c ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow j/\psi k^{+ } k^{-}$\end{document } and ( d ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow \psi(2s ) k^{+ } k^{-}$\end{document } for resonant component ( full circles ) and non - resonant component ( open circles ) . the total fitted function ( solid ) and the combinatorial background ( dashed ) the fit is described in the text summary of the signal yields for the six b modes considered and the ratios of the number of j/ and (2s ) decays : n is the summed signal yield for resonant and non - resonant modes , n is the signal yield for non - resonant modes only and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$n^{\mathrm{res}}_{\psi x}$\end{document } is the signal yield for resonant decays ( through k or ) . the branching fraction ratio is calculated using 1 where n is the number of signal candidates and is the overall efficiency . the overall efficiency is the product of the geometrical acceptance of the detector , the combined reconstruction and selection efficiency , and the trigger efficiency . the simulation samples used are based on the pythia 6.4 generator configured with the parameters detailed in ref . . the evtgen and geant4 packages are used to generate hadron decays and simulate interactions in the detector , respectively . the digitized output is passed through a full simulation of both the hardware and software trigger and then reconstructed in the same way as the data . the overall efficiency ratio is 0.9010.016 , 1.0110.014 and 0.9940.014 for the b , the b and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } channels respectively . since the selection criteria for bj/x and b(2s)x decays are identical , the ratio of efficiencies is expected to be close to unity . the deviation of the overall efficiency ratio from unity in the case of the bk decays is due to the difference between the pt spectra of muons for the j/ and (2s ) decays . for the b and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } channels this difference is small . it has been checked that the behaviour of the efficiencies of all selection criteria is consistent in the data and simulation . since the decay products in each of the pairs of channels considered have similar kinematics , most uncertainties cancel in the ratio . the different contributions to the systematic uncertainties affecting this analysis are discussed in the following and summarized in table 2 . table 2systematic uncertainties ( in % ) on the relative branching fractionssource b channel b channel \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } channelnon - resonant decays1.53.4data - simulation agreement1.70.52.0magnet polarity1.40.60.7finite simulation sample size0.30.50.6trigger1.11.11.1background shape0.60.20.2signal shape0.70.80.5angular distribution<0.10.6particle misidentification0.4<0.1<0.1sum in quadrature2.72.24.3 systematic uncertainties ( in % ) on the relative branching fractions the dominant source of systematic uncertainty arises from the subtraction of the non - resonant components in the b and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } decays . first , determining the number of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{(s)}\rightarrow \psi k^{*0}(\phi)$\end{document } decays directly using the splot technique by unfolding and fitting the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{(s)}$\end{document } mass distribution of candidates containing genuine k( ) resonances . second , using the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{(s)}$\end{document } mass distribution as the discriminating variable to unfold the k(kk ) mass distribution of genuine \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{(s)}$\end{document } candidates and fitting this distribution to determine the number of non - resonant decays . the corresponding uncertainties are found to be 1.5 % in the b channel and 3.4 % in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } channel . the other important source of uncertainty arises from the estimation of the efficiencies due to the potential disagreement between data and simulation . the corresponding uncertainties are found to be 1.7 % in the b channel , 0.5 % in the b channel and 2.0 % in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } channel . the observed difference in the efficiency ratios for the two magnet polarities is conservatively taken as an estimate of the systematic uncertainty . this is 1.4 % in the b channel , 0.6 % in the b channel and 0.7 % in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } channel . the trigger is highly efficient in selecting b meson decays with two muons in the final state . for this analysis the di - muon pair is required to trigger the event . differences in the trigger efficiency between data and simulation are studied in the data using events which were triggered independently on the di - muon pair . a further uncertainty arises from the imperfect knowledge of the shape of the signal and background in the b meson mass distribution . to estimate this effect , a linear and a quadratic function are considered as alternative models for the background mass distribution . in addition , a double gaussian shape and a sum of double - sided crystal ball and gaussian shapes are used as alternative models for the signal shape . the maximum observed change in the ratio of yields in the (2s ) and j/ modes is taken as systematic uncertainty . the central value of the relative efficiency is determined by assuming that the angular distribution of the b(2s)x decay is the same as that of the bj/x . the systematic uncertainty due to the unknown polarization of the (2s ) in the b meson decays is estimated as follows . the simulation samples were re - weighted to match the angular distributions found from the data and the relative efficiency was recalculated . the difference between the baseline analysis and the re - weighted simulation is taken as the systematic uncertainty , as shown in table 2 . finally , the uncertainty due to potential contribution from the cabibbo - suppressed mode with a misidentified as k is found to be 0.4 % in the b channel and negligible in the b and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } channels . the uncertainty due to the cross - feed between b and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } channels with a misidentified as k ( or a k misidentified as ) is negligible . since the di - electron branching fractions are measured more precisely than those of the di - muon decay modes , we assume lepton universality and take \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_{\psi}=\mathcal{b } ( j/\psi \rightarrow \mu ^{+}\mu ^{-})/\mathcal { b } ( \psi(2s)\rightarrow \mu ^{+}\mu ^{- } ) = \mathcal{b } ( j/\psi \rightarrow e^{+}e^{-})/ \mathcal{b } ( \psi(2s)\rightarrow e^{+}e^{- } ) = 7.69\pm0.19$\end{document } . the results are combined using eq . ( 1 ) to give where the first uncertainty is statistical , the second is systematic and the third is the uncertainty on the r value . the resulting branching fraction ratios are compatible with , but significantly more precise than , the current world averages of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{b } ( b^{+}\rightarrow \psi ( 2s ) k^{+})/\mathcal{b } ( b^{+}\rightarrow j/\psi k^{+})=0.60\pm0.07$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{b } ( b^{0}_{s } \rightarrow \psi ( 2s)\phi)/\mathcal{b } ( b^{0}_{s } \rightarrow j/\psi \phi)=0.53\pm 0.10$\end{document } and the cdf result of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{b } ( b^{0}\rightarrow \psi ( 2s ) k^{*0})/\mathcal{b } ( b^{0}\rightarrow j/\psi k^{*0 } ) = 0.515\pm0.113\pm0.052$\end{document } . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow \psi(2s)\phi$\end{document } decay is particularly interesting since , with more data , it can be used for the measurement of cp violation in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } mixing .

the relative rates of b - meson decays into j/ and (2s ) mesons are measured for the three decay modes in pp collisions recorded with the lhcb detector . the ratios of branching fractions ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{b } $ \end{document } ) are measured to be where the third uncertainty is from the ratio of the (2s ) and j/ branching fractions to +.

Introduction Detector description Event selection Measurement of Efficiencies and systematic uncertainties Results

the relative branching fractions of b , b and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s}$\end{document } mesons into j/ and (2s ) mesons have previously been studied by both the cdf and d0 collaborations [ 24 ] . since the current experimental results for the study of cp violation in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } mixing using the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow j/\psi \phi$\end{document } decay [ 57 ] are statistically limited , it is important to establish other channels where this analysis can be done . in this paper , measurements of the ratios of the branching fractions of b mesons decaying to (2s)x and j/x are reported , where b denotes a b , b or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s}$\end{document } meson ( charge conjugate decays are implicitly included ) and x denotes a k , k or meson . the data were collected by the lhcb experiment in pp collisions at the centre - of - mass energy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt{s } = 7~\mathrm{tev}$\end{document } during 2011 and correspond to an integrated luminosity of 0.37 fb . the reduced of this fit ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\chi^{2}_{\mathrm{dtf}}/\mathrm{ndf}$\end{document } ) is required to be less than 5 , where the dtf algorithm takes into account the number of decay products to determine the number of degrees of freedom . the fit is described in the texttable 1summary of the signal yields for the six b modes considered and the ratios of the number of j/ and (2s ) decays : n is the summed signal yield for resonant and non - resonant modes , n is the signal yield for non - resonant modes only and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$n^{\mathrm{res}}_{\psi x}$\end{document } is the signal yield for resonant decays ( through k or ) . the total fitted function ( solid ) and the combinatorial background ( dashed ) the fit is described in the text summary of the signal yields for the six b modes considered and the ratios of the number of j/ and (2s ) decays : n is the summed signal yield for resonant and non - resonant modes , n is the signal yield for non - resonant modes only and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$n^{\mathrm{res}}_{\psi x}$\end{document } is the signal yield for resonant decays ( through k or ) . the overall efficiency ratio is 0.9010.016 , 1.0110.014 and 0.9940.014 for the b , the b and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } channels respectively . for the b and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } channels this difference is small . table 2systematic uncertainties ( in % ) on the relative branching fractionssource b channel b channel \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } channelnon - resonant decays1.53.4data - simulation agreement1.70.52.0magnet polarity1.40.60.7finite simulation sample size0.30.50.6trigger1.11.11.1background shape0.60.20.2signal shape0.70.80.5angular distribution<0.10.6particle misidentification0.4<0.1<0.1sum in quadrature2.72.24.3 systematic uncertainties ( in % ) on the relative branching fractions the dominant source of systematic uncertainty arises from the subtraction of the non - resonant components in the b and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } decays . the corresponding uncertainties are found to be 1.5 % in the b channel and 3.4 % in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } channel . the corresponding uncertainties are found to be 1.7 % in the b channel , 0.5 % in the b channel and 2.0 % in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } channel . finally , the uncertainty due to potential contribution from the cabibbo - suppressed mode with a misidentified as k is found to be 0.4 % in the b channel and negligible in the b and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } channels . since the di - electron branching fractions are measured more precisely than those of the di - muon decay modes , we assume lepton universality and take \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_{\psi}=\mathcal{b } ( j/\psi \rightarrow \mu ^{+}\mu ^{-})/\mathcal { b } ( \psi(2s)\rightarrow \mu ^{+}\mu ^{- } ) = \mathcal{b } ( j/\psi \rightarrow e^{+}e^{-})/ \mathcal{b } ( \psi(2s)\rightarrow e^{+}e^{- } ) = 7.69\pm0.19$\end{document } . the resulting branching fraction ratios are compatible with , but significantly more precise than , the current world averages of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{b } ( b^{+}\rightarrow \psi ( 2s ) k^{+})/\mathcal{b } ( b^{+}\rightarrow j/\psi k^{+})=0.60\pm0.07$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{b } ( b^{0}_{s } \rightarrow \psi ( 2s)\phi)/\mathcal{b } ( b^{0}_{s } \rightarrow j/\psi \phi)=0.53\pm 0.10$\end{document } and the cdf result of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{b } ( b^{0}\rightarrow \psi ( 2s ) k^{*0})/\mathcal{b } ( b^{0}\rightarrow j/\psi k^{*0 } ) = 0.515\pm0.113\pm0.052$\end{document } . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } \rightarrow \psi(2s)\phi$\end{document } decay is particularly interesting since , with more data , it can be used for the measurement of cp violation in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b^{0}_{s } $ \end{document } mixing .

water - mediated interactions play critical roles in biomolecular recognition processes , such as protein folding and protein ligand binding . in these processes , hydrophobic regions and apolar molecules are often driven together by the surrounding aqueous solution which has been noted since the famous publication by kauzmann in 1959 . usually , increasing the apolar binding surface area decreases its dissociation constant ( increase the strength of binding ) . during the process of optimizing lead molecules to drug , one of the important strategies for medicinal chemists is to utilize these hydration properties of ligands and receptors . the importance of hydrophobic effects in quantitative structure activity relationship ( qsar ) has been documented since the first publication of qsar in 1962 . detailed understanding of the hydration properties for each important biological system is important for the rational design of better drugs . theoretical approaches for modeling the hydrophobic effect , such as void volume theories , interpret that the hydrophobic effect is length - scale dependent . at small length scales ( e.g. , solute radius smaller than roughly 0.5 nm ) , the solvation of solutes is mainly an entropically driven process in which the water hydrogen bonding network rearranges itself to adapt for the intercalation of solutes . on the other hand , at large length scales , a significant number of water hydrogen bonds around the solute are broken due to the restructuring at extended , relatively flat surfaces of the solutes , resulting into a domination of enthalpic contributions to the solvation process . unlike generic theoretical models with uniform chemical detail , for proteins with both polar and apolar residues , the solute in such a heterogeneous and dynamical environment , water and protein structures are sensitive to the local solute solvent interface geometry , as well as van der waals ( vdw ) and electrostatic interactions . the coupling between interface geometry and structural polarity is crucial to protein biological function . characterizing the hydration properties surrounding the biomolecules and comparing them with those of bulk water significant progress has been made through the combination of various experiments , theoretical techniques , and models . computer simulations , such as molecular dynamics ( md ) simulation with explicit solvent models , can shed light on the delicate balance among these factors and illustrate the hydrophobic effects associated with ligand molecules and induced polarization . however , one drawback of molecular dynamics simulations is the relatively high computational demand for large systems such as biomolecules . furthermore , the intrinsic fluctuations in all atom md simulations can obscure the underlying intuitive physical pictures . in addition to the explicit solvent simulations , various implicit - solvent models are also developed as efficient alternatives to study the biomolecular hydration behaviors and the values of hydration free energy . to date , most commonly used implicit - solvent models ( e.g. , poisson solvent interfaces ( e.g. , solvent accessible surface ( sas ) , solvent excluded surface ( ses ) , or van der waals ( vdw ) surface ) , in which polar and apolar interactions are assumed to be additive and decoupled . recently , a gaussian - based approach was developed to obtain the distribution of the dielectric constant for a protein in solution . by using the local dielectric constant values , the implicit - solvent models lead to better results than the ones using a uniform dielectric constant . there are also other microscopic theoretical treatments of water without predefined surface and based on the statistical mechanics theory of molecular liquid . the most notable one is the three - dimensional reference interaction site model ( 3d - rism ) . in this model , the solvent distribution function and correlation functions are self - consistently solved with a proper closure relation . it avoids the explicit molecular sampling of instantaneous solvent configurations because it works on average distribution function for the particular atoms . with recent advancement , 3d - rism is able to calculate hydration free energy , equilibrium solvent distributions , and other thermodynamic quantities efficiently for biomolecular systems . the variational implicit - solvent model ( vism ) developed by dzubiella et al . provides a self - consistent description of the molecular solvation with the contributions from the solute solvent interface geometry and van der waals and electrostatic interactions coupled together in a physical based free - energy functional within the purely surface - based implicit - solvent model framework . solvent interface is determined by minimizing the solvation free - energy functional that balances the interface local geometry with various hydration free energy contributions . a similar approach has also been put forward by wei et al . in their model , both theoretical and experimental studies over the past decades showed a complicated picture of hydration behaviors around biomolecules . in this study , we apply a joint md and vism analysis to gain certain quantitative and qualitative insights into the heterogeneous hydration , the solute most previous studies of hydration behavior focused on either solute geometrical complexity with uniform chemical details or heterogeneous chemical details with a simple domain interface . hua et al . modeled parallel plates consisting of both hydrophobic and hydrophilic particles . they showed that the behavior of water between the two plates strongly depends on the distribution of the hydrophobic and hydrophilic particles on the plates . in the studies presented here , we choose a biologically important and realistic system , the p53/mdm2 protein complex with both geometrical and chemical detail complexities , as an example to investigate hydration properties near complex biomolecular binding sites . the receptor protein mdm2 acts as a negative regulator of the tumor suppressor protein p53 , and loss of the p53 function is observed in 50% of human malignancy . to understand the hydration properties of the p53/mdm2 complex during the process of association is very important for the understanding of the p53/mdm2 binding mechanism . furthermore , inhibitor design to reactivate p53 as tumor suppressor protein will also benefit from this study . this system exhibits a strong hydrophobic character at the p53/mdm2 binding interface ( 70% of the residues at the binding interface are apolar ) . however , the edge of the binding pocket is decorated by polar hydrophilic residues . therefore , it provides an excellent example to study the heterogeneous hydration properties in a protein the red color represents charged hydrophilic residues , the pink color represents neutral hydrophilic residues , and the blue color represents hydrophobic residues . in figure 1a , it is apparent that the hydrophobic areas of the two domains are in contact with each other in the bound state . in figure 1b one can clearly see that the binding interfaces are essentially hydrophobic ( blue ) . the domain interface is buried with hydrophobic residues in blue , charged residues in red , and neutral hydrophilic residues in pink . ( b ) two domains of the complex are separated and rotated in such a way that the binding interfaces face the reader . in this work , md simulations and vism calculations are used to study the heterogeneous hydration behaviors around the protein during the p53/mdm2 binding . through md simulations , we construct water density profiles from which we observe high water density solvation shells near the hydrophilic residues and depleted water density regions near the hydrophobic binding pocket of mdm2 . furthermore , we identify a bimodal state for the water occupancy behavior in the binding pocket when the interdomain distance is between 4 and 6 . a solute solvent hydration energy density map is used to illustrate the relation between the water density and the various solute solvent interactions . as of now , six clinical trials we hope our study can further the understanding of the binding affinity and kinetics between p53 and mdm2 , which eventually could help to design next generation p53/mdm2 inhibitors . the initial structure of the p53/mdm2 complex is taken from the protein data bank ( pdb code : 1ycr.pdb ) . hydrogen atoms are added at ph = 7.0 using the protein preparation workflow in maestro . acetyl ( ace ) and n - methyl amide ( nma ) are incorporated to cap the n- and c - termini , respectively . a series of configurations for the protein complex are produced by separating the two domains along the axis through their geometrical centers . the value of d = 0 corresponds to the native crystal structure of the complex . for md simulations , each configuration is then placed in an orthorhombic box with a minimum distance of 10.0 to the boundary of box and hydrated with a pre - equilibrated box of tip3p water using the system builder module of the desmond package . all overlapping solvent molecules are removed , and counterions are added to maintain charge neutrality . for vism calculations , the same input solute structures with partial charges as in the md simulations are used , so that comparisons between explicit and implicit modeling can be made . all molecular dynamics ( md ) simulations are performed using the desmond package . the opls 2005 force field is used to model the protein interactions , and the tip3p model is used for the water . particle - mesh ewald method ( pme ) is used to calculate the long - range electrostatic interactions with grid spacing of 0.8 . van der waals and short - range electrostatic interactions are smoothly truncated at 9.0 . the nose - hoover thermostat is used to maintain the constant simulation temperature and the martina - tobias - klein method is used to control the pressure . the equations of motion are integrated using the multistep respa integrator with an inner time step of 2.0 fs for bonded interactions and nonbonded interactions within the short - range cutoff . an outer time step of 6.0 fs is used for nonbonded interactions beyond the cutoff . periodic boundary conditions are applied . to focus on the water behavior around the p53/mdm2 surface , we constrain the protein molecules so that their conformational fluctuations are not convoluted with the process . these constraints will also facilitate the grid - based water density mapping around the protein and inside of the binding pocket and the direct comparison with the vism calculations . the systems are equilibrated with the default protocol provided in desmond . after the equilibration , a 40 ns npt production simulation is performed for each configuration at temperature 300 k and pressure 1.01 bar with a 20 kcal/(mol ) harmonic potential restraint on solute atoms , and the simulation trajectories are saved in 4-ps intervals for analysis . the details of vism with the coulomb - field approximation ( cfa ) are described extensively in the previous publications . in short , we optimize the free energy of the solvation system as a functional of all possible solute solvent interfaces .1where n solute atoms are located at x1 , ... , xn inside m and with point charges q1 , ... , qn , respectively . ( in our system of p53/mdm2 , n = 1688 . ) the first term pvol(m ) is the volumetric part of the energy for creating the solute cavity m with p being the pressure difference between the solvent liquid and solute vapor . the second term is the surface energy , where (x ) is the surface tension given by (x ) = 0(1 2h(x ) ) , where 0 is the constant macroscopic surface tension for a planar solvent liquid vapor interface which is set as 0.127 kbt / at 300 k according to the tip3p water simulation . is the first order correction coefficient often termed as the tolman coefficient which is set as 1.0 , and h(x ) is the mean curvature defined as the average of the two principal curvatures . the third term is the energy of the van der waals interaction between the solute atoms and the continuum solvent . the parameter w is the solvent density which is set as bulk water density 0 = 0.0333/ . it is defined by the born cycle as the difference of the energies of two states , where 0 is the vacuum permittivity , m is the relative permittivity of the solute molecule , and w is the relative permittivity of the solvent which are defined by the vism solute solvent interface by (x ) = m if x m and (x ) = w if x m . now the free energy g[ ] determines the effective boundary force , g[ ] , acting on the vism solute solvent surface , where is the variational derivative with respect to the location change of . it is only the normal component of this force that can affect the motion of such a solute we denote by n = n(x ) the unit normal vector at a point x on the solute solvent surface , pointing from the solute region m to the solvent region m . k = k(x ) is the gaussian curvature , defined as the product of the two principal curvatures , at a point x on . this force will be used as the normal velocity in our level - set numerical calculations . to minimize the free - energy functional ( 1 ) , we choose two different types of initial surfaces , a loose and a tight initial surface . both of them enclose all the solute atoms located at x1 , ... , xn . the tight initial surface is defined by the van der waals ( vdw ) surface . the loose initial surface is often set to be a large sphere enclosing all the solute atoms . in this study , it is chosen so that the closest solute atom ( from the vdw sphere edge ) is at least 1.5 water diameters away form the surface . the initial interface can have a very large value of the free energy . it is subsequently moved in the direction of steepest descent of the free energy by the level - set method until a minimum is reached . the starting point of the level - set method is the representation of a surface using the ( zero ) level - set of a function = (x ) : = { x : (x ) = 0}. the motion of a moving surface = (t ) with t denoting the time is then tracked by the evolution of the level - set function = (x , t ) whose zero level - set is (t ) at each t. such evolution is determined by the level - set equation3 to study the water density profiles around the p53/mdm2 complex , a lattice with grid size of 0.8 is constructed and tip3p water oxygen atoms from md simulations are assigned to the nearest grid points . to understand the origin of the inhomogeneous water distribution from the solute solvent interaction perspective , a solute solvent hydration energy density map is constructed . from this energy density map , we directly identify the hydrophobic and hydrophilic regions around the protein . in this study , we use the vism free energy functional to fulfill this purpose . in principle , the solute solvent hydration energy density map can also be obtained from md results . however , it is much more desirable to calculate the solute solvent hydration energy density from efficient methods than long time md simulations . from the vism solvation free energy functional ( eq 1 ) , the van der waals and electrostatic solvation free energy contributions can be considered as the integration of solute , we define a solute solvent hydration energy density as4where56 the vdw solute solvent hydration energy density vdw(x ) and electrostatic solute solvent hydration energy densities elec(x ) are obtained by using the same formula as the individual component of the solvation free energy functional ( i.e. , eq 1 ) in vism without the volume integration . in order to study the aqueous behavior only in the hydrophobic binding pocket of mdm2 , we used the differences between the contracted level - set vism surface with a loose initial surface and the molecular surface to define the binding pocket ( figure 2 ) . binding pocket region is defined by the differences between molecular surface ( green ) and a contracted vism surface with loose initial surface ( red ) . the water density profile is constructed from a 40 ns md simulation with tip3p water . figure 3 shows the cross - section of water density profile across the target protein mdm2 binding pocket when interdomain distance d = 12 . in this figure , the protein complex is represented by its molecular surface with the same color code as figure 1 . the value of the local water density is represented by colors in the legend . white color indicates zero density , blue represents half of the bulk water density , yellow represents the bulk water density , and red represents twice of the bulk water density . ( a ) cross - section of water density profile through the binding pocket based on the md simulation at interdomain distance d = 12 . the molecular surface is represented with the same color code as figure 1 ( blue for hydrophobic residues , red for charged residues , pink for neutral hydrophilic residues ) . the colors in the legend represent the average water density ( range from 0 to 2 and in units of bulk density 0 ) . ( b ) the same cross - section water density profile as ( a ) without showing the protein . it is observed that high density hydration layers are formed around most parts of the solute molecules as shown by the red color . the water density of the hydration layer close to hydrophilic residues ( red molecular surface ) is much higher and more discrete than that near hydrophobic residues ( blue molecular surface ) . when near the convex protein surface , even around the hydrophobic residues , high density and continued hydration layers are observed . when the protein surface is concave and residues are hydrophobic near the binding pocket at the interdomain region , the hydration layers form at the entrance of the pocket away from the molecular surface and water density drops to zero at the bottom of the hydrophobic pocket . these hydration shells behave differently according to local protein surface geometry and chemical details . such a phenomenon as high density layers formed far away from the molecular surface is also observed in several simpler model systems previously and is attributed to the liquid vapor interface in the hydrophobic binding pocket , and the water density fluctuating between higher than bulk to zero . these behaviors are explained by capillary evaporation and capillary condensation driven by solute solvent interactions . to understand quantitatively the relations between the hydration water density distribution around the protein and the solute solvent interactions , we analyze and compare the water density profile w(x ) and the solute solvent hydration energy density vdw+elec(x ) . the solute solvent interactions contributed hydration energy density consists of vdw and electrostatic components between solute and solvent . details can be found in the theory and methods part . in figure 4a , it shows the cross - section of water density profile w(x ) across the binding pocket with the location of molecular surface ( black line inside ) . the blue color represents vdw+elec(x ) smaller than 0.1 kbt / . by comparing figure 3a and figure 4b , we find that the high density water layers form at the region with the solute solvent hydration energy density less than 0.1 kbt / shown as deep blue in figure 4b . solvent hydration energy density ( vdw and electrostatic contributions ) are shown in figure 4c , d , respectively . ( a ) cross - section of water density profile w(x ) ( range from 0 to 2 and in units of bulk density 0 ) through the mdm2 binding pocket , black line inside is the location of molecular surface ( ms ) from md simulations , ( b ) solute solvent hydration energy density map ( vdw+elec(x ) ) and ( c ) vdw ( vdw(x ) ) , ( d ) electrostatic ( elec(x ) ) ( range from 0.1 to 0.1 and in units of kbt / ) individual parts derived from vism free energy functional . most hydrophilic areas shown in red in figure 4a originate from the favorable electrostatic solute this is demonstrated in figure 4d where the blue color depicts the strongly attractive solute solvent vdw repulsion ( shown red in figure 4c ) counter - balances the condensation . thus water densities in these solvation shells are much higher near the polar hydrophilic residues . in contrast , the white color at the binding pocket region indicates that the electrostatic contribution is nearly zero , i.e. , an apolar region . the vdw force is the dominant interaction between solute and solvent at this region . the lack of strong competition between attractive and repulsive solute solvent interactions leads to a lower water density solvation shell minimizing the surface energy and the high density hydration layers form far from the molecular surface at this concave region . the comparison between water density distribution from extensive md simulations and the hydration energy density from vism free energy functional shows that we can qualitatively identify the hydrophobic and hydrophilic regions from the solute solvent hydration energy density map without extra - simulations and it can be achieved in minutes just based on the information of 3d protein structure . solvent interactions in a free energy functional and is solved self - consistently . in the following study , we investigate the interplay between solute solvent interactions and the solute solvent interface geometry by numeric minimization of the vism free energy functional ( i.e. , eq 1 ) with respect to the solute figure 5 shows the superposition of equilibrium vism surfaces from both loose and tight initial surfaces with the water density profile and the solute the two equilibrium vism surfaces correspond to two stable states of solvation and are the results of the complex relations between polar , apolar , and surface energy contributions to the hydration process . when superimposing the equilibrium vism surfaces on the md water density profile w(x ) in figure 5a , we find that most part of the vism surface corresponds to the high density hydration shell regardless of the initial conditions except for the interdomain region . in figure 5b solvent hydration energy density map vdw+elec(x ) to show the relation between water density and solute water density profile ( range from 0 to 2 and in units of bulk density 0 ) ( a ) from md simulations and solute solvent hydration energy density map ( range from 0.1 to 0.1 and in units of kbt / ) ( b ) from vism free energy functional are superimposing with the equilibrium vism surfaces ( depicted by the thick orange white blue lines ) which are obtained from loose and tight initial surfaces , respectively . furthermore , in figure 6a , it shows the average water density ( in units of average bulk water density 0 ) vs the signed distance to the p53/mdm2 equilibrium vism surface ( a tight initial surface was used here ) . a negative value represents the distance to the equilibrium vism surface from inside and vice versa . the water density peak at zero suggests that the equilibrium vism surface is largely located at the first hydration shell and the water density vanishes to nearly zero inside the vism surface ( < 1.4 ) . however , in the mdm2 binding pocket , the vism surface captures the high energy density shell much further away from molecular surface ( figure 5b ) . comparing md results with vism surfaces , we can identify the delicate hydration balance for hydrophobic and hydrophilic effects near protein surfaces with both geometrical and chemical complexities . we find vism is able to properly describe hydrophobic binding pockets which are of ultimate interest in most cases . in contrast , traditional implicit - solvent models based on predetermined surfaces , such as pbsa , are only able to account for the geometrical shape defined by the vdw radii . solvent hydration energy density map vdw+elec(x ) ( black line ) and the individual components vdwc(x ) ( green line ) and elec(x ) ( red line ) as a function of signed distance to the equilibrium vism surface . by comparison of these two figures , one can see that the solute solvent interactions are essentially the main driving forces for the formation of high density hydration layer . ( a ) water density profile w(x ) ( in units of average bulk water density 0 ) in 1d obtained from the md simulation vs the distance to the vism surface of a tight initial . ( b ) solute solvent hydration energy density and individual components from the vism free energy functional vs the distance to the vism surfaces obtained by a tight initial surface . a negative distance represents the distance from the vism surface to the solute direction , and a positive value represents the other way . , it takes about a week on average for 40 ns md simulation with 8 cpus in parallel for each configuration . in contrast , it takes about 1 h for tight initial surface and 3 to 4 h for loose initial surface with single cpu processor for the same configuration in md simulations . the calculation speed of vism strongly depends on the initial surfaces and convergence threshold . we are working on speeding up the vism calculation through numerical algorithm improvement , better initial conditions , and parallelization . interesting hydration behaviors happen inside of the hydrophobic mdm2 binding pocket . in the following study , we define the region of mdm2 binding pocket as the area between the molecular surface and a contracted vism surface from a loose initial surface of mdm2 ( details in methods of analysis section ) . in figure 7a , it shows the water density distribution inside of the binding pocket of mdm2 . in this figure , the blue color represents density lower than half of the average bulk density 0 and the red color represents regions with water density 1.5 times higher than the average bulk density 0 . when the transactivation domain of p53 binds to mdm2 , the key residue l26 occupies the left region in the figure , w23 occupies the middle , and f19 occupies the right . the left pocket is formed by hydrophobic residues ile99 , leu57 , and leu54 . the middle one is formed by val75 , leu57 , ile61 , val93 , and ile99 , and the right pocket is formed by val93 , val75 , and il61 . in figure 7a , we find three isolated high water density regions in the p53-l26 binding pocket , four high water density regions in the w23 binding pocket , and two high water density regions in the f19 binding pocket . these high density regions will be occupied by the three p53 residues when they bind . in figure 7b , we show vdw+elec(x ) inside of the binding pocket , the blue color represents hydration energy density lower than 0.1 kbt / indicating hydrophilic regions , the red color represents energy density higher than + 0.1 kbt / indicating hydrophobic regions , and the white color represents the zero hydration energy density level . when p53 binds to this pocket and water molecules are replaced by ligand atoms , a substantial amount of entropy can be gained by occupying the red high water density with ordered water regions ( red in figure 7a ) . enthalpic gain is more pronounced at the blue region where water molecules are relatively close to the surface of the protein . in figure 7b , this figure illustrates that the low vdw+elec(x ) regions locate near the critical binding regions for the three p53 key residues . ( a ) water density profile inside of the mdm2 binding pocket from md simulations with p53 peptide absence . the entrance of the binding pocket is at the front and the bottom is at the back . in part a , the blue color represents density lower than 0.50 and the red color represents density higher than 1.50 . ( b ) solute solvent hydration energy density map vdw+elec(x ) from vism free energy functional inside of the binding pocket . in part b , the blue color represents the value lower than 0.1 kbt / , the red color represents values higher than + 0.1 kbt / , and the white color represents the zero energy level . in figure 8a , we show the distribution of water densities inside of the binding pocket in a range of interdomain distances from 4 to 12 . in this figure , when the interdomain distance is larger than 6 , the water density inside of the cavity has a one - state distribution , and the peaks are located around 0.50.60 . when the interdomain distance decreases to 5 , an interesting two - state water density profile emerges with peaks at 0.20 and 0.50 . they correspond to partially dry and wet states for this pocket . as the interdomain distance decreases further to 4 , the water density profile peaks at zero , corresponding to a completely dry state . ( a ) water density distribution inside of the binding pocket obtained from md simulations when different interdomain distances range from 4 to 12 . blue line represents the bulk water distribution in the same volume as d = 12 . ( b ) normalized autocorrelation function ( acf ) cnn(t ) of occupancy fluctuations in the pocket with the p53/mdm2 interdomain distance increasing from 4 to 12 . the blue line represents the bulk water autocorrelation in the same volume as d = 12 . in figure 8b , we show the water occupancy autocorrelation function ( acf ) , cnn(t ) = n(t)n(0)/n. bulk water acf is shown with the blue line as a comparison . in the recent work of setny et al . , a more than 1 order of magnitude shift in time scale for the acf in a purely hydrophobic pocket was reported . here , we find that the acf of occupancy fluctuation in the mdm2 binding pocket depends on the interdomain distances . a very long correlation time exists for a particular interdomain distance . when the separation deviates from there , the correlation time reduces quickly . in the case of p53/mdm2 , the distance is around 5 . when the interdomain distance is larger than that value , the binding pocket begins to get flooded by bulk water giving shorter correlation times . when the interdomain distance is smaller than 5 , the water also fluctuates faster . under this condition , the waters inside the pocket prefer to evacuate the binding site quickly . interestingly but not surprisingly , when around d = 5 , the drying and wetting processes compete and result in long correlation times ( 400 ps ) . as a result , a bimodal distribution with two peaks at 0.20 and 0.50 arises at this distance as shown in figure 7a with population ratio of 0.41/0.59 which is obtained by gaussian distribution fitting of the bimodal density probability . in order to interpret the population of dry and wet states in 40 ns md simulation at certain distance , we artificially define the dry state as the pocket water density smaller than 0.20 and the wet state as pocket water density larger than 0.50 . figure 9 shows the water density profile by averaging md frames which are selected by the dry ( a ) and wet ( b ) state criteria . in figure 8a , a water depleted dry region is observed in the interdomain region which is flooded in figure 8b . during the 40 ns simulation , we find 19% frames in which the pocket water density is smaller than 0.20 and 24% frames with larger than 0.50 pocket water density . in this interdomain distance , ( a ) water density profile by averaging md trajectories in which the binding pocket water density is less than 0.20 . ( b ) water density profile by averaging md trajectories in which the binding pocket water density is larger than 0.50 . figures are for the p53/mdm2 at an interdomain distance of 5 . in figure 10 , we also calculated the relative population of dry and wet states for different interdomain distances ranging from 4 to 12 . from these p53/mdm2md simulations , we find that the binding pocket will prefer a flooded wet state when the interdomain distance is larger than 6 and a depleted dry state when the interdomain distance is smaller than 4 . the interesting dry wet transition likely takes place when the p53/mdm2 interdomain distance is between 4 and 6 . the dehydration at the hydrophobic interface has profound implications to the kinetics of p53/mdm2 binding . mdm2 has positively charged residues ( k51 and h73 ) at the edge of the binding pocket , and p53 transactivation domain ( tad ) has two corresponding negatively charged partners ( e17 and e28 ) . the electrostatic interactions help to orientate ligand and receptor during binding . however , schon et al . , showed that the association rate of p53/mdm2 was independent of the ionic strength and the truncation of e17 and e28 did not reduce the binding affinity . on the basis of these results , they hypothesized that the binding of p53/mdm2 is dominated by the dehydration of the hydrophobic interface , hydrophobic interactions , and interface rearrangement while electrostatic contribution is less pronounced . probability of having a dry and wet pocket when interdomain distances ranging from 4 to 12 in md simulations . water density smaller than 0.20 for dry pocket ( blue line ) and larger than 0.50 for wet pocket ( red line ) . in the current study , the binding process of the p53 transactivation domain ( tad ) and mdm2 is modeled by conformational constrained protein domains . although the p53 tad has been shown as predominantly disordered before binding in solution , a high percentage of -helical secondary structure is required for stable binding with mdm2 . the high affinity of stapled p53 peptide also demonstrates that preorganization of tad is beneficial to the binding . therefore , the current frozen conformational modeling system can provide important insights to the binding process from the hydration perspective . it should be noted that the static protein simulations and analysis can not capture every aspect of the actual binding process , such as the long relaxation time for the pocket water due to large scale protein conformational dynamics . the coupling of protein dynamics to the surrounding water dynamics is of immense interest , and a completed picture will be undoubtedly much more complex than that presented in this study . in summary , we have studied the aqueous behavior around the p53/mdm2 complex in detail using md simulations and vism calculations . we show that water density is significantly lower in regions near the concave hydrophobic binding pocket and the hydrophobic ligand residues . near the hydrophobic cavity , the first solvation layer forms at the entrance of the pocket . the water density drops from higher than bulk to zero at the pocket bottom . the water in the hydrophobic binding pocket behaves like the liquid vapor interface and the water density fluctuates between high and low values . in addition , we show that the local water density is correlated with the local solute solvent hydration energy density . the interplay among polar , apolar , and geometric factors forms the energy minimum at the solute solvent interface . inside of the hydrophobic binding cavity , the averaged water density is lower than the bulk , and water molecules are heterogeneously distributed inside the pocket . the water occupancy in the concave binding pocket fluctuates between dry and wet states as a function of interdomain distance . this fluctuation has important implications to the kinetics of the p53/mdm2 binding process . as one of the most studied tumor proliferation pathways , detailed molecular binding mechanism and kinetics are of ultimate interest for developing therapeutics for activating p53 . although it is generally known for the hydrophobic nature of binding , we illustrate that the balance of hydrophobicity , shape , and polarity plays a key role in hydration . numerous successful drugs have also demonstrated that balanced hydrophilic and hydrophobic properties are critical for drug development . our results hopefully can provide a rational strategy to the design of new drugs with balanced hydrophobic and polar properties and taking advantage of the bimodal solvation process in the p53/mdm2 complex . for instance , one can optimize molecules that minimize the probability of bimodal solvation to gain fast onset kinetics . this study also illustrates the physical connection between the newly developed vism model and explicit water simulations . the vism surface mostly corresponds to the first high water density shell around the protein . in a protein binding site with complex geometry and charge distributions , it often defines the interface where bulk waters are greatly perturbed by the protein residues . in the case of the mdm2 binding site , it encloses the region that has 40% less water than the surrounding bulk . there have been many attempts to locate hot spots for water on the protein surface to find the potential binding sites , from both explicit and implicit - solvent aspects . the kinetics of hydration sites ( both drying and wetting processes ) strongly depends on the protein surface geometrical and physiochemical properties . for instance , the water molecules can be trapped and take a longer time to escape in deep and polar cavities than the ones in shallow hydrophobic areas . the variety of protein surfaces leads to relatively broad distributions of thermodynamics properties for hydration sites . in explicit solvent md simulation studies , pure water solvents as well as mixed solvents are used to investigate the solvation of protein ligand binding sites . correct hydration site predictions are only possible if the bound solvent molecules can be exchanged at a reasonably faster speed relative to the simulation time scale . have studied the exchange frequency by counting all the occurrences of solute solvent contacts for eight proteins including p53 peptide and mdm2 . they have shown that solvent interaction withthe p53 binding site is mostly short - lived ( up to 9 ns at most ) . beuming et al . have produced consistent convergence in the hydration site - free energies across a broad range of targets in their internal studies with 5.0 kcal / mol harmonic restraint and 2 ns production simulations . of course , sufficient sampling is not always warranted in every system by md simulation . for example , water molecule permeation through the na / glucose cotransporter ( sglt1 ) under equilibrium require hundreds of nanosecond simulations due to cotransporting partners , long transporting channel , and protein conformational change . in p53/mdm2 , sampling is less problematic as the pocket is relatively shallow so that the time scale to reach equilibrium hydration is shorter than sglt1 . although an extremely long kinetic trap for water can not be completely ruled out , we believe that 40 ns production md simulation after equilibration provides us sufficient hydration information for this particular system . all our calculations are based on the constrained protein surfaces to emphasize the role of water fluctuations during the hydration processes . it is similar to the early bagchi - zewail model . on the basis of their theoretical models , they revealed that the hydration dynamics directly relate to the water residence time near this protein surface . the hydration dynamics at the protein surface was characterized by two time scales , an ultrafast bulk like time scale followed by a slow one 10 times longer . the fast time scale arises mainly due to the reorientation and translation of hydrating water . it indicates that the water response is not qualitatively modified by a constrained protein . although the constrained protein surface limits rearrangements of local water networks , it does not alter the hydration dynamics qualitatively . of course , protein flexibility is still required to observe the slow component . in the current static model , important hydration effects , such as the different relaxation time scales for the pocket water due to the protein chemical and geometrical environment , the fluctuations between bound and bulk water , can be captured . it has also been shown that constrained protein simulations facilitate the grid mapping of water density . incorporating protein full flexibility will undoubtedly offer a richer and more complex picture of actual water behaviors around protein . we are actively pursuing this direction and hope to report our results in a future publication .

water - mediated interactions play critical roles in biomolecular recognition processes . explicit solvent molecular dynamics ( md ) simulations and the variational implicit - solvent model ( vism ) are used to study those hydration properties during binding for the biologically important p53/mdm2 complex . unlike simple model solutes , in such a realistic and heterogeneous solute solvent system with both geometrical and chemical complexity , the local water distribution sensitively depends on nearby amino acid properties and the geometric shape of the protein . we show that the vism can accurately describe the locations of high and low density solvation shells identified by the md simulations and can explain them by a local coupling balance of solvent solute interaction potentials and curvature . in particular , capillary transitions between local dry and wet hydration states in the binding pocket are captured for interdomain distance between 4 to 6 , right at the onset of binding . the underlying physical connection between geometry and polarity is illustrated and quantified . our study offers a microscopic and physical insight into the heterogeneous hydration behavior of the biologically highly relevant p53/mdm2 system and demonstrates the fundamental importance of hydrophobic effects for biological binding processes . we hope our study can help to establish new design rules for drugs and medical substances .

Introduction Theory and Methods Results and Discussion Conclusions

water - mediated interactions play critical roles in biomolecular recognition processes , such as protein folding and protein ligand binding . the importance of hydrophobic effects in quantitative structure activity relationship ( qsar ) has been documented since the first publication of qsar in 1962 . detailed understanding of the hydration properties for each important biological system is important for the rational design of better drugs . unlike generic theoretical models with uniform chemical detail , for proteins with both polar and apolar residues , the solute in such a heterogeneous and dynamical environment , water and protein structures are sensitive to the local solute solvent interface geometry , as well as van der waals ( vdw ) and electrostatic interactions . computer simulations , such as molecular dynamics ( md ) simulation with explicit solvent models , can shed light on the delicate balance among these factors and illustrate the hydrophobic effects associated with ligand molecules and induced polarization . furthermore , the intrinsic fluctuations in all atom md simulations can obscure the underlying intuitive physical pictures . in addition to the explicit solvent simulations , various implicit - solvent models are also developed as efficient alternatives to study the biomolecular hydration behaviors and the values of hydration free energy . by using the local dielectric constant values , the implicit - solvent models lead to better results than the ones using a uniform dielectric constant . the variational implicit - solvent model ( vism ) developed by dzubiella et al . provides a self - consistent description of the molecular solvation with the contributions from the solute solvent interface geometry and van der waals and electrostatic interactions coupled together in a physical based free - energy functional within the purely surface - based implicit - solvent model framework . in this study , we apply a joint md and vism analysis to gain certain quantitative and qualitative insights into the heterogeneous hydration , the solute most previous studies of hydration behavior focused on either solute geometrical complexity with uniform chemical details or heterogeneous chemical details with a simple domain interface . they showed that the behavior of water between the two plates strongly depends on the distribution of the hydrophobic and hydrophilic particles on the plates . in the studies presented here , we choose a biologically important and realistic system , the p53/mdm2 protein complex with both geometrical and chemical detail complexities , as an example to investigate hydration properties near complex biomolecular binding sites . to understand the hydration properties of the p53/mdm2 complex during the process of association is very important for the understanding of the p53/mdm2 binding mechanism . this system exhibits a strong hydrophobic character at the p53/mdm2 binding interface ( 70% of the residues at the binding interface are apolar ) . however , the edge of the binding pocket is decorated by polar hydrophilic residues . therefore , it provides an excellent example to study the heterogeneous hydration properties in a protein the red color represents charged hydrophilic residues , the pink color represents neutral hydrophilic residues , and the blue color represents hydrophobic residues . in figure 1a , it is apparent that the hydrophobic areas of the two domains are in contact with each other in the bound state . ( b ) two domains of the complex are separated and rotated in such a way that the binding interfaces face the reader . in this work , md simulations and vism calculations are used to study the heterogeneous hydration behaviors around the protein during the p53/mdm2 binding . through md simulations , we construct water density profiles from which we observe high water density solvation shells near the hydrophilic residues and depleted water density regions near the hydrophobic binding pocket of mdm2 . furthermore , we identify a bimodal state for the water occupancy behavior in the binding pocket when the interdomain distance is between 4 and 6 . a solute solvent hydration energy density map is used to illustrate the relation between the water density and the various solute solvent interactions . as of now , six clinical trials we hope our study can further the understanding of the binding affinity and kinetics between p53 and mdm2 , which eventually could help to design next generation p53/mdm2 inhibitors . the initial structure of the p53/mdm2 complex is taken from the protein data bank ( pdb code : 1ycr.pdb ) . for vism calculations , the same input solute structures with partial charges as in the md simulations are used , so that comparisons between explicit and implicit modeling can be made . all molecular dynamics ( md ) simulations are performed using the desmond package . the opls 2005 force field is used to model the protein interactions , and the tip3p model is used for the water . these constraints will also facilitate the grid - based water density mapping around the protein and inside of the binding pocket and the direct comparison with the vism calculations . it is defined by the born cycle as the difference of the energies of two states , where 0 is the vacuum permittivity , m is the relative permittivity of the solute molecule , and w is the relative permittivity of the solvent which are defined by the vism solute solvent interface by (x ) = m if x m and (x ) = w if x m . it is subsequently moved in the direction of steepest descent of the free energy by the level - set method until a minimum is reached . the motion of a moving surface = (t ) with t denoting the time is then tracked by the evolution of the level - set function = (x , t ) whose zero level - set is (t ) at each t. such evolution is determined by the level - set equation3 to study the water density profiles around the p53/mdm2 complex , a lattice with grid size of 0.8 is constructed and tip3p water oxygen atoms from md simulations are assigned to the nearest grid points . to understand the origin of the inhomogeneous water distribution from the solute solvent interaction perspective , a solute solvent hydration energy density map is constructed . from the vism solvation free energy functional ( eq 1 ) , the van der waals and electrostatic solvation free energy contributions can be considered as the integration of solute , we define a solute solvent hydration energy density as4where56 the vdw solute solvent hydration energy density vdw(x ) and electrostatic solute solvent hydration energy densities elec(x ) are obtained by using the same formula as the individual component of the solvation free energy functional ( i.e. in order to study the aqueous behavior only in the hydrophobic binding pocket of mdm2 , we used the differences between the contracted level - set vism surface with a loose initial surface and the molecular surface to define the binding pocket ( figure 2 ) . the value of the local water density is represented by colors in the legend . ( a ) cross - section of water density profile through the binding pocket based on the md simulation at interdomain distance d = 12 . when the protein surface is concave and residues are hydrophobic near the binding pocket at the interdomain region , the hydration layers form at the entrance of the pocket away from the molecular surface and water density drops to zero at the bottom of the hydrophobic pocket . such a phenomenon as high density layers formed far away from the molecular surface is also observed in several simpler model systems previously and is attributed to the liquid vapor interface in the hydrophobic binding pocket , and the water density fluctuating between higher than bulk to zero . to understand quantitatively the relations between the hydration water density distribution around the protein and the solute solvent interactions , we analyze and compare the water density profile w(x ) and the solute solvent hydration energy density vdw+elec(x ) . by comparing figure 3a and figure 4b , we find that the high density water layers form at the region with the solute solvent hydration energy density less than 0.1 kbt / shown as deep blue in figure 4b . ( a ) cross - section of water density profile w(x ) ( range from 0 to 2 and in units of bulk density 0 ) through the mdm2 binding pocket , black line inside is the location of molecular surface ( ms ) from md simulations , ( b ) solute solvent hydration energy density map ( vdw+elec(x ) ) and ( c ) vdw ( vdw(x ) ) , ( d ) electrostatic ( elec(x ) ) ( range from 0.1 to 0.1 and in units of kbt / ) individual parts derived from vism free energy functional . in contrast , the white color at the binding pocket region indicates that the electrostatic contribution is nearly zero , i.e. the lack of strong competition between attractive and repulsive solute solvent interactions leads to a lower water density solvation shell minimizing the surface energy and the high density hydration layers form far from the molecular surface at this concave region . the comparison between water density distribution from extensive md simulations and the hydration energy density from vism free energy functional shows that we can qualitatively identify the hydrophobic and hydrophilic regions from the solute solvent hydration energy density map without extra - simulations and it can be achieved in minutes just based on the information of 3d protein structure . in the following study , we investigate the interplay between solute solvent interactions and the solute solvent interface geometry by numeric minimization of the vism free energy functional ( i.e. when superimposing the equilibrium vism surfaces on the md water density profile w(x ) in figure 5a , we find that most part of the vism surface corresponds to the high density hydration shell regardless of the initial conditions except for the interdomain region . in figure 5b solvent hydration energy density map vdw+elec(x ) to show the relation between water density and solute water density profile ( range from 0 to 2 and in units of bulk density 0 ) ( a ) from md simulations and solute solvent hydration energy density map ( range from 0.1 to 0.1 and in units of kbt / ) ( b ) from vism free energy functional are superimposing with the equilibrium vism surfaces ( depicted by the thick orange white blue lines ) which are obtained from loose and tight initial surfaces , respectively . the water density peak at zero suggests that the equilibrium vism surface is largely located at the first hydration shell and the water density vanishes to nearly zero inside the vism surface ( < 1.4 ) . however , in the mdm2 binding pocket , the vism surface captures the high energy density shell much further away from molecular surface ( figure 5b ) . comparing md results with vism surfaces , we can identify the delicate hydration balance for hydrophobic and hydrophilic effects near protein surfaces with both geometrical and chemical complexities . in contrast , traditional implicit - solvent models based on predetermined surfaces , such as pbsa , are only able to account for the geometrical shape defined by the vdw radii . by comparison of these two figures , one can see that the solute solvent interactions are essentially the main driving forces for the formation of high density hydration layer . ( b ) solute solvent hydration energy density and individual components from the vism free energy functional vs the distance to the vism surfaces obtained by a tight initial surface . in figure 7a , it shows the water density distribution inside of the binding pocket of mdm2 . in this figure , the blue color represents density lower than half of the average bulk density 0 and the red color represents regions with water density 1.5 times higher than the average bulk density 0 . in figure 7b , we show vdw+elec(x ) inside of the binding pocket , the blue color represents hydration energy density lower than 0.1 kbt / indicating hydrophilic regions , the red color represents energy density higher than + 0.1 kbt / indicating hydrophobic regions , and the white color represents the zero hydration energy density level . enthalpic gain is more pronounced at the blue region where water molecules are relatively close to the surface of the protein . ( a ) water density profile inside of the mdm2 binding pocket from md simulations with p53 peptide absence . the entrance of the binding pocket is at the front and the bottom is at the back . ( b ) solute solvent hydration energy density map vdw+elec(x ) from vism free energy functional inside of the binding pocket . in figure 8a , we show the distribution of water densities inside of the binding pocket in a range of interdomain distances from 4 to 12 . in this figure , when the interdomain distance is larger than 6 , the water density inside of the cavity has a one - state distribution , and the peaks are located around 0.50.60 . ( a ) water density distribution inside of the binding pocket obtained from md simulations when different interdomain distances range from 4 to 12 . ( b ) normalized autocorrelation function ( acf ) cnn(t ) of occupancy fluctuations in the pocket with the p53/mdm2 interdomain distance increasing from 4 to 12 . here , we find that the acf of occupancy fluctuation in the mdm2 binding pocket depends on the interdomain distances . when the interdomain distance is larger than that value , the binding pocket begins to get flooded by bulk water giving shorter correlation times . in order to interpret the population of dry and wet states in 40 ns md simulation at certain distance , we artificially define the dry state as the pocket water density smaller than 0.20 and the wet state as pocket water density larger than 0.50 . in this interdomain distance , ( a ) water density profile by averaging md trajectories in which the binding pocket water density is less than 0.20 . in figure 10 , we also calculated the relative population of dry and wet states for different interdomain distances ranging from 4 to 12 . from these p53/mdm2md simulations , we find that the binding pocket will prefer a flooded wet state when the interdomain distance is larger than 6 and a depleted dry state when the interdomain distance is smaller than 4 . mdm2 has positively charged residues ( k51 and h73 ) at the edge of the binding pocket , and p53 transactivation domain ( tad ) has two corresponding negatively charged partners ( e17 and e28 ) . , showed that the association rate of p53/mdm2 was independent of the ionic strength and the truncation of e17 and e28 did not reduce the binding affinity . on the basis of these results , they hypothesized that the binding of p53/mdm2 is dominated by the dehydration of the hydrophobic interface , hydrophobic interactions , and interface rearrangement while electrostatic contribution is less pronounced . probability of having a dry and wet pocket when interdomain distances ranging from 4 to 12 in md simulations . in the current study , the binding process of the p53 transactivation domain ( tad ) and mdm2 is modeled by conformational constrained protein domains . it should be noted that the static protein simulations and analysis can not capture every aspect of the actual binding process , such as the long relaxation time for the pocket water due to large scale protein conformational dynamics . in summary , we have studied the aqueous behavior around the p53/mdm2 complex in detail using md simulations and vism calculations . we show that water density is significantly lower in regions near the concave hydrophobic binding pocket and the hydrophobic ligand residues . near the hydrophobic cavity , the first solvation layer forms at the entrance of the pocket . the water in the hydrophobic binding pocket behaves like the liquid vapor interface and the water density fluctuates between high and low values . in addition , we show that the local water density is correlated with the local solute solvent hydration energy density . the water occupancy in the concave binding pocket fluctuates between dry and wet states as a function of interdomain distance . although it is generally known for the hydrophobic nature of binding , we illustrate that the balance of hydrophobicity , shape , and polarity plays a key role in hydration . our results hopefully can provide a rational strategy to the design of new drugs with balanced hydrophobic and polar properties and taking advantage of the bimodal solvation process in the p53/mdm2 complex . in a protein binding site with complex geometry and charge distributions , it often defines the interface where bulk waters are greatly perturbed by the protein residues . there have been many attempts to locate hot spots for water on the protein surface to find the potential binding sites , from both explicit and implicit - solvent aspects . the kinetics of hydration sites ( both drying and wetting processes ) strongly depends on the protein surface geometrical and physiochemical properties . in explicit solvent md simulation studies , pure water solvents as well as mixed solvents are used to investigate the solvation of protein ligand binding sites . the hydration dynamics at the protein surface was characterized by two time scales , an ultrafast bulk like time scale followed by a slow one 10 times longer . in the current static model , important hydration effects , such as the different relaxation time scales for the pocket water due to the protein chemical and geometrical environment , the fluctuations between bound and bulk water , can be captured .

the numbers of eosinophils and basophils were found to be increased in tissue , blood , and bone marrow and also correlated with disease severity [ 13 ] . additionally , several studies showed that eosinophil / basophil progenitor cells , which develop from bone marrow cd34 cells under the influence of specific chemokines and cytokines , are increased in the peripheral blood of atopic subjects with asthma [ 5 , 6 ] , allergic rhinitis [ 68 ] , nasal polyposis , or atopic skin manifestations which was also reviewed in denburg and keith and gauvreau and denburg . in children , cord blood eo / b progenitors differ in phenotype and function among infants at risk for atopy . cord blood progenitors were further seen to be predictive for frequency and characteristics of acute respiratory illness in infants during the first year of life . next to these quantitative relations to disease outcomes , denburg et al . demonstrated that maternal diet during pregnancy can interfere with the number and function of progenitor cells and subsequently with disease outcomes such as atopic dermatitis and wheeze at one year of age . in agreement , our own group contributed data showing that eo / b progenitor cells of one - year - old children with cradle cap were increased in association with environmental pollutants . so far , data showing a responsiveness of eo / b progenitors to lifestyle and environmental factors are limited to the infant hematopoietic system . within the lina study ( lifestyle and environmental factors and their influence on newborns allergy risk ) we already demonstrated that cord blood but not maternal th1/th2 cytokine levels depends on chemical exposure during pregnancy . this result may point to an increased vulnerability of infant compared to adult t cells to environmental exposure . the aim of the present study was to investigate whether allergy relevant eosinophil / basophil progenitor cells from infants also respond with more sensitivity to environmental pollutants compared to progenitor cells from their mothers living in the same environment . the mother - child study lina was designed to investigate how environmental factors in the pre- and postnatal period influence the immune system and whether they are determinants of increased allergy risk later in children 's life . for this study , 629 mother - child pairs ( including 7 twins ) six hundred six mother - child pairs participated in the one - year and 546 pairs in the two - year follow - up ( one scheduled visit / year around the child 's birthday ) . blood samples from 397 mothers and 340 children were obtained at children 's age of two ( mean age : 2 years and 26 days , min max : 1 year and 343 days2 years and 161 days ) as part of the scheduled visit . sufficient peripheral blood mononuclear cells ( pbmcs ) for methylcellulose cultures were available for a subset of 68 corresponding mother - child pairs ( 66 mothers , 68 children ; 2 sets of twins ) . all relevant n - numbers are shown in figure a.1 in supplementary data available online at http://dx.doi.org/10.1155/2016/5293932 . participation in the lina study was voluntary and written informed consent was obtained from all participants . the study was approved by the ethics committee of the university of leipzig ( file ref . information concerning general aspects of life and environmental conditions during pregnancy was collected by detailed questionnaires during the 36th week of pregnancy . further , information about respiratory outcomes of the child in the last 12 months as well as information about housing and environmental conditions ( e.g. , environmental tobacco smoke ( ets ) exposure , and usage of cleaning agents ) was obtained annually . all questionnaires were self - administered by the parents . for more detailed information , please see the methods section of supplementary data . pbmcs were isolated within six hours after collection of about 3 ml ( child ) to 5 ml ( mother ) fresh heparinised peripheral blood via ficoll paque density centrifugation . pbmcs were frozen in 1 ml aliquots of 90% fetal bovine serum and 10% dimethylsulfoxide with 1030 10 cells . the cell thawing protocol and isolation of nonadherent mononuclear cells ( namncs ) were performed according to reece et al . . viability of pbmcs after thawing and namncs after 2 h incubation averaged 93.4% . in the present paper a well - established and prevalidated method of functional methylcellulose assay was used to assess eo / b cfus [ 12 , 14 , 16 ] . to assess eosinophil / basophil progenitor cell differentiation by colony formation 5 10 namncs were incubated in duplicate in the presence of recombinant human cytokine il-3 ( 1 ng / ml ) , il-5 ( 1 ng / ml ) , or gm - csf ( 10 ng / ml ) ( r&d systems europe ltd . , abingdon , oxon , uk ) over 14 days . eosinophil / basophil colony forming units ( cfus ) were detected and enumerated by their characteristic morphology using a light inverted microscope . the investigator , who performed the progenitor assays and analyses , was blinded to the identity of subjects and other data collected . due to insufficient cell numbers after thawing , methylcellulose assays could not be performed for all three cytokines in certain samples . case numbers for il-3- , il-5- , and gm - csf - stimulated cultures resulted in a total of 67 , 66 , and 56 analysable samples for the children and in 63 , 36 , and 11 samples for the mothers , respectively . to measure the individual exposure to volatile organic compounds ( vocs ) in the homes , passive samplers ( 3 m monitors , type ovm 3500 ; 3 m gmbh , neuss , germany ) were placed in the child 's bedroom ( or alternatively in the room where the child preferentially spent most of their time ) around the first birthday of the child . concentrations of vocs were analysed as described earlier and adjusted for seasonal variations as described in schlink et al . . the chi squared test was performed to compare parameters of the analysed subcohort with the remaining lina cohort ( n : 546 68 = 478 ) . analyses related to eo / b cfus are calculated within the described subcohort ( n = 68 mother - child pairs , including two sets of twins ) . calculations for general associations independent of eo / b cfu analyses ( e.g. , ets exposure versus voc concentrations ) were performed for the entire lina cohort ( n = 546 ) . when statistical analyses required a division into subgroups ( e.g. , exposure to ets : yes versus no ) data were not presented for n - numbers < 5 . it was tested whether the available sample size for eo / b analyses has the power to detect expected effect of indoor pollutants ( in particular vocs ) . with a type one error rate ( alpha ) of 0.05 , a power goal of 0.9 , and an expected population correlation of 0.460 ( according to our earlier paper the eo / b cfus and the total sum of all vocs correlated with r = 0.460 ) , the required sample size for such analyses was 45 . since the majority of parameters were not normally distributed , analyses were performed using nonparametric tests in general . to address the relationship between the numbers of eo / b cfus and atopic outcomes or exposure to indoor ets or disinfectants , medians were compared using the mann - whitney u test . to verify these results , multiple logistic regression models were used to determine adjusted odds ratios ( or ) with a 95% confidence interval . the following confounders were used : month of birth ( for seasonal changes ) , gender of the child ( for potential differences between boys and girls ) , parental school education ( as a marker for the social status ) , and family history of atopy ( to consider the individual genetic background ) , as well as exposure to indoor ets during pregnancy , maternal cotinine level at the child 's first birthday , the sum of all measured vocs at the child 's first birthday ( both cotinine and vocs as a marker for ets exposure in the second year of life ) , dampness in the dwelling during second year of life ( as a marker for early airway allergen exposure ) , and children with infections of the airways in the second year of life ( to consider a potential nonallergic origin of wheezing ) . spearman 's rank correlation test was applied to analyse the association between maternal and infant number of eo / b cfus and between the number of smoked cigarettes and eo / b cfus as well as between voc concentrations and the number of eo / b cfus . adjustments due to multiple testing were not performed since our analyses were based on an a priori hypothesis . there were no differences in the distribution of considered parameters in the analysed subcohort ( n = 68 ) compared with the remaining lina cohort ( n = 478 ) . in general , a positive correlation was found between corresponding maternal and infant il-3- ( p < 0.001 , r = 0.447 , n = 65 ) as well as gm - csf- ( p = 0.028 , r = 0.686 , n = 10 ) stimulated eo / b cfus ( table 2 ) . all shown significant associations between eo / b cfus , environmental exposures , and clinical outcomes are summarized in figure 1 . during the second year of life 61.5% of the families in the analysed subcohort declared their usage of disinfectants in the household as once a week or more , once a month or more , or occasionally , with no differences when compared to the remaining lina cohort ( p = 0.608 , table 1(b ) ) . within the analysed subcohort , 10.4% of the participants were exposed to indoor ets ( almost ) daily , once a week or more , or occasionally , with no differences when compared to the remaining lina cohort ( p = 0.222 , table 1(b ) ) . ets exposed mothers had higher urine cotinine levels ( median : 42.15 g / g , iqr : 2.02308.30 ) compared to mothers without ets exposure ( median : 1.22 g / g , iqr : 0.433.92 ; p < 0.001 ) . further , a positive correlation between maternal urine cotinine levels and the number of smoked cigarettes per day in the dwellings was observed ( p < 0.001 , r = 0.171 ) . smoking at home was related to enhanced indoor concentrations of the aromatic vocs benzene ( p = 0.002 ) and m + p - xylene ( p = 0.048 ) . in addition the number of smoked cigarettes per day was correlated with indoor benzene concentrations ( p = 0.008 , r = 0.117 ) . two - year - old children exposed to ets at home had significant higher numbers of il-3- ( p = 0.010 ) and gm - csf - stimulated eo / b cfus ( p = 0.014 , table 3 and supplementary table a.1 ) compared to children without ets exposure . coincident with these findings , children 's il-3- and gm - csf - stimulated eo / b cfus correlated positively with the number of smoked cigarettes per day ( table 4 ) . in contrast , maternal il-3-stimulated eo / b cfus showed no association either with ets exposure at home or with the number of smoked cigarettes per day . with respect to vocs , positive correlations were found between children 's il-3- , il-5- , or gm - csf - stimulated eo / b cfus and the sum of all measured vocs as well as for the single smoking related vocs benzene , m + p - xylene , and o - xylene ( p < 0.05 , table 5 ) . in mothers , only one correlation was seen for il-3-stimulated eo / b cfus and benzene . the usage of disinfectants was found to be associated with increased numbers of gm - csf - stimulated eo / b cfus among infants ( p = 0.031 , table 3 ) , while maternal eo / b cfus did not vary significantly . within the analysed subcohort , 22.7% of the children were positive for wheezing ever , 19.0% for recurrent wheezing , and 10.3% for wheezing requiring medical treatment during the second year of life . furthermore a physician - diagnosed bronchitis was seen in 28.6% of the children and obstructive bronchitis in 9.4% . there were no differences in the distribution of considered respiratory outcomes in the analysed subcohort ( n = 68 ) compared to the remaining cohort ( n = 478 , p > 0.05 , table 1(c ) ) . children who suffered from wheezing requiring medical treatment during the second year of life had significantly more il-3- ( p = 0.015 ) and gm - csf - stimulated eo / b cfus ( p = 0.023 , figure 2 ) at the age of two . the association between il-3-stimulated eo / b cfus and wheezing with medical treatment remains stable after adjustment for possible confounding factors ( month of birth , gender of the child , parental school education , family history of atopy , and exposure to indoor ets during pregnancy as well as maternal cotinine level , the sum of all measured vocs , dampness , and infections of the airways in the second year of life ) . we considered airway infections as an additional factor since wheezing episodes can also occur together with airway ( and in particular virus ) infection . no significant association was found between eo / b cfus and the occurrence of wheezing symptoms ever ( il-3 : p = 0.926 ; il-5 : p = 0.379 ; gm - csf : p = 0.943 ) , recurrent wheezing ( il-3 : p = 0.574 ; il-5 : p = 0.415 ; gm - csf : p = 0.909 ) , bronchitis ( il-3 : p = 0.281 ; il-5 : p = 0.067 ; gm - csf : p = 0.095 ) , or obstructive bronchitis ( il-3 : p = 0.308 ; il-5 : p = 0.495 ; gm - csf : p = 0.663 ) . within earlier studies , we showed that there are increases in blood eosinophil / basophil progenitor cells in one - year - old children in association with exposure to environmental chemicals . in the current work we wanted to clarify whether this progenitor cell responsiveness is specific to the infant hematopoietic system or can also be seen in adults . therefore , we analysed mother - child pairs living under the same environmental conditions for their differentiation of eo / b progenitor cells . to our knowledge absolute eo / b colony numbers of mothers and their infants have not been compared before . it is well known that numbers of progenitor cells , except in bone marrow , are highest in cord blood and decrease in peripheral blood later in life . our data suggest that the absolute number of eosinophil / basophil progenitor cells in peripheral blood of two - year - old children is already comparable to adult levels , either when compared with their own mothers or with peripheral blood samples from former studies . according to our hypothesis we could demonstrate with the present data that infant 's progenitor cells seem to respond with more sensitivity to environmental pollutants ( ets , vocs , and disinfectants ) compared to maternal progenitor cells . this is in agreement with results shown earlier within the lina study : vocs emitted due to renovation activities were observed to influence the child 's but not the mother 's immune response . in cord blood but not in peripheral blood of the mothers increased il-4 and il-5 serum levels were seen in relation to chemical exposure due to renovation activities during pregnancy . the current study provides further evidence that under similar exposure scenarios the infant 's immune system is more susceptible to the influence of environmental exposure compared to the maternal immune system . we hypothesize that this increased sensitivity goes back to the still not fully mature infant 's immune system . compensation mechanisms which might lower / negate the adverse effects of environmental exposure in adults might not yet be fully developed in young children . in addition , our data support results showing that enhanced numbers of eo / b progenitor cells as a consequence of environmental pollutants may increase the risk for wheeze and skin manifestations in early infancy [ 13 , 14 ] . however , these earlier studies based their findings of lifestyle- or environment - dependent differentiation of eo / b progenitors on a selected high - risk study population [ 13 , 14 ] . in the present paper we provide strong evidence that the impact of environmental pollutants on stem cell differentiation is also seen in the general population . the fact that exposure to tobacco smoke / vocs seems to influence the development of respiratory diseases has been shown before . there are several epidemiological studies demonstrating that exposure to prenatal smoke or passive tobacco smoke early in life is associated with an increased risk of wheezing in early infancy [ 2226 ] . for example , passive household smoke exposure enhanced the risk for wheeze in children 2 years of age ( or = 1.35 ) . similarly , pattenden et al . demonstrated that smoking exposure within the first 2 years of life was associated with increased risk for wheeze ( or : 1.17 ) . there is furthermore evidence that exposure to tobacco smoke / vocs provokes an immunological imbalance . newborn children from smoking mothers were reported to have fewer cord blood regulatory t cell numbers and an enhanced susceptibility to microbial infections through alterations of toll - like receptor- ( tlr- ) signalling as well as a weak th1 stimulation capacity . as demonstrated in an earlier study by our group , exposure to vocs may also direct the child 's immune system towards a th2 phenotype [ 17 , 30 ] . a th2 milieu caused by external stimuli has been shown to induce trafficking of il-5-producing th2 lymphocytes to the bone marrow where they promote eosinophilopoiesis through il-5r signalling [ 11 , 31 ] . in addition , the th2 cytokines like il-4 and il-13 were described to regulate the transmigration of eosinophils from bone marrow to the tissues . in children with skin manifestations a correlation between il-4 blood levels and stimulated eo / b progenitor cells thus , considering the fact that environmental pollutants such as tobacco smoke or vocs induce a th2 response in the child , we hypothesize that this may favour eo / b progenitor cell differentiation , which could in turn contribute to an enhanced development of respiratory outcomes . finally , the present data demonstrate that eo / b progenitors of two - year - old children which correlated positively with wheezing in early childhood were progenitors stimulated by il-3 and gm - csf : cytokines which are known to influence early eosinophil / basophil lineage differentiation . fernandes et al . also showed that il-3- and gm - csf - stimulated eo / b cfus in cord blood are predictive for acute respiratory illnesses with fever or wheeze in the first year of life . this group and others discussed the hypothesis that immature progenitors are key determinants of atopic risk [ 10 , 12 ] . our data confirm this hypothesis by showing a correlation between severe wheeze ( requiring medical treatment ) and early - stage eo / b cfus in two - year - old infants . furthermore , within the lina study , we have also found a positive association between eo / b progenitor cells in cord blood and respiratory outcomes during the first two years . we could not include data on asthma in our paper , since asthma prevalence at this age is comparably low . however , wheezing may favour asthma development later in life : it was shown , for example , that wheeze present in high - risk infants may be transient or remain persistent through childhood . it was also suggested that 15% of infants who wheeze progress to chronic asthma , mostly those associated with family history of atopy . the strength of our study derives from the fact that the analyses of eo / b progenitor cells were performed in mother - child pairs which are well characterised regarding their immune parameters , atopic outcomes , and indoor air exposure to environmental chemicals . for example , individual ets exposure was assessed not only by questionnaire data , which are always dependent on honesty and compliance of the participants , but in addition by analysis of maternal urine cotinine levels and moreover the measurement of voc concentrations in the homes . all of these parameters highly support each other and represent an objective ets exposure scenario , which shows a consistent positive association with infant eo / b cfus . by coupling progenitor cell measurements with environmental exposures and disease outcomes we were able to address the question of possible mechanisms responsible for the environmentally triggered increase in allergic outcomes . a weakness of the lina study in general is the potential bias by high rates of participating atopic parents ( about 65% ) . we addressed this point by including family history of atopy as a confounding variable in the regression models . the high prevalence of atopic parents ( who are already aware of their atopic disease and probably avoid potential hazards more than others ) might also be one reason for the quite low prevalence of children exposed to ets during the second year of life ( 10% versus 18.7% shown earlier for germany ) . one other limitation of the lina study is that measurements of voc and cotinine concentrations are only available at the child 's first birthday due to missing home visits in the second year of life . however , we could demonstrate an almost consistent smoking behaviour of the study participants within the first and second year of life ( 85.7% ) . therefore , we assume that voc and cotinine levels measured at the child 's first birthday also represent the child 's exposure around the second birthday . this was confirmed by significant associations between ets exposure within the second year of life and voc or cotinine concentrations measured at the child 's first birthday . another limitation of the study is the restricted number of cases with stem cell analyses due to the very high experimental effort resulting in low numbers of children in certain outcomes . however , to our knowledge , the number of eo / b progenitor cell analyses included in the present paper ( in total almost 140 ) is higher than in any other earlier published study focused on health effects in relation to eo / b progenitor cell function . also some conditions of stimulated eo / b progenitor cells ( especially maternal il-5 and gm - csf cfu ) resulted in small numbers of cases due to low number of available pbmcs . therefore , the presented results have to be interpreted with caution and need further validation . in the present study we could confirm our earlier published data showing that infant eo / b progenitor cell differentiation is associated with indoor chemical exposure . therefore , at least in infants , an increase of these hematopoietic cells by environmental exposure could contribute to an enhanced risk of the development of respiratory outcomes . the association of indoor chemical exposure and the differentiation of eo / b progenitors appears to be mainly restricted to the infant 's hematopoietic system . this is consistent with earlier results from the lina study showing that cord blood but not maternal th1/th2 cytokine levels depends on chemical exposure during pregnancy . taken together , we can state that children 's immune and hematopoietic cells seem to be more sensitive to environmental exposure compared to maternal cells . these results further support the hypothesis of a highly vulnerable and exposure sensitive time window in the perinatal period with consequences for children 's disease risk . protection against harmful environmental exposure and lifestyle conditions is therefore of much higher relevance for young children compared to adults . however , data needs to be confirmed in a larger cohort to verify the results based on the present small sample size .

purpose . enhanced eosinophil / basophil ( eo / b ) progenitor cell levels are known to be associated with allergic inflammation and atopy risk . the aim of the present study was to investigate the influence of different indoor exposures on the recruitment and differentiation of eo / b progenitors in mother - child pairs . methods . in 68 mother - child pairs of the lina study peripheral blood mononuclear cells were used to assess eo / b colony forming units ( cfus ) . information about disease outcomes and indoor exposures was obtained from questionnaires . indoor concentrations of volatile organic compounds ( vocs ) were measured by passive sampling . results . infant 's eo / b cfus were positively associated with exposure to tobacco smoke , disinfectants , or vocs . in contrast , for maternal eo / b cfus , only a few associations were seen . higher numbers of infant eo / b cfus were observed in children with wheezing symptoms within the second year of life . conclusions . we demonstrate that infant 's hematopoietic cells seem to respond with more sensitivity to environmental exposure compared to maternal cells . at least in infants , an activation of these hematopoietic cells by environmental exposure could contribute to an enhanced risk for the development of respiratory outcomes .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

the numbers of eosinophils and basophils were found to be increased in tissue , blood , and bone marrow and also correlated with disease severity [ 13 ] . additionally , several studies showed that eosinophil / basophil progenitor cells , which develop from bone marrow cd34 cells under the influence of specific chemokines and cytokines , are increased in the peripheral blood of atopic subjects with asthma [ 5 , 6 ] , allergic rhinitis [ 68 ] , nasal polyposis , or atopic skin manifestations which was also reviewed in denburg and keith and gauvreau and denburg . in children , cord blood eo / b progenitors differ in phenotype and function among infants at risk for atopy . cord blood progenitors were further seen to be predictive for frequency and characteristics of acute respiratory illness in infants during the first year of life . next to these quantitative relations to disease outcomes , denburg et al . demonstrated that maternal diet during pregnancy can interfere with the number and function of progenitor cells and subsequently with disease outcomes such as atopic dermatitis and wheeze at one year of age . in agreement , our own group contributed data showing that eo / b progenitor cells of one - year - old children with cradle cap were increased in association with environmental pollutants . so far , data showing a responsiveness of eo / b progenitors to lifestyle and environmental factors are limited to the infant hematopoietic system . within the lina study ( lifestyle and environmental factors and their influence on newborns allergy risk ) we already demonstrated that cord blood but not maternal th1/th2 cytokine levels depends on chemical exposure during pregnancy . this result may point to an increased vulnerability of infant compared to adult t cells to environmental exposure . the aim of the present study was to investigate whether allergy relevant eosinophil / basophil progenitor cells from infants also respond with more sensitivity to environmental pollutants compared to progenitor cells from their mothers living in the same environment . the mother - child study lina was designed to investigate how environmental factors in the pre- and postnatal period influence the immune system and whether they are determinants of increased allergy risk later in children 's life . for this study , 629 mother - child pairs ( including 7 twins ) six hundred six mother - child pairs participated in the one - year and 546 pairs in the two - year follow - up ( one scheduled visit / year around the child 's birthday ) . sufficient peripheral blood mononuclear cells ( pbmcs ) for methylcellulose cultures were available for a subset of 68 corresponding mother - child pairs ( 66 mothers , 68 children ; 2 sets of twins ) . participation in the lina study was voluntary and written informed consent was obtained from all participants . the study was approved by the ethics committee of the university of leipzig ( file ref . information concerning general aspects of life and environmental conditions during pregnancy was collected by detailed questionnaires during the 36th week of pregnancy . further , information about respiratory outcomes of the child in the last 12 months as well as information about housing and environmental conditions ( e.g. , environmental tobacco smoke ( ets ) exposure , and usage of cleaning agents ) was obtained annually . pbmcs were isolated within six hours after collection of about 3 ml ( child ) to 5 ml ( mother ) fresh heparinised peripheral blood via ficoll paque density centrifugation . the cell thawing protocol and isolation of nonadherent mononuclear cells ( namncs ) were performed according to reece et al . in the present paper a well - established and prevalidated method of functional methylcellulose assay was used to assess eo / b cfus [ 12 , 14 , 16 ] . to assess eosinophil / basophil progenitor cell differentiation by colony formation 5 10 namncs were incubated in duplicate in the presence of recombinant human cytokine il-3 ( 1 ng / ml ) , il-5 ( 1 ng / ml ) , or gm - csf ( 10 ng / ml ) ( r&d systems europe ltd . eosinophil / basophil colony forming units ( cfus ) were detected and enumerated by their characteristic morphology using a light inverted microscope . to measure the individual exposure to volatile organic compounds ( vocs ) in the homes , passive samplers ( 3 m monitors , type ovm 3500 ; 3 m gmbh , neuss , germany ) were placed in the child 's bedroom ( or alternatively in the room where the child preferentially spent most of their time ) around the first birthday of the child . analyses related to eo / b cfus are calculated within the described subcohort ( n = 68 mother - child pairs , including two sets of twins ) . calculations for general associations independent of eo / b cfu analyses ( e.g. , ets exposure versus voc concentrations ) were performed for the entire lina cohort ( n = 546 ) . it was tested whether the available sample size for eo / b analyses has the power to detect expected effect of indoor pollutants ( in particular vocs ) . with a type one error rate ( alpha ) of 0.05 , a power goal of 0.9 , and an expected population correlation of 0.460 ( according to our earlier paper the eo / b cfus and the total sum of all vocs correlated with r = 0.460 ) , the required sample size for such analyses was 45 . to address the relationship between the numbers of eo / b cfus and atopic outcomes or exposure to indoor ets or disinfectants , medians were compared using the mann - whitney u test . to verify these results , multiple logistic regression models were used to determine adjusted odds ratios ( or ) with a 95% confidence interval . the following confounders were used : month of birth ( for seasonal changes ) , gender of the child ( for potential differences between boys and girls ) , parental school education ( as a marker for the social status ) , and family history of atopy ( to consider the individual genetic background ) , as well as exposure to indoor ets during pregnancy , maternal cotinine level at the child 's first birthday , the sum of all measured vocs at the child 's first birthday ( both cotinine and vocs as a marker for ets exposure in the second year of life ) , dampness in the dwelling during second year of life ( as a marker for early airway allergen exposure ) , and children with infections of the airways in the second year of life ( to consider a potential nonallergic origin of wheezing ) . spearman 's rank correlation test was applied to analyse the association between maternal and infant number of eo / b cfus and between the number of smoked cigarettes and eo / b cfus as well as between voc concentrations and the number of eo / b cfus . in general , a positive correlation was found between corresponding maternal and infant il-3- ( p < 0.001 , r = 0.447 , n = 65 ) as well as gm - csf- ( p = 0.028 , r = 0.686 , n = 10 ) stimulated eo / b cfus ( table 2 ) . all shown significant associations between eo / b cfus , environmental exposures , and clinical outcomes are summarized in figure 1 . during the second year of life 61.5% of the families in the analysed subcohort declared their usage of disinfectants in the household as once a week or more , once a month or more , or occasionally , with no differences when compared to the remaining lina cohort ( p = 0.608 , table 1(b ) ) . within the analysed subcohort , 10.4% of the participants were exposed to indoor ets ( almost ) daily , once a week or more , or occasionally , with no differences when compared to the remaining lina cohort ( p = 0.222 , table 1(b ) ) . smoking at home was related to enhanced indoor concentrations of the aromatic vocs benzene ( p = 0.002 ) and m + p - xylene ( p = 0.048 ) . two - year - old children exposed to ets at home had significant higher numbers of il-3- ( p = 0.010 ) and gm - csf - stimulated eo / b cfus ( p = 0.014 , table 3 and supplementary table a.1 ) compared to children without ets exposure . coincident with these findings , children 's il-3- and gm - csf - stimulated eo / b cfus correlated positively with the number of smoked cigarettes per day ( table 4 ) . in contrast , maternal il-3-stimulated eo / b cfus showed no association either with ets exposure at home or with the number of smoked cigarettes per day . with respect to vocs , positive correlations were found between children 's il-3- , il-5- , or gm - csf - stimulated eo / b cfus and the sum of all measured vocs as well as for the single smoking related vocs benzene , m + p - xylene , and o - xylene ( p < 0.05 , table 5 ) . in mothers , only one correlation was seen for il-3-stimulated eo / b cfus and benzene . the usage of disinfectants was found to be associated with increased numbers of gm - csf - stimulated eo / b cfus among infants ( p = 0.031 , table 3 ) , while maternal eo / b cfus did not vary significantly . within the analysed subcohort , 22.7% of the children were positive for wheezing ever , 19.0% for recurrent wheezing , and 10.3% for wheezing requiring medical treatment during the second year of life . furthermore a physician - diagnosed bronchitis was seen in 28.6% of the children and obstructive bronchitis in 9.4% . there were no differences in the distribution of considered respiratory outcomes in the analysed subcohort ( n = 68 ) compared to the remaining cohort ( n = 478 , p > 0.05 , table 1(c ) ) . children who suffered from wheezing requiring medical treatment during the second year of life had significantly more il-3- ( p = 0.015 ) and gm - csf - stimulated eo / b cfus ( p = 0.023 , figure 2 ) at the age of two . the association between il-3-stimulated eo / b cfus and wheezing with medical treatment remains stable after adjustment for possible confounding factors ( month of birth , gender of the child , parental school education , family history of atopy , and exposure to indoor ets during pregnancy as well as maternal cotinine level , the sum of all measured vocs , dampness , and infections of the airways in the second year of life ) . no significant association was found between eo / b cfus and the occurrence of wheezing symptoms ever ( il-3 : p = 0.926 ; il-5 : p = 0.379 ; gm - csf : p = 0.943 ) , recurrent wheezing ( il-3 : p = 0.574 ; il-5 : p = 0.415 ; gm - csf : p = 0.909 ) , bronchitis ( il-3 : p = 0.281 ; il-5 : p = 0.067 ; gm - csf : p = 0.095 ) , or obstructive bronchitis ( il-3 : p = 0.308 ; il-5 : p = 0.495 ; gm - csf : p = 0.663 ) . within earlier studies , we showed that there are increases in blood eosinophil / basophil progenitor cells in one - year - old children in association with exposure to environmental chemicals . in the current work we wanted to clarify whether this progenitor cell responsiveness is specific to the infant hematopoietic system or can also be seen in adults . therefore , we analysed mother - child pairs living under the same environmental conditions for their differentiation of eo / b progenitor cells . to our knowledge absolute eo / b colony numbers of mothers and their infants have not been compared before . it is well known that numbers of progenitor cells , except in bone marrow , are highest in cord blood and decrease in peripheral blood later in life . our data suggest that the absolute number of eosinophil / basophil progenitor cells in peripheral blood of two - year - old children is already comparable to adult levels , either when compared with their own mothers or with peripheral blood samples from former studies . according to our hypothesis we could demonstrate with the present data that infant 's progenitor cells seem to respond with more sensitivity to environmental pollutants ( ets , vocs , and disinfectants ) compared to maternal progenitor cells . this is in agreement with results shown earlier within the lina study : vocs emitted due to renovation activities were observed to influence the child 's but not the mother 's immune response . in cord blood but not in peripheral blood of the mothers increased il-4 and il-5 serum levels were seen in relation to chemical exposure due to renovation activities during pregnancy . the current study provides further evidence that under similar exposure scenarios the infant 's immune system is more susceptible to the influence of environmental exposure compared to the maternal immune system . in addition , our data support results showing that enhanced numbers of eo / b progenitor cells as a consequence of environmental pollutants may increase the risk for wheeze and skin manifestations in early infancy [ 13 , 14 ] . however , these earlier studies based their findings of lifestyle- or environment - dependent differentiation of eo / b progenitors on a selected high - risk study population [ 13 , 14 ] . in the present paper we provide strong evidence that the impact of environmental pollutants on stem cell differentiation is also seen in the general population . the fact that exposure to tobacco smoke / vocs seems to influence the development of respiratory diseases has been shown before . there are several epidemiological studies demonstrating that exposure to prenatal smoke or passive tobacco smoke early in life is associated with an increased risk of wheezing in early infancy [ 2226 ] . for example , passive household smoke exposure enhanced the risk for wheeze in children 2 years of age ( or = 1.35 ) . demonstrated that smoking exposure within the first 2 years of life was associated with increased risk for wheeze ( or : 1.17 ) . there is furthermore evidence that exposure to tobacco smoke / vocs provokes an immunological imbalance . newborn children from smoking mothers were reported to have fewer cord blood regulatory t cell numbers and an enhanced susceptibility to microbial infections through alterations of toll - like receptor- ( tlr- ) signalling as well as a weak th1 stimulation capacity . in children with skin manifestations a correlation between il-4 blood levels and stimulated eo / b progenitor cells thus , considering the fact that environmental pollutants such as tobacco smoke or vocs induce a th2 response in the child , we hypothesize that this may favour eo / b progenitor cell differentiation , which could in turn contribute to an enhanced development of respiratory outcomes . finally , the present data demonstrate that eo / b progenitors of two - year - old children which correlated positively with wheezing in early childhood were progenitors stimulated by il-3 and gm - csf : cytokines which are known to influence early eosinophil / basophil lineage differentiation . also showed that il-3- and gm - csf - stimulated eo / b cfus in cord blood are predictive for acute respiratory illnesses with fever or wheeze in the first year of life . our data confirm this hypothesis by showing a correlation between severe wheeze ( requiring medical treatment ) and early - stage eo / b cfus in two - year - old infants . furthermore , within the lina study , we have also found a positive association between eo / b progenitor cells in cord blood and respiratory outcomes during the first two years . the strength of our study derives from the fact that the analyses of eo / b progenitor cells were performed in mother - child pairs which are well characterised regarding their immune parameters , atopic outcomes , and indoor air exposure to environmental chemicals . all of these parameters highly support each other and represent an objective ets exposure scenario , which shows a consistent positive association with infant eo / b cfus . by coupling progenitor cell measurements with environmental exposures and disease outcomes we were able to address the question of possible mechanisms responsible for the environmentally triggered increase in allergic outcomes . a weakness of the lina study in general is the potential bias by high rates of participating atopic parents ( about 65% ) . the high prevalence of atopic parents ( who are already aware of their atopic disease and probably avoid potential hazards more than others ) might also be one reason for the quite low prevalence of children exposed to ets during the second year of life ( 10% versus 18.7% shown earlier for germany ) . one other limitation of the lina study is that measurements of voc and cotinine concentrations are only available at the child 's first birthday due to missing home visits in the second year of life . however , we could demonstrate an almost consistent smoking behaviour of the study participants within the first and second year of life ( 85.7% ) . this was confirmed by significant associations between ets exposure within the second year of life and voc or cotinine concentrations measured at the child 's first birthday . another limitation of the study is the restricted number of cases with stem cell analyses due to the very high experimental effort resulting in low numbers of children in certain outcomes . however , to our knowledge , the number of eo / b progenitor cell analyses included in the present paper ( in total almost 140 ) is higher than in any other earlier published study focused on health effects in relation to eo / b progenitor cell function . also some conditions of stimulated eo / b progenitor cells ( especially maternal il-5 and gm - csf cfu ) resulted in small numbers of cases due to low number of available pbmcs . therefore , the presented results have to be interpreted with caution and need further validation . in the present study we could confirm our earlier published data showing that infant eo / b progenitor cell differentiation is associated with indoor chemical exposure . therefore , at least in infants , an increase of these hematopoietic cells by environmental exposure could contribute to an enhanced risk of the development of respiratory outcomes . the association of indoor chemical exposure and the differentiation of eo / b progenitors appears to be mainly restricted to the infant 's hematopoietic system . this is consistent with earlier results from the lina study showing that cord blood but not maternal th1/th2 cytokine levels depends on chemical exposure during pregnancy . taken together , we can state that children 's immune and hematopoietic cells seem to be more sensitive to environmental exposure compared to maternal cells . protection against harmful environmental exposure and lifestyle conditions is therefore of much higher relevance for young children compared to adults . however , data needs to be confirmed in a larger cohort to verify the results based on the present small sample size .

cellular adaptation to o2 deprivation hypoxia is a physiological response during the course of normal development and aging in metazoans , and is critical for continued growth and survival of the organism . central to this adaptive response is hypoxia - inducible factor 1 ( hif-1 ) , a transcription factor that orchestrates global responses to hypoxia in gene expression.1 hif-1 is a heterodimer composed of hif-1 ( gene symbol hif1a ) and aryl hydrocarbon receptor nuclear translocator ( gene symbol arnt , and also known as hif-1).2 both subunits possess a basic helix - loop - helix domain capable of dimerization and binding to the promoters of hundreds of target genes through the recognition of hypoxia - responsive elements that contain the core sequence rcgtg.37 as the regulatory subunit of hif-1 , the o2-labile hif-1 stabilizes in response to hypoxia , leading to heterodimerization , dna binding , and recruitment of the transcription co - activators p300 and cbp.810 hif-1 uses a hydrophobic interface including cys800 to interact with p300 and cbp.1114 interestingly , in normoxia , hif-1 target genes are transcriptionally active but paused for rna polymerase ii elongation ; hypoxia - activated hif-1 employs cdk8-mediator and the super elongation complex to alleviate the pausing for elongation.15 numerous hif-1 target genes are activated by this canonical mechanism of transcription , such as those involved in angiogenesis , metabolic reprogramming , cell survival and proliferation , and migration and metastasis ( figure 1).1618 also contributing to these cancer biological processes is hif-2 ( gene symbol epas1),19,20 a paralog of hif-1 , which shares similar biochemical properties , regulatory mechanisms , and target genes , even though distinct , and even opposing , functions of hif-2 have begun to be appreciated.21 hif-1 is sensitive to o2 , owing to the presence of an o2-dependent degradation domain that mediates ubiquitin - proteasomal proteolysis.22 the von hippel - lindau ( vhl ) protein is a part of the e3 ubiquitin ligase,23,24 which recognizes two highly conserved proline residues ( pro402 and pro564 in hif-1 , pro405 and pro531 in hif-2 ) for polyubiquitination ; however , hydroxyl modification of these proline residues is a prerequisite for vhl recognition.2527 prolyl hydroxylation is catalyzed by three prolyl hydroxylase domain - containing ( phd ) proteins of the fe(ii)- and 2-oxoglutarate ( 2og)-dependent dioxygenase family : phd1 , phd2 , and phd3 ( gene symbols egln2 , egln1 , and egln3 , respectively ) , which use molecular o2 for sensing and signaling.2834 likewise , the factor inhibiting hif-1 ( fih1 , gene symbol hif1an)35 is another type of hydroxylase that modifies asn803 in the hif-1 transactivation domain to block p300 interaction , thereby providing an additional mechanism for modulating hif-1 activity.36,37 although fih1 is a dioxygenase that uses molecular o2 for substrate modification , its in vivo role for hif-1 regulation requires further investigation because mice with a hif1an - null mutation demonstrated no alterations of hif-1 function but exhibited a hypermetabolic state with hyperventilation and decreased body mass , which was regulated systemically by the nervous system.38 besides hydroxylation , numerous modulatory mechanisms have been identified , adding the complexity of hif regulation . these include oncogenes and tumor - suppressor genes , acetylation and sumoylation , reactive oxygen species and nitric oxide , and micrornas . these regulatory mechanisms have been extensively reviewed.39 in addition , both hif-1 and hif-2 employ crosstalk mechanisms to regulate cell proliferation , dna repair , mitochondrial biology , and cell stemness.40,41 these noncanonical mechanisms of hif-1 and hif-2 actions seem independent of arnt and direct dna binding ; rather , they require protein protein interactions ( figure 1 ) . hif-1 and hif-2 regulate cell - cycle genes in an opposing fashion ; hif-1 inhibits cell - cycle progression by suppressing c - myc activity , whereas hif-2 does the reverse.4244 by similar mechanisms , hif-1 represses , whereas hif-2 stimulates , dna repair genes.4547 hif-1 engages in the notch signaling pathway to block cell differentiation , and the -catenin signaling pathway to regulate embryonic and adult cell proliferation , albeit in an opposite way.4851 finally , hif-1 also inhibits dna replication through direct interaction with the adenosine triphosphatase cdc6.52 further studies are required to elucidate detailed mechanisms of noncanonical actions of hif-1 and hif-2. solid cancers frequently harbor hypoxic regions where hif-1 and hif-2 become overexpressed and activated.53 furthermore , oncogenic signaling and growth factor stimulation lead to the activation of the phosphotidylinositol 3-kinase ( pi3k)/akt / mammalian target of rapamycin ( mtor ) and mitogen - activated protein kinase ( mapk ) pathways , thereby stimulating hif-1 and hif-2 synthesis.16,17,5456 as a result , increased expression of both hif-1 and hif-2 has been observed in a wide variety of human cancers and , in general , is associated with poor prognosis.17,57 likewise , numerous studies support the notion that hif-1 and hif-2 promote tumor angiogenesis and growth.17,18,57 furthermore , rare somatic , gain - of - function mutations in epas1 have been identified to be associated with paraganglioma with polycythemia58 and pheochromocytoma.59 it has been recognized , however , that hif-1 expression is also associated with favorable prognosis , eg , in patients with neuroblastoma and renal cell carcinomas.21,60 forced expression of hif-1 in renal cell carcinoma cell lines retarded tumor growth in xenografts,61,62 suggesting a tumor - suppressing role for hif-1. conversely , it is suggested that hif-1 is not required for renal cyst development in conditional vhl - null mice.63 furthermore , rare somatic , loss - of - function hif1a mutations have been identified in the specimens of clear - cell renal carcinoma,64,65 and common focal , homozygous deletions have been detected in cell lines of vhl - deficient renal cell carcinomas.66 despite this cogent evidence , the mechanism by which hif-1 acts as a tumor suppressor remains unclear , and how cancer cells might escape from hif-1 suppression needs to be addressed , especially in majority of renal cell carcinomas where hif-1 is commonly overexpressed . what may explain retarded tumor growth in the xenograft studies , however , is that hif-1 inhibits cell proliferation.40 consistently , renal cell carcinomas with hif-1 expression are statistically much smaller than those without , and intriguingly seem more metastatic.47 therefore , the role of hif-1 in malignant progression and metastasis can not be ruled out in vhl - deficient renal cell carcinomas . hif-2 , on the other hand , is believed to be a major contributor to vhl - deficient renal carcinogenesis.33 however , in other experimental settings , hif-2 seems to be a tumor suppressor ; hif-2 increases apoptosis in glioma67 and inhibits oncogenic signaling and activates a tumor - suppressor gene in non - small - cell lung cancer.68 clearly , the role of hif-1 and hif-2 in cancer biology is complex , and their biological functions are likely context dependent . among the first recognized biological functions of hif-1 was metabolic adaptation to decreased o2 availability , resulting from transcriptional upregulation of multiple genes involved in glucose transport and glycolysis , such as those encoding aldolase a ( aldoa ) , phosphoglycerate kinase 1 ( pgk1 ) , lactate dehydrogenase a ( ldha ) , and glucose transporters ( slc2a1 and slc2a3).1 genetic deletion of hif1a gene in mouse embryonic cells confirmed the critical role for hif-1 in glycolysis and lactate production.69,70 under normal o2 tensions , glycolysis catabolizes glucose to pyruvate , which is converted to acetyl - coa by pyruvate dehydrogenase for oxidative phosphorylation in the tricarboxylic acid ( tca ) or krebs cycle . under hypoxia , cells decrease oxidative phosphorylation in the mitochondria and adopt anaerobic glycolysis and conversion of pyruvate to lactate . as a key regulator of this process , hif-1 actively suppresses mitochondrial o2 consumption by transcriptionally upregulating both pyruvate dehydrogenase kinases 1 and 3 ( pdk1 and pdk3 , respectively),7173 which subsequently inactivate pyruvate dehydrogenase to block pyruvate conversion and flux of acetyl - coa into the tca cycle . moreover , hif-1 has been shown to inhibit oxidative phosphorylation by inhibiting mitochondrial biogenesis.74 in addition to the critical role in the metabolic switch from oxidative phosphorylation to anaerobic fermentation the pasteur effect , hif also stimulates energy storage for hypoxic cell survival through the induction of glycogen and lipid synthesis.75 primarily , hif-1 promotes glycogen accumulation through transcriptional activation of several genes involved in glycogen biosynthesis , such as gys1 , ppp1r3c , and pgm1.7678 hif-1 also upregulates expression of the peroxisome proliferator - activated receptor ( gene symbol pparg ) , thereby increasing fatty acid uptake and triglyceride biosynthesis in cardiac hypertrophy.79 additional hif-1 target genes responsible for lipid accumulation include hilpda and lpin1.80,81 interestingly , conditional deletion of murine vhl in hepatocytes indicates a role for hif-2 , rather than hif-1 , in lipid synthesis , oxidation , and storage,82 another example of context dependence for hif function . consistent with the role of hif-1 in energy maintenance and conservation,83 it stands to reason that cancer cells adopt glycolysis under low o2 tensions for survival . however , cancer cells generally manifest characteristics of increased proliferation associated with high glucose uptake and lactate production even in the presence of o2 aerobic glycolysis or the warburg effect.8486 until recently , the significance of aerobic glycolysis remained debatable because it was unclear why cancer cells prefer energy - inefficient glycolysis to support proliferation , notwithstanding the recognition of the role of hif-1 in aerobic glycolysis.87 a reinterpretation of the warburg effect is that aerobic glycolysis is not merely employed for bioenergetics but , more importantly , for biosynthesis of macromolecules ( nucleotides , amino acids , and lipids ) necessary for cell proliferation.85,86,88 this view has integrated oncogenic signaling ( pi3k - akt - mtor and c - myc ) with regulation of metabolic pathways , biosynthesis , and cell proliferation . recent studies have pointed out the m2 splice isoform of pyruvate kinase ( pkm2 , a splice variant of the pkm gene ) as a metabolic switch for aerobic glycolysis and tumorigenesis.89,90 in normal adult tissues , the splice variant pkm1 catalyzes the final step of glycolysis by transferring the phosphate from phosphoenolpyruvate to adenosine diphosphate , thereby yielding pyruvate and adenosine triphosphate , whereas pkm2 is expressed primarily in lung tissues and proliferating cells including stem cells and cancer cells.91 the transcription factor c - myc promotes pkm2 splicing by inducing the expression of three heterogeneous nuclear ribonucleoproteins ( hnrnps).92 it has been shown that oncogenic signaling converges on the activation of mtor to increase hif-1 levels for pkm transcription and concomitantly c - myc levels for pkm2 splicing ( figure 2).90 in addition to being a glycolytic enzyme , pkm2 can function as a protein kinase for gene transcription in the nucleus.9395 activation of epithelial growth factor receptor induces extracellular signal - regulated kinase 2 ( gene symbol mapk1)-mediated phosphorylation and translocation of pkm2 into the nucleus . nuclear pkm2 interacts with -catenin to activate target genes such as ccnd1 and myc through phosphorylation of histone h3 , which leads to the dissociation of hdac3 and acetylation of histone h3 . accordingly , nuclear pkm2 promotes cell - cycle progression and cell proliferation by stimulating cyclin d1 and c - myc expression . c - myc further enhances pkm2 expression in a positive - feedback loop and also induces glycolytic gene expression.9395 pkm2 can also phosphorylate signal transducer and activator of transcription 3 ( gene symbol stat3 ) to drive gene expression.96 in addition , pkm2 is a transcriptional coactivator of hif-1 transcriptional activity.97 pkm2 can be hydroxylated by phd3 , and hydroxylated pkm2 enhances hif-1 activity through direct interaction . taken together , mice with a conditional allele that abolishes pkm2 but not pkm1 expression show accelerated mammary tumor formation and heterogeneous pkm1 expression.98 pkm1 was detected in non - proliferating cells but not in those in proliferation , which suggests that active pyruvate kinase is required for non - proliferating , but not necessarily for proliferating , tumor cells . interestingly , pkm2 found in tumor cells is usually dimeric with low enzymatic activity for glycolysis , whereas the tetrameric form in normal cells has a high activity.91 furthermore , oncogenic signaling inhibits the tetrameric form of pkm2 through direct and selective binding to tyrosine - phosphorylated peptides , thereby enabling anabolism from glucose metabolites for tumor growth.99 thus , hif-1-mediated pkm2 expression provides a regulated , bifunctional protein that can act on the one hand as a tetrameric glycolytic enzyme for bioenergetics and lactate production but on the other as a dimeric transcriptional coactivator / protein kinase for cell proliferation . most importantly , loss of glycolytic activity resulting from either a dimer or a tyrosine - phosphorylated peptides - bound tetramer diverts glycolytic metabolites into the anabolic pathways ( figure 2 ) . neither hif-1 nor hif-2 is known to transcriptionally regulate genes in the tca cycle ; rather , it is the intermediate metabolites including fumarate and succinate that have been shown to increase the activities of these transcription factors.100 whereas germline mutation in the fh gene ( encoding fumarate hydratase ) predisposes individuals to hereditary leiomyomatosis and renal cell carcinoma , hereditary mutations in the genes encoding four subunits of succinate dehydrogenase ( sdha , sdhb , sdhc , and sdhd ) and one cofactor ( sdhaf2 ) are linked to familial pheochromocytomas and paragangliomas.100102 these tumors are characterized by the induction of pseudo - hypoxia , ie , increased hif activities and target gene expression in normoxia . at the molecular level , inactivation of fumarate hydratase and succinate dehydrogenase leads to the accumulation of fumarate and succinate , respectively,103 resulting in allosteric inhibition of prolyl hydroxylase phds and induction of hif-1 and hif-2.104106 targeted inactivation of mouse fh1 confirmed the resultant activation of hif-1 and hif-2 and development of proliferative renal cysts.107 fh - deficient renal cancer cells also exhibit aerobic glycolysis and increased expression of hif-1.108 additionally , accumulated fumarate produces succinated glutathione , resulting in increased mitochondrial reactive oxygen species and hif-1 activation.109 in fact , it has been shown that fumarate can modify numerous proteins including mitochondrial aconitase 2 for the inhibition of aconitase activity in fh1-null mouse embryonic fibroblasts,110 even though reduced cytosolic , but not mitochondrial , aconitase activity is observed in fh - deficient renal cancer cells associated with iron deficiency.108 despite these findings , whether activation of hif pathway has a causal effect on renal cyst formation was questioned.111 indeed , in mouse genetic studies , fh1-associated renal cyst formation was independent of hif-1 and hif-2 , and was further exacerbated by inactivation of hif-1 but not hif-2.112 in fact , fh - deficient cysts and tumors were associated with upregulation of the keap1-nrf2 antioxidant pathway ( figure 3a ) , resulting from derepression of nuclear factor erythroid 2-like 2 ( nrf2 , gene symbol nfe2l2 ) upon fumarate inhibition of the negative regulator kelch - like ech - associated protein 1 ( gene symbol keap1 ) by succination . consistently , upregulation of antioxidant response genes through the keap1-nrf2 axis is a distinct feature of type 2 papillary renal cell carcinoma caused by fh mutation , in contrast to those of clear cell carcinomas arising from sdh or vhl mutation.113 furthermore , nrf2 apparently decreases hif-1 levels by reducing reactive oxygen species in fh - deficient cells.109 the nrf2-mediated antioxidant and detoxification program is involved in oncogene - induced tumorigenesis.114 intriguingly , nrf2 has also been shown to promote biosynthesis by redirecting glucose and glutamine into the anabolic pathways in the presence of activated pi3k - akt signaling.115 despite the lack of involvement of hif-1 in fh1-associated renal cyst development , upregulation of hif-1 remains essential to glycolytic metabolism and oncogenic growth of fh - deficient renal cancer,108 consistent with the aforementioned role of hif-1 in glycolysis and anabolism . although mutations in sdhd is linked to hereditary paraganglioma,116 heterozygous deletion of sdhd in mice develops no tumor but subtle glomus cell hypertrophy and hyperplasia.117 succinate accumulation has been shown to specifically inhibit phd3 apoptotic activity , thereby blocking the apoptosis of sympathetic neuronal precursor cells during development and contributing to the pathogenesis of familial pheochromocytoma ( figure 3a).118 interestingly , mice with germline deletion of egln3 also exhibited reduced apoptosis and increased cell numbers in the superior cervical ganglia , adrenal medulla , and carotid body.119 furthermore , a combined heterozygous deletion of epas1 rendered sympathetic neurons more sensitive to induced apoptosis , indicating an anti - apoptotic role of hif-2 in this setting . conversely , as mentioned above , rare somatic , gain - of - function mutations in epas1,58,59 as well as germline mutations in fh120 have been associated with malignant paragangliomas and pheochromocytomas . methylome analysis of a large cohort of paraganglioma patients has revealed a hypermethylator phenotype similar to that in gliomas.121 sdhb deletion in mouse chromaffin cells led to dna hypermethylation , resulting from succinate inhibition of the jmjc domain - containing histone demethylases and dna hydroxylases ( figure 3b ) . there has been rapid expansion of knowledge in recent years of single somatic mutations in cytosolic isocitrate dehydrogenase 1 ( gene symbol idh1 ) , and these mutations were identified initially in > 70% who grade ii iii gliomas and secondary glioblastomas.122 tumors lacking mutations in idh1 often had single mutations in idh2 , a mitochondrial gene in the tca cycle for interconversion of isocitrate and 2og . similar mutations have also been found in < 23% acute myeloid leukemia , albeit mainly in idh2.123125 interestingly , all the mutations are heterozygous and affect the same active site of the enzyme at a single arginine residue ( arg132 of idh1 and arg172 of idh2 ) , thereby reducing the intracellular concentration of 2og,122 a cosubstrate of the hif prolyl hydroxylases . accordingly , it was found that mutant idh1 forms a catalytically inactive heterodimer with the wild type , resulting in a decrease of phd activity and an increase of hif-1 levels in human gliomas harboring an idh1 mutation.126 this loss - of - function theory was only part of the story , however , because a wild type allele of idh1 or idh2 always remains in these tumors , raising the possibility of gain of function as result of mutations . indeed , both mutant idh1 and mutant idh2 acquire the ability to catalyze the nicotinamide adenine dinucleotide phosphate - dependent reduction of 2og to ( r)-enantiomer of 2-hydroxyglutarate ( ( r)-2hg).124,127,128 accordingly , elevated levels of ( r)-2hg are detectable in idh mutant gliomas and acute myeloid leukemias . so , is ( r)-2hg an oncometabolite stimulating hypoxic signaling for tumorigenesis ? on the contrary , it has been suggested that ( r)-2hg downregulates hif-1 and hif-2 levels by increasing phd1 and phd2 activity to promote transformation of immortalized human astrocytes ( figure 3a).129 this finding was supported by the evidence that ( r)-2hg can substitute 2og for phd enzymatic activity in a cell - free reaction , and this finding is correlated with diminished expression of hif-1 and hif-2 in idh1 mutant cells and reduced hif target gene expression in idh mutant gliomas . furthermore , decreased hif-1 expression is conducive to transformation by mutant idh1 . moreover , ( r)-2hg is sufficient to promote leukemogenesis by inducing cytokine independence and blocking hematopoietic differentiation , whereas the ( s)-enantiomer of 2-hydroxyglutarate , despite being a more potent inhibitor of 2og - dependent dioxygenases,129131 fails to do so owing to its inhibition of phd activity to increase , rather than decrease , hif-1 levels.132 the notion that ( r)-2hg inhibits hif signaling seems at odds with the genetic evidence of mouse studies ; increased hif-1 protein levels and target gene transcription were observed in the embryo of brain idh1 ( r132h ) knock - in mice where high levels of ( r)-2hg were produced,133 and no alteration of hif-1 signaling was found in the hematopoietic stem cells and progenitor cells from the knock - in mice of the myeloid lineage.134 although the mechanisms by which hif-1 prevents astrocyte transformation and leukemogenesis remain unclear , a lower level of hif-1 expression in idh1 mutant cells might be conducive to tumor initiation because hif-1 also upregulates histone demethylases,135,136 which have been shown to be inhibited by ( r)-2hg for transformation . in addition to phds , ( r)-2hg is a potent inhibitor of other 2og - dependent dioxygenases,137 including the tet family of 5-methylcytosine hydroxylases , which convert 5-methyl - cytosine to 5-hydroxymethylcytosine for dna demethylation.138140 in fact , idh1 somatic mutations in glioblastomas and lower - grade gliomas have a cpg island methylator phenotype , displaying dna hypermethylation at a large number of loci.141,142 similarly , acute myeloid leukemias with idh1 or idh2 mutations also exhibit genome - wide dna hypermethylation with a specific signature shared with those harboring mutations in tet2,125 a member of the tet protein family . consistently , expression of idh mutants was found to impair tet2 catalytic function , and mutations in idh1 and idh2 were mutually exclusive with those in tet2 in a large cohort of acute myeloid leukemias.125 although both forms of 2-hg have been shown to be competitive inhibitors of multiple 2og - dependent dioxygenases , including the jmjc domain - containing histone demethylases and the tet family of 5-methylcytosine hydroxylases as well as phds,130 the half - maximal inhibitory concentration of ( r)-2hg for histone demethylases is 200-fold less than for phd2 , indicating more important effects of idh mutations on chromatin remodeling.131 in keeping with this , idh mutation has been shown to inhibit histone demethylation and induce dna hypermethylation in cell culture and animal model , thereby blocking cell differentiation.134,143,144 furthermore , targeted inhibition of mutant idh2 with the small molecule agi-6780 induces differentiation of established erythroleukemia cells and primary human acute myeloid leukemia cells in vitro.145 the small - molecule inhibitor of mutant idh1 ( agi-5198 ) also promotes differentiation of glioma cells harboring idh1 mutation and inhibits the growth of tumor xenografts.146 interestingly , agi-5198 induces histone demethylation and expression of genes associated with glial differentiation without appreciable changes in genome - wide dna methylation , suggesting additional mechanisms of idh1 mutation for glioma growth . it took nearly two decades to appreciate that hif-1 stimulation of glycolysis is not merely to maintain bioenergetics for cell survival , but equally importantly is to promote biosynthesis of macromolecules for cell proliferation . pkm2 engages in various mechanisms as a dimer to drive cell proliferation147 and as a glycolytic enzyme in tetramer is further suppressed by oncogenic signaling . although how pkm2 oscillates between dimer and tetramer is not well understood , receptor tyrosine kinase - mediated activation of the pi3k - akt - mtor pathway seems essential in orchestrating cellular biosynthesis of nucleotides , amino acids , and lipids that involves multiple pathways including hif-1 and c - myc signaling , and glutamine - dependent anaplerosis for cell proliferation.148 yet , rapid cell proliferation induces hypoxia , which is known to suppress mtorc1 through multiple mechanisms.21 in fact , hif-1 is known to inhibit mtorc1 activity through transcriptional upregulation of ddit4 ( encoding redd1 ) , a negative regulator of mtorc1 activity,149,150 whereas hif-2 , unrelated to aerobic glycolysis , stimulates mtorc1 activity by transcriptionally upregulating slc7a5 ( encoding an amino acid transporter).151 one possible answer to this conundrum is to inactivate hif-1 , as mentioned above , albeit rarely , which implies the benefit of keeping hif-1 expression in cancer cells . alternatively , cancer cells may decrease expression of the stress - sensor protein atm ( ataxia telangiectasia mutated , gene symbol atm ) , which is required for transcriptional activation of redd1 through hif-1 phosphorylation,152 or they may attempt to maintain mtorc1 activity by relying on exogenous desaturated lipids from serum for survival.153 mechanism by entering cell - cycle arrest while promoting an angiogenic response to alleviate hypoxic stress for continued proliferation.154 by altering the intracellular or intercellular context , cancer cells can adopt aberrant ways of maintaining survival and proliferation . therefore , the selection of nrf2 signaling for detoxification and anabolism , rather than anti - proliferative hif-1 signaling , might be beneficial to renal cyst development at the early stage in fh - mutant cells . similarly , an initial decrease in hif-1 levels may facilitate dna hypermethylation in cells harboring fh , sdh , or idh mutations , because hif-1 does the opposite by inducing histone demethylases and dna demethylation , as also observed in metastasis of vhl - deficient renal cancer.155 it is also noteworthy that none of the fh , sdh , and idh mouse models has recapitulated a corresponding phenotype of the human diseases , suggesting the requirement of additional genetic / epigenetic alterations for tumorigenesis . further studies are warranted to elucidate the mechanisms underlying these changes and the logistics of utilizing various signaling pathways to cancer s best advantage . understanding of these dynamic processes is expected to provide more unexpected answers to the mystery of cancer .

tumor hypoxia has long been recognized as a driving force of malignant progression and therapeutic resistance . the discovery of hypoxia - inducible transcription factors ( hifs ) has greatly advanced our understanding of how cancer cells cope with hypoxic stress by maintaining bioenergetics through the stimulation of glycolysis . until recently , however , it remained perplexing why proliferative cancer cells opt for aerobic glycolysis , an energy - inefficient process of glucose metabolism . furthermore , the role of hif in cancer has also become complex . in this review , we highlight recent groundbreaking findings in cancer metabolism , put forward plausible explanations to the complex role of hif , and underscore remaining issues in cancer biology .

Introduction HIF-1, HIF-2, and cancer Critical role of HIF-1 in metabolic switch from oxidative phosphorylation to anaerobic glycolysis HIF-1 diverts glycolytic metabolites into biosynthesis by blunting pyruvate production Hereditary mutations in fumarate hydratase and succinate dehydrogenase Somatic mutations in isocitrate dehydrogenase Conclusion

central to this adaptive response is hypoxia - inducible factor 1 ( hif-1 ) , a transcription factor that orchestrates global responses to hypoxia in gene expression.1 hif-1 is a heterodimer composed of hif-1 ( gene symbol hif1a ) and aryl hydrocarbon receptor nuclear translocator ( gene symbol arnt , and also known as hif-1).2 both subunits possess a basic helix - loop - helix domain capable of dimerization and binding to the promoters of hundreds of target genes through the recognition of hypoxia - responsive elements that contain the core sequence rcgtg.37 as the regulatory subunit of hif-1 , the o2-labile hif-1 stabilizes in response to hypoxia , leading to heterodimerization , dna binding , and recruitment of the transcription co - activators p300 and cbp.810 hif-1 uses a hydrophobic interface including cys800 to interact with p300 and cbp.1114 interestingly , in normoxia , hif-1 target genes are transcriptionally active but paused for rna polymerase ii elongation ; hypoxia - activated hif-1 employs cdk8-mediator and the super elongation complex to alleviate the pausing for elongation.15 numerous hif-1 target genes are activated by this canonical mechanism of transcription , such as those involved in angiogenesis , metabolic reprogramming , cell survival and proliferation , and migration and metastasis ( figure 1).1618 also contributing to these cancer biological processes is hif-2 ( gene symbol epas1),19,20 a paralog of hif-1 , which shares similar biochemical properties , regulatory mechanisms , and target genes , even though distinct , and even opposing , functions of hif-2 have begun to be appreciated.21 hif-1 is sensitive to o2 , owing to the presence of an o2-dependent degradation domain that mediates ubiquitin - proteasomal proteolysis.22 the von hippel - lindau ( vhl ) protein is a part of the e3 ubiquitin ligase,23,24 which recognizes two highly conserved proline residues ( pro402 and pro564 in hif-1 , pro405 and pro531 in hif-2 ) for polyubiquitination ; however , hydroxyl modification of these proline residues is a prerequisite for vhl recognition.2527 prolyl hydroxylation is catalyzed by three prolyl hydroxylase domain - containing ( phd ) proteins of the fe(ii)- and 2-oxoglutarate ( 2og)-dependent dioxygenase family : phd1 , phd2 , and phd3 ( gene symbols egln2 , egln1 , and egln3 , respectively ) , which use molecular o2 for sensing and signaling.2834 likewise , the factor inhibiting hif-1 ( fih1 , gene symbol hif1an)35 is another type of hydroxylase that modifies asn803 in the hif-1 transactivation domain to block p300 interaction , thereby providing an additional mechanism for modulating hif-1 activity.36,37 although fih1 is a dioxygenase that uses molecular o2 for substrate modification , its in vivo role for hif-1 regulation requires further investigation because mice with a hif1an - null mutation demonstrated no alterations of hif-1 function but exhibited a hypermetabolic state with hyperventilation and decreased body mass , which was regulated systemically by the nervous system.38 besides hydroxylation , numerous modulatory mechanisms have been identified , adding the complexity of hif regulation . solid cancers frequently harbor hypoxic regions where hif-1 and hif-2 become overexpressed and activated.53 furthermore , oncogenic signaling and growth factor stimulation lead to the activation of the phosphotidylinositol 3-kinase ( pi3k)/akt / mammalian target of rapamycin ( mtor ) and mitogen - activated protein kinase ( mapk ) pathways , thereby stimulating hif-1 and hif-2 synthesis.16,17,5456 as a result , increased expression of both hif-1 and hif-2 has been observed in a wide variety of human cancers and , in general , is associated with poor prognosis.17,57 likewise , numerous studies support the notion that hif-1 and hif-2 promote tumor angiogenesis and growth.17,18,57 furthermore , rare somatic , gain - of - function mutations in epas1 have been identified to be associated with paraganglioma with polycythemia58 and pheochromocytoma.59 it has been recognized , however , that hif-1 expression is also associated with favorable prognosis , eg , in patients with neuroblastoma and renal cell carcinomas.21,60 forced expression of hif-1 in renal cell carcinoma cell lines retarded tumor growth in xenografts,61,62 suggesting a tumor - suppressing role for hif-1. conversely , it is suggested that hif-1 is not required for renal cyst development in conditional vhl - null mice.63 furthermore , rare somatic , loss - of - function hif1a mutations have been identified in the specimens of clear - cell renal carcinoma,64,65 and common focal , homozygous deletions have been detected in cell lines of vhl - deficient renal cell carcinomas.66 despite this cogent evidence , the mechanism by which hif-1 acts as a tumor suppressor remains unclear , and how cancer cells might escape from hif-1 suppression needs to be addressed , especially in majority of renal cell carcinomas where hif-1 is commonly overexpressed . what may explain retarded tumor growth in the xenograft studies , however , is that hif-1 inhibits cell proliferation.40 consistently , renal cell carcinomas with hif-1 expression are statistically much smaller than those without , and intriguingly seem more metastatic.47 therefore , the role of hif-1 in malignant progression and metastasis can not be ruled out in vhl - deficient renal cell carcinomas . hif-2 , on the other hand , is believed to be a major contributor to vhl - deficient renal carcinogenesis.33 however , in other experimental settings , hif-2 seems to be a tumor suppressor ; hif-2 increases apoptosis in glioma67 and inhibits oncogenic signaling and activates a tumor - suppressor gene in non - small - cell lung cancer.68 clearly , the role of hif-1 and hif-2 in cancer biology is complex , and their biological functions are likely context dependent . among the first recognized biological functions of hif-1 was metabolic adaptation to decreased o2 availability , resulting from transcriptional upregulation of multiple genes involved in glucose transport and glycolysis , such as those encoding aldolase a ( aldoa ) , phosphoglycerate kinase 1 ( pgk1 ) , lactate dehydrogenase a ( ldha ) , and glucose transporters ( slc2a1 and slc2a3).1 genetic deletion of hif1a gene in mouse embryonic cells confirmed the critical role for hif-1 in glycolysis and lactate production.69,70 under normal o2 tensions , glycolysis catabolizes glucose to pyruvate , which is converted to acetyl - coa by pyruvate dehydrogenase for oxidative phosphorylation in the tricarboxylic acid ( tca ) or krebs cycle . moreover , hif-1 has been shown to inhibit oxidative phosphorylation by inhibiting mitochondrial biogenesis.74 in addition to the critical role in the metabolic switch from oxidative phosphorylation to anaerobic fermentation the pasteur effect , hif also stimulates energy storage for hypoxic cell survival through the induction of glycogen and lipid synthesis.75 primarily , hif-1 promotes glycogen accumulation through transcriptional activation of several genes involved in glycogen biosynthesis , such as gys1 , ppp1r3c , and pgm1.7678 hif-1 also upregulates expression of the peroxisome proliferator - activated receptor ( gene symbol pparg ) , thereby increasing fatty acid uptake and triglyceride biosynthesis in cardiac hypertrophy.79 additional hif-1 target genes responsible for lipid accumulation include hilpda and lpin1.80,81 interestingly , conditional deletion of murine vhl in hepatocytes indicates a role for hif-2 , rather than hif-1 , in lipid synthesis , oxidation , and storage,82 another example of context dependence for hif function . consistent with the role of hif-1 in energy maintenance and conservation,83 it stands to reason that cancer cells adopt glycolysis under low o2 tensions for survival . however , cancer cells generally manifest characteristics of increased proliferation associated with high glucose uptake and lactate production even in the presence of o2 aerobic glycolysis or the warburg effect.8486 until recently , the significance of aerobic glycolysis remained debatable because it was unclear why cancer cells prefer energy - inefficient glycolysis to support proliferation , notwithstanding the recognition of the role of hif-1 in aerobic glycolysis.87 a reinterpretation of the warburg effect is that aerobic glycolysis is not merely employed for bioenergetics but , more importantly , for biosynthesis of macromolecules ( nucleotides , amino acids , and lipids ) necessary for cell proliferation.85,86,88 this view has integrated oncogenic signaling ( pi3k - akt - mtor and c - myc ) with regulation of metabolic pathways , biosynthesis , and cell proliferation . recent studies have pointed out the m2 splice isoform of pyruvate kinase ( pkm2 , a splice variant of the pkm gene ) as a metabolic switch for aerobic glycolysis and tumorigenesis.89,90 in normal adult tissues , the splice variant pkm1 catalyzes the final step of glycolysis by transferring the phosphate from phosphoenolpyruvate to adenosine diphosphate , thereby yielding pyruvate and adenosine triphosphate , whereas pkm2 is expressed primarily in lung tissues and proliferating cells including stem cells and cancer cells.91 the transcription factor c - myc promotes pkm2 splicing by inducing the expression of three heterogeneous nuclear ribonucleoproteins ( hnrnps).92 it has been shown that oncogenic signaling converges on the activation of mtor to increase hif-1 levels for pkm transcription and concomitantly c - myc levels for pkm2 splicing ( figure 2).90 in addition to being a glycolytic enzyme , pkm2 can function as a protein kinase for gene transcription in the nucleus.9395 activation of epithelial growth factor receptor induces extracellular signal - regulated kinase 2 ( gene symbol mapk1)-mediated phosphorylation and translocation of pkm2 into the nucleus . interestingly , pkm2 found in tumor cells is usually dimeric with low enzymatic activity for glycolysis , whereas the tetrameric form in normal cells has a high activity.91 furthermore , oncogenic signaling inhibits the tetrameric form of pkm2 through direct and selective binding to tyrosine - phosphorylated peptides , thereby enabling anabolism from glucose metabolites for tumor growth.99 thus , hif-1-mediated pkm2 expression provides a regulated , bifunctional protein that can act on the one hand as a tetrameric glycolytic enzyme for bioenergetics and lactate production but on the other as a dimeric transcriptional coactivator / protein kinase for cell proliferation . neither hif-1 nor hif-2 is known to transcriptionally regulate genes in the tca cycle ; rather , it is the intermediate metabolites including fumarate and succinate that have been shown to increase the activities of these transcription factors.100 whereas germline mutation in the fh gene ( encoding fumarate hydratase ) predisposes individuals to hereditary leiomyomatosis and renal cell carcinoma , hereditary mutations in the genes encoding four subunits of succinate dehydrogenase ( sdha , sdhb , sdhc , and sdhd ) and one cofactor ( sdhaf2 ) are linked to familial pheochromocytomas and paragangliomas.100102 these tumors are characterized by the induction of pseudo - hypoxia , ie , increased hif activities and target gene expression in normoxia . at the molecular level , inactivation of fumarate hydratase and succinate dehydrogenase leads to the accumulation of fumarate and succinate , respectively,103 resulting in allosteric inhibition of prolyl hydroxylase phds and induction of hif-1 and hif-2.104106 targeted inactivation of mouse fh1 confirmed the resultant activation of hif-1 and hif-2 and development of proliferative renal cysts.107 fh - deficient renal cancer cells also exhibit aerobic glycolysis and increased expression of hif-1.108 additionally , accumulated fumarate produces succinated glutathione , resulting in increased mitochondrial reactive oxygen species and hif-1 activation.109 in fact , it has been shown that fumarate can modify numerous proteins including mitochondrial aconitase 2 for the inhibition of aconitase activity in fh1-null mouse embryonic fibroblasts,110 even though reduced cytosolic , but not mitochondrial , aconitase activity is observed in fh - deficient renal cancer cells associated with iron deficiency.108 despite these findings , whether activation of hif pathway has a causal effect on renal cyst formation was questioned.111 indeed , in mouse genetic studies , fh1-associated renal cyst formation was independent of hif-1 and hif-2 , and was further exacerbated by inactivation of hif-1 but not hif-2.112 in fact , fh - deficient cysts and tumors were associated with upregulation of the keap1-nrf2 antioxidant pathway ( figure 3a ) , resulting from derepression of nuclear factor erythroid 2-like 2 ( nrf2 , gene symbol nfe2l2 ) upon fumarate inhibition of the negative regulator kelch - like ech - associated protein 1 ( gene symbol keap1 ) by succination . consistently , upregulation of antioxidant response genes through the keap1-nrf2 axis is a distinct feature of type 2 papillary renal cell carcinoma caused by fh mutation , in contrast to those of clear cell carcinomas arising from sdh or vhl mutation.113 furthermore , nrf2 apparently decreases hif-1 levels by reducing reactive oxygen species in fh - deficient cells.109 the nrf2-mediated antioxidant and detoxification program is involved in oncogene - induced tumorigenesis.114 intriguingly , nrf2 has also been shown to promote biosynthesis by redirecting glucose and glutamine into the anabolic pathways in the presence of activated pi3k - akt signaling.115 despite the lack of involvement of hif-1 in fh1-associated renal cyst development , upregulation of hif-1 remains essential to glycolytic metabolism and oncogenic growth of fh - deficient renal cancer,108 consistent with the aforementioned role of hif-1 in glycolysis and anabolism . although mutations in sdhd is linked to hereditary paraganglioma,116 heterozygous deletion of sdhd in mice develops no tumor but subtle glomus cell hypertrophy and hyperplasia.117 succinate accumulation has been shown to specifically inhibit phd3 apoptotic activity , thereby blocking the apoptosis of sympathetic neuronal precursor cells during development and contributing to the pathogenesis of familial pheochromocytoma ( figure 3a).118 interestingly , mice with germline deletion of egln3 also exhibited reduced apoptosis and increased cell numbers in the superior cervical ganglia , adrenal medulla , and carotid body.119 furthermore , a combined heterozygous deletion of epas1 rendered sympathetic neurons more sensitive to induced apoptosis , indicating an anti - apoptotic role of hif-2 in this setting . accordingly , it was found that mutant idh1 forms a catalytically inactive heterodimer with the wild type , resulting in a decrease of phd activity and an increase of hif-1 levels in human gliomas harboring an idh1 mutation.126 this loss - of - function theory was only part of the story , however , because a wild type allele of idh1 or idh2 always remains in these tumors , raising the possibility of gain of function as result of mutations . on the contrary , it has been suggested that ( r)-2hg downregulates hif-1 and hif-2 levels by increasing phd1 and phd2 activity to promote transformation of immortalized human astrocytes ( figure 3a).129 this finding was supported by the evidence that ( r)-2hg can substitute 2og for phd enzymatic activity in a cell - free reaction , and this finding is correlated with diminished expression of hif-1 and hif-2 in idh1 mutant cells and reduced hif target gene expression in idh mutant gliomas . it took nearly two decades to appreciate that hif-1 stimulation of glycolysis is not merely to maintain bioenergetics for cell survival , but equally importantly is to promote biosynthesis of macromolecules for cell proliferation . although how pkm2 oscillates between dimer and tetramer is not well understood , receptor tyrosine kinase - mediated activation of the pi3k - akt - mtor pathway seems essential in orchestrating cellular biosynthesis of nucleotides , amino acids , and lipids that involves multiple pathways including hif-1 and c - myc signaling , and glutamine - dependent anaplerosis for cell proliferation.148 yet , rapid cell proliferation induces hypoxia , which is known to suppress mtorc1 through multiple mechanisms.21 in fact , hif-1 is known to inhibit mtorc1 activity through transcriptional upregulation of ddit4 ( encoding redd1 ) , a negative regulator of mtorc1 activity,149,150 whereas hif-2 , unrelated to aerobic glycolysis , stimulates mtorc1 activity by transcriptionally upregulating slc7a5 ( encoding an amino acid transporter).151 one possible answer to this conundrum is to inactivate hif-1 , as mentioned above , albeit rarely , which implies the benefit of keeping hif-1 expression in cancer cells . alternatively , cancer cells may decrease expression of the stress - sensor protein atm ( ataxia telangiectasia mutated , gene symbol atm ) , which is required for transcriptional activation of redd1 through hif-1 phosphorylation,152 or they may attempt to maintain mtorc1 activity by relying on exogenous desaturated lipids from serum for survival.153 mechanism by entering cell - cycle arrest while promoting an angiogenic response to alleviate hypoxic stress for continued proliferation.154 by altering the intracellular or intercellular context , cancer cells can adopt aberrant ways of maintaining survival and proliferation . understanding of these dynamic processes is expected to provide more unexpected answers to the mystery of cancer .

current biomedical nanotechnology has been aimed at effectively imaging and characterizing abnormal biological processes at the cellular or even molecular level in living subjects . successful progresses in the fundamental molecular imaging research including instrumentations and image construction / registration techniques greatly motivate the innovations of imaging probes . as a relatively new molecular imaging technique , photoacoustic imaging ( pai ) pai takes advantage of individual strengths of both optical and acoustic imaging while largely overcoming the weaknesses associated with each modality , providing functional and molecular information of abnormalities with deep tissue penetration , high sensitivity , and excellent spatial resolution . in addition to several endogenous contrasts ( such as melanin and hemoglobin ) , various light - absorbing nanoparticles ( including au - based nanostructures , carbon nanotubes , and nanodroplets ) have been developed for pai contrast enhancement . although there are some nanoparticle - based exogenous agents particularly promising for pai , their potential toxicity is still under investigation , and the improvement of their in vivo behavior remains a challenge since most attempts to enhance the contrast effect compromise their pharmacokinetic profile . as one class of strong optical - absorption nanomaterials , colloidal metallic nanostructures ( nps ) have recently attracted significant attention from diverse disciplines for many biomedical applications . many synthetic methods have been developed for the construction of a variety of plasmonic nanostructures with controllable size and uniform shape ( for instance , triangles , prisms , rods , cubes , shells , stars , and cages ) , largely because the effect of induced - shape anisotropy of these nanostructures results in the splitting of the underlying surface plasmon enhancement ( spr ) into several shape - dependent modes , which could be accordingly tuned within the near - infrared window ( nir , 650950 nm ) . as representative anisotropic nanogeometries , au - nanorods and -nanocages have shown great promise as pai contrasts ; however , they generally have relatively large particle sizes ( 50 nm ) , which could result in unfavorable in vivo behavior and severely limit their application for targeted cancer imaging . by making a compromise by trading contrast effects for the ability to have preferable in vivo behaviors , strenuous efforts have been made recently to improve their targeting efficiency , and to control pharmacokinetics and biodistributions through variations of their sizes , shapes , and surface properties . although some metallic nanostructures with complex shapes showed desired tumor targeting efficiency without compromising optical properties , great challenges still remain in the precise control of their geometries and monodispersity . recently , many colloidal hybrid nanostructures with sophisticated architectures have been constructed from multiple functional components either assembled using linkers or fused together by solid - state interfaces . in order to build novel hybrid nanostructures with optimal structure and morphology for spr , we herein developed a synthetic strategy to construct a series of anisotropic gold - based nanomultipods , including dipods , tripods , and tetrapods ( figure 1 ) , with improved yield and excellent quality in a predictable , controlled , and stepwise manner . among them , the gold tripods ( au tripods ) have relatively small sizes with a narrow size distribution and display stringently controlled morphology and , more importantly , have well - defined absorptions in visible and nir regions . as a proof of concept , we further conjugated v3 integrins targeting the peptide , cyclic c(rgdfc ) peptide , to pegylated au - tripods ( rgd - au - tripods ) and used this novel nanoprobe as a pai contrast agent to image tumor angiogenesis . the tumor targeting efficacy and in vivo profile of pegylated au - tripods ( modified with different sizes of pegs and/or rgd ) labeled with radionuclide cu were evaluated in a subcutaneous v3-positive u87 mg glioblastoma xenograft model using small animal positron emission tomography ( pet ) . finally , pai was performed to investigate the targeting and imaging performance of rgd - au - tripods as photoacoustic contrast agents . pt dumbbell nps , au - dipods , au - tripods and au - tetrapods ) . ( a ) schematic showing the stepwise syntheses of various gold multipods via a set of known nucleation reactions and epitaxial growth processes . various gold multipods are modeled by lumerical fdtd solution ( lumerical solution inc . ) . considering the regioselectivity , several possible regioisomers are shown in the bottom - left panel . hrtem images of representative gold multipods are shown in the bottom - right panels . by sequentially applying a set of nucleation reactions and epitaxial growth processes , we successfully built up four gold - based nanostructures , comprising au pt dumbbell , dipods , tripods , and tetrapods , with predetermined composition and morphology ( figure 1a ) . in this structure - guided approach , we chose colloidal platinum ( pt ) nps as core seeds ( or starting materials ) because they are highly monodisperse and their sizes can be facilely tuned from 3 to 7 nm , and more importantly , they have definite and rigid cubic shapes ( supporting information [ si ] , figure s4 ) . in order to tune the sizes of pt nanocrystals , we applied stepwise seed - mediated growth processes to obtain monodisperse pt nanocubes ( si section c.1.1 . and 4.9 nm pt nps with truncated cubic shapes could preferably grew into thermodynamically more stable morphologies ( cubic 6.5 nm pt nps ) after an extra seed - mediated growth process . similarly , 7.5 nm pt nps were obtained when 5.8 nm pt nps were used as seeds . all pt nanocubes have a very narrow size and shape distribution ( figure s5 and table s2 ) . due to excellent lattice coherence between pt and au crystals in the fcc phase , the epitaxial growth of gold preferentially occurs at the vertices of cubic pt seeds ( si section c.1.2 ) , resulting in the formation of various au heterostructures ( figure 1 ) . typically , the epitaxial growth of au on 4.9 nm pt seeds produced dumbbell - like au pt nps . there are two geometrical isomers ; one is linear and the other is bent , corresponding to para and meta configurations , respectively . interestingly , we found that , unlike 7.5 nm pt nps as seeds resulting in a mixture of tripods and tetrapods , 6.5 nm pt seeds exclusively led to the formation of tripods with a narrow distribution of size and shape ( figure 1b ) . the statistical analysis suggested that a majority of resultant nps were au - tripods ( si figure s7s9 ) . to the best of our knowledge , it is the first time for constructing novel au tripods with high yield and improved quality by sequentially applying a set of known nucleation reactions and epitaxial growth processes . there are two geometrical isomers identified in the resultant tripods ; namely one with c3v symmetry is called as tripod - a and the other with c2v symmetry is tripod - t ( figure 1a ) . the steric accessibility determines the spatial distribution of au crystals on the pt nanocubes ( si scheme s2 and s3 ) . there are two meta positions favorable for the nucleation of incoming au crystals when an intermediate has a bent shape , thereby resulting in the formation of tripod - a ; there are six nucleation positions equal to the incoming au crystals in the case of a linear intermediate , producing the other type of tripods ( tripod - t ) . the statistical analysis results showed that an amount of tripod - t was finally obtained slightly higher than that of tripod - a ( 60% vs 40% in the resultant tripods , the sizes and shapes of various au - multipods and each component within nps were determined by dynamic light scattering ( dls ) and tem ( si table s3 and figure s9 ) . the au - tripods showed the lowest circularity among the branched nps , largely due to their anisotropic nanostructures and surface roughness . the representative tem and scanning transmission electron microscope ( stem ) images ( figure 2b e ) of au - tripods showed that well - crystallized interfaces were formed between the pt seeds and grown au branches . the lattice fringes between pt seeds and au branches shown in si figure s11 are 0.23 nm and related to ( 111 ) planes of either pt or au in the fcc phase [ 2.260 for ( 111 ) plane of pt , 2.355 for ( 111 ) plane of au ] . stem images clearly showed that each au branch epitaxially grew at one of the vertices of cubic pt seeds . two types of tripods , tripod - t and tripod - a , were definitively identified in the stem images ( figure 2d e ) . the forward and inverse fast fourier transforms ( fft ) were further applied to their hr - tem images to distinguish au branches and pt core . the right panel of figure 2 shows the hr - tem images of three typical tripods ( the first two belong to the type tripod - t ; the third one is the inset in diffractogram patterns shows the splits of the ( 222 ) and ( 331 ) peaks into two spots : one ( blue ) for pt and the other ( black ) for au crystal , because of slightly different planar distances of the pt and au crystals . inverse fft reconstructions of the pt ( bottom ) or au ( top ) nps using only their own reflections , , , , and , provided the real - spatial distributions of pt and au in tripods , respectively . therefore , the lattice images coupled with the forward and inverse fast fourier transform further confirmed the spatial configuration of au branches and pt core within au - tripods . hrtem and stem images of au - tripods , and their fourier transform and inverse fourier transform analyses . there are two types of tripods : tripod - a ( b ) and tripod - t ( c ) . ( f h ) hrtem images of typical tripod - t and its fast fourier transform ( fft ) and inverse fast fourier transform ( inverse fft ) analyses . the insets in ( g ) show the splits of the ( 222 ) and ( 331 ) peaks into two spots : one for pt and the other for au crystal . inverse fft reconstructions of the pt ( bottom ) or au ( top ) nps using only the superlattice reflections , , , , and , are shown in ( h ) . ( i k ) hrtem , fft , and inverse fft reconstruction of the other tripod - t with different orientation . inverse fft reconstruction of the pt ( bottom ) or au ( top ) nps using the superlattice reflections , , , and . ( l n ) hrtem , fft , and inverse fft reconstruction of tripod - a . the split of the ( 222 ) peaks is attributed to the difference between pt and au crystals and is shown in the inset of ( m ) . inverse fft reconstructions of the pt ( bottom ) or au ( top ) nps using only the superlattice reflections , , and are shown in ( n ) . a conventional uv vis nir spectrometer can measure the extinction spectra of nanostructures , which comprise two components : scattering and absorption , eventually providing the extinction cross section ( e , e = a + s , where a is absorption cross section and s is scattering cross section ) . as seen in figure 3a , the au - tripods have a much stronger extinction peak in the nir region , compared with the other au - multipods . there are two plasmon resonances at the 540 and 700 nm peaks in the region of 400 to 1000 nm . the 540 nm feature corresponds to a quadrupole resonance out of the plane of the gold tripods , and the 700 nm feature is attributed to a dipole resonance in the plane of the au - tripods . optical properties of au - tripods , and measurement and simulation of optical absorption cross sections of tripods . ( a ) uv vis extinction curves of various gold multipods ( including au pt dumbbell nps , au - dipods , au - tripods , and au - tetrapods ) at the same sample weight ( based on icp - ms ) . ( b ) uv vis extinction curves of gold nanospheres , cubic platinum nps , and gold nanorods ( 54 nm length and 18 nm diameter , more information in the si , sections c.2 and c.3 ) . ( c ) the calculated absorption cross section of tripod - t as a function of x ( the incident beam is polarized along the z - axis , and the tripod - t is rotated around the x - axis . x is the angle between the e - field and the long axis of tripod - t . ( d ) the calculated absorption cross section of tripod - a as a function of x ( the incident beam is polarized along the z - axis , and the tripod - a is rotated around the x - axis . x is the angle between the e - field and the side of tripod - a . polarization dependence of the average electric field intensity of tripod - t ( e ) and tripod - a ( f ) . electric field intensity contours in xz plane , xy plane , and yz plane at 700 nm were obtained from the fdtd calculations on both tripod - t and tripod - a . the long axis of tripod - t is parallel to the z - axis ; one side of tripod - a is parallel to the z - axis . the excitation polarization relative to the z - axis is 0. x and y represent the horizontal and vertical lengths of the calculated area . the a of au - tripods was calculated using combined uv vis nir spectrometer and photoacoustic measurements . while uv vis nir spectrometer measures the total extinction coefficient , in photoacoustic imaging , the detected signal is directly proportional to the absorption coefficient ( a ) of the nps . because the spr peak of the tripods is tuned to 700 nm , methylene blue is suitable as a reference dye to obtain the calibration curve . on the basis of the linear relationship between a and photoacoustic signal amplitude and the known a of methylene blue ( si figure s15 ) , the photoacoustic signals from au - tripods with different concentrations were converted into a , and the a of au - tripods was calculated by dividing the a by the corresponding concentration of the au - tripods . in the calculation , the a and e of the tripods at 670 nm were 2.02 0.03 10 and 2.06 0.03 10 m , respectively ( a/e = 0.98 ) . as a result , on a per - weight basis , the au - tripods are generally able to generate more contrast ( 33% ) on pa images within the nir region compared to gold rods ( si table s4 ) . to gain more insight into the localized surface plasmon resonance ( lspr ) spectra and to obtain better structural optimization , we performed numerical analysis on various gold multipods using a commercial finite difference time domain ( fdtd ) simulation package ( lumerical solution inc . , the au - multipods are modeled as a cubic pt core with au spheres ( see detailed modeling in the si sections b.3 and c.4 ) . the corresponding geometrical parameters were obtained from the previous results in si tables s2 and s3 . the contributions of absorption cross sections of two types of au - tripods ( tripod - t and tripod - a ) as a function of the rotated angle around the x - axis were obtained using the fdtd simulation and are shown in c and d of figure 3 . the calculated absorption cross section maxima of tripod - t were at 530 and 710 nm , in good agreement with experimental data ( a and c of figure 3 ) , while theoretical absorption maximum peaks of tripod - a occurred at 520 and 900 nm ( very broad peak centered at 900 nm ) . using the fdtd method , we have further studied the near - field optical properties of all gold multipods , gold rods , and nanospheres ( si section c.4 , figures s16s21 ) . the electric field intensity contours were calculated at the excitation wavelength of 700 nm as the excitation polarization was varied gradually from the longitudinal to transverse directions . similar to gold rods , the au - tripods have a strong polarization - dependent cross section . it was clear that under the resonance excitation , the maximum field enhancement regions were observed to rotate away from vertical tripod as the excitation polarization rotated around the y - axis ( si figure s18 ) . it is worth noting that the electric fields on the surface of au - tripods ( especially au - tripod - t ) are 23 orders of magnitude higher when compared to fields around spherical gold nanoparticles at the same weight in the nir range . as seen in the figure 3e , f and si figure s18 , the edges of tripods and the junctions between two au au nps on the pt cores are locations of enhanced fields ( also called hot spots ) , due to the occurrence of coupled plasmons . in order to stabilize the au - tripods in the aqueous solution and provide the capability for subsequent surface modification , we developed a facile , versatile pegylation strategy which can significantly increase in vivo circulation time of resultant nps and reduce their reticuloendothelial system ( res ) accumulation versus uncoated counterparts . thiolate bonds at the gold sulfur interface and the self - assembly of a monolayer on gold surface ( si scheme s1 ) . the bidentate thiol - terminated polyethylene glycol ( peg ) chains facilitated subsequent immobilization of various biological molecules via bioconjugation chemistry . the c(rgdfc ) was efficiently and site - specifically conjugated on the maleimide - terminated nps in an oriented and homogeneous fashion . in order to track the rgd - au - tripods in vivo by pet , the radioactive metal chelator , 2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid ( p - scn - bn - nota ) was further conjugated to the surface of rgd - au - tripods in a well - defined manner for cu radiolabeling . the hydrodynamic size change and the corresponding change in potentials of modified au - tripods clearly confirmed the efficient conjugation of the c(rgdfc ) peptide to the nanoparticles ( si figure s25 and table s5s8 ) . there were no significant size changes or aggregation in the presence of the mouse serum after 48 h at 37 c ( si figure s27 ) . we also validated the targeting ability of rgd - au - tripods to v3-positive u87 mg cells in vitro by determining their cellular uptake using tem and inductively coupled plasma mass spectrometry ( icp - ms ) analysis ( si figures s28 and s29 ) . in order to obtain a better understanding of the in vivo behavior of au - tripods and study their potential toxicity , we carried out a pilot preclinical animal toxicity study to assess the potential toxicity of tripods to pave the way for their clinical translation . both hematology and serum biochemistry analyses , and histologic and microscopic examination revealed that no evidence of significant acute toxicity was observed and the tripods are likely highly biocompatible in small living subjects ( si figures s35s37 ) . the pharmacokinetics , biodistribution , and tumor - targeting ability of au - tripods were investigated in small living animals . as described in the previous section , the water - soluble au - tripods were coated with a layer of functional peg chains . quantification of grafted peg density on pegylated au - tripods was performed using spectrophotometric analyses of free amines on the pegylated au - tripods ( si table s6 ) . typically , the density of amine groups on au - tripod ( peg 3400 coating ) was 0.966 0.07 number / nm ; the ratio of amine groups to a single tripod was 757 56 . the chelating agent nota was attached to the terminal of peg chains for pet radionuclide cu . importantly , the cu radiolabels on the tripods remained intact on the tripods even though they were incubated in the mouse serum over 24 h. the u87 mg tumor - bearing mice ( n = 4 ) were tail - vein injected with cu au - tripod , followed by small animal pet scans at different time points ( 1 , 2 , 4 , 24 , and 48 h ) . pet imaging results revealed nonspecific uptake of pegylated tripods by the liver , spleen , and even kidney ( figure 4a and si figures s30s32 ) , but minimum accumulation in the muscle or other major organs . although most of the tripods were eliminated through hepatic excretion , the kidney retention of radio - labeled tripods over time suggested that the renal excretion could be an additional clearance route for tripods in mice . since the heart acted as a cardiac blood pool , the signals from the heart were used as indicators to calculate the blood circulation time . the pegylated tripods showed slow systemic clearance because of a long blood circulation time ( t1/2 = 2.29 h ) , making themselves more available for distribution to target tissues via the enhanced permeability and retention ( epr ) effect . small animal pet images and pet quantification of intravenous injected tripods with different surface functionalization in mice bearing the u87 mg human glioblastroma tumor . ( a c ) targeting of integrin v3-postitive u87 mg tumor in mice by rgd - functionalized tripods . decay - corrected whole - body coronal pet images of nude mice bearing human u87 mg tumors at 1 , 4 , 24 , and 48 h after injection of 3.7 mbq of cu - rgd - au - tripod , cu - rgd - au - tripod with a blocking dose of c(rgdfc ) ( 21 mol of c(rgdfc)/kg of mouse body weight ) , and cu au - tripod ( 200 pmol / kg of mouse body weight , or 2 mg / kg of mouse body weight ) . ( d , f ) pet quantification of tumors and major organs after intravenous injection to mice bearing subcutaneous u87 mg glioma xenografts ( n = 4 per group , data represent means sd ) . ( g i ) , comparison of tumor and major organ uptake of cu - rgd - au - tripod , cu - rgd - au - tripod with a blocking dose of c(rgdfc ) , and cu au - tripod for a time period up to 48 h after intravenous injection to u87 mg tumor - bearing mice ( n = 4 per group ) . 0.01 , * p < 0.05 ( two - sided student s t - test ) . in order to target the integrin v3 for imaging tumor angiogenesis and metastasis , the au - tripods were conjugated with both p - scn - bn - nota for cu labeling and c(rgdfc ) for tumor targeting . the numbers of nota on the rgd - tripod were determined by an isotope dilution assay and typically equaled 54.5 3.3 . the conjugated rgd was actually determined by measuring the differences in concentration of rgd using hplc before and after addition of au - tripods during the coupling reaction . the ratio of rgd to nota on au - tripods was 8.2 0.5 . the targeting ability and specificity of rgd - au - tripods for the v3 integrin were evaluated in the u87 mg tumor - bearing mice ( n = 4 ) . the representative coronal and transverse pet images of a mouse acquired at 1 , 4 , 24 , and 48 h after tail vein injection of cu - rgd - au - tripods , cu au - tripods , or cu - rgd - au - tripods coinjected with a blocking dose of c(rgdfc ) are shown in figure 4 . quantitative analysis showed that tumor uptake of cu - rgd - au - tripods gradually accumulated in the tumor between 1 and 24 h , reaching a plateau at about 24 h post - injection , and then leveled off in the next 24 h. the cu - rgd - au - tripods exhibited a significantly high tumor uptake of 7.9% id / g after 24 h post - injection , which was more than 3 times higher than that of plain cu au - tripods ( 2.6% id / g ) . such high tumor accumulation was attributed to the tumor specific binding affinity of rgd functionalization on the tripods and their long blood circulation time . in the control group , the tumor uptake was significantly blocked when a blocking dose of c(rgdfc ) was injected into tumor - bearing mice along with cu - rgd - au - tripods . the reduced tumor uptake was observed ( 3.8% id / g after 24 h post - injection ) , which is significantly lower than that of unblocked one ( p < 0.05 ) . interestingly , compared to plain tripods , cu - rgd - au - tripods displayed much longer blood circulation time ( t1/2 = 10.3 h for cu - rgd - au - tripods vs t1/2 = 6.4 h for cu au - tripods ) and less res uptake . cu - rgd - au - tripods were also found to be excreted by both hepatobiliary and renal routes , as indicated by the deposition of radioactivity in the kidney and the gastrointestinal tract ( b and e of figure 4 ) , which were also confirmed by the tissue histology following in vivo imaging ( si figure s40 ) . to confirm the photoacoustic detection of tripods in tumor cells , we incubated u87 mg cells with rgd - au - tripods for 2 h under standard conditions . the cells with internalized rgd - au - tripods after incubation were washed and placed in agarose phantom at increasing cell concentrations from 11.5 10 to 368 10 cells per well ( n = 3 per group ) . quantitative analysis of the photoacoustic signals from the phantom revealed that the minimum detectable number of tumor cells exposed to rgd - au - tripods could be as low as 11.5 10 ( a and b of figure 5 , and figure s17 of si ) . we observed a linear correlation between the number of rgd - au - tripod - loaded cells and the corresponding photoacoustic signal . the photoacoustic signal from the phantom when excited at 700 nm provided the best sensitivity to detect tumor cells . even at 900 nm , ( a ) the top view of three - dimensional ( 3d ) volume rendering of photoacoustic images of an agarose phantom containing decreasing number of u87 mg cancer cells exposed to rgd - au - tripod at different wavelengths ( 670 , 700 , 725 , 750 , 800 , 850 , 900 nm ) . ( b ) quantitative analysis of the photoacoustic signal ( relative to the background signal ) from the phantom ( n = 3 ) . ( c ) rgd - au - tripod ranging in concentrations from 390 pm to 12.5 nm were injected subcutaneously into the flank of living mice ( n = 3 ) and scanned with photoacoustic instrument . ( d , e ) picomolar photoacoustic detection of tripods in living mice . the coronal view ( d ) of 3d volume rendering of photoacoustic images of subcutaneous inclusions . the skin is visualized in the ultrasound image ( gray - scale images ) , which is overlaid with photoacoustic images ( green - scale images ) . ( f , g ) three - dimensional volume rendering of photoacoustic images ( green ) and ultrasound images ( brown ) of subcutaneous inclusion . ( h ) photoacoustic signals recorded in vivo increased linearly with the tripod concentration ( r = 0.96 , n = 3 mice , data represent mean sd ) . ( i ) quantitative analysis of the photoacoustic signal ( relative to the background signal ) ( mice n = 3 ) . to test the tripod s pai sensitivity in living mice , we subcutaneously injected the right hind side of a mouse with 30 l of rgd - au - tripods mixed with matrigel ( n = 3 ) at increasing concentrations of 0.39 , 0.78 , 1.56 , 3.125 , 6.25 , and 12.5 nm . after the incision was solidified in place , the mouse was placed on its left side ( left lateral recumbent ) and partially embedded in the agarose gel covered with a water bath , and was then scanned under the photoacoustic system . while the ultrasound signals were used to reconstruct the mouse anatomy , including skin and inclusion edges , the photoacoustic signals showed the tripods contrast in the mice ( figure 5c h ) . quantitative analysis of the photoacoustic signals from each inclusion using a 3d region of interest drawn over the inclusion revealed a linear correlation between the tripod concentration and the corresponding photoacoustic signal . compared to the tissue background , about 200 pm of rgd - au - tripods extrapolated from the signal - concentration curve provided the equivalent photoacoustic signal as the tissue background . similar to pet imaging , the pai imaging ability of rgd - au - tripods to v3 integrin - positive tumor was evaluated in the u87 mg tumor - bearing mice ( n = 3 ) ( see figure 6 ) . before the injection , the photoacoustic and ultrasound images of the mice were taken . photoacoustic images with lateral step size of 0.25 mm were acquired at 700 nm wavelength . following the photoacoustic scan , an ultrasound image of the entire tumor area was acquired . the u87 mg tumor - bearing mice were then injected with 100200 l of rgd - au - tripods ( 200 pmol per kg mouse body weight ) in pbs through the tail vein . for the receptor - blocking experiment , mice were coinjected with 21 mol of c(rgdfc)/kg of mouse body weight and 100200 l of rgd - au - tripods ( 200 pmol per kg mouse body weight ) in pbs through the tail vein . after injection , photoacoustic images were acquired at 700 nm at 0.5 , 1 , 2 , and 4 h post - injection . quantitative analysis of the photoacoustic signal from the tumor was done by drawing three - dimensional ( 3d ) regions - of - interest ( rois ) around tumors on the basis of the ultrasound images . since there was the background blood signal in each photoacoustic scan , a subtraction image calculated at the 2-h post - injection image minus the preinjection image is shown in si figure s39 and was used to subtract out the background hemoglobin signal and examine the differences between groups . mice injected with rgd - au - tripods showed significantly higher photoacoustic signal in the tumor compared with the blocking group coinjected with rgd after 2 h post - injection . quantitative analysis showed that tumor uptake of rgd - au - tripods gradually accumulated in the tumor between 1 and 4 h , reaching a plateau at about 2 h post - injection , and then leveled off in the next 2 h. the mice injected with rgd - au - tripods at 2 h post - injection showed more than 3 times higher photoacoustic signal in the tumor than the mice coinjected with rgd and rgd - au - tripods . the tumor tissue histology combined with silver staining further confirmed the specific targeting ability of rgd - au - tripods toward u87 mg tumors ( si figure s40 ) . targeting of integrin v3-postitive u87 mg tumors in mice by rgd - au - tripod . ( a ) the coronal , sagittal , and transverse views of 3d volume rendering of photoacoustic images and ultrasound images of nude mice bearing u87 mg tumors were obtained before injection or at 1 , 4 , 24 , and 48 h after intravenous injection of rgd - au - tripod ( 200 pmol / kg of mouse body weight , or 2 mg / kg of mouse body weight ) . ( b ) the coronal , sagittal , and transverse views of 3d volume rendering of photoacoustic images and ultrasound images of nude mice bearing u87 mg tumors were obtained before injection or at 1 , 4 , 24 , and 48 h after coinjection of a blocking dose of c(rgdfc ) ( 21 mol of c(rgdfc)/kg of mouse body weight ) and rgd - au - tripod ( 200 pmol / kg of mouse body weight , or 2 mg / kg of mouse body weight ) . subtraction images were calculated at the 2-h post - injection image minus the preinjection image ( si figure s34 ) . ( c ) mice injected with rgd - au - tripod showed significantly higher photoacoustic signal than mice with coinjection of a blocking dose of c(rgdfc ) and the same amount of rgd - au - tripod ( p < 0.001 , two - sided student s t - test ) . ( d ) the perspective views of 3d volume rendering of photoacoustic images ( green ) and ultrasound images ( brown ) of tumors . we showed the ability to engineer and manipulate constitutional nanocrystals at the nanometer - scale to build novel nanotripods in a predictable and controlled manner . the construction of au - tripods involved a set of nucleation reactions and epitaxial growth processes , which are controlled by surface diffusion . as solid - state analogues of multifunctional organic molecules , pt nanocubes could be used as building blocks to construct sophisticated hybrid architectures . although sun and co - workers provided an important insight in the formation of various branched nps , the stochastic simulation in two dimensions inevitably overlooked certain configurations of constructed nanostructures . the selective growth of a heterogeneous nanocrystal phase onto certain regions of nanocrystal seeds depends on the surface potential and lattice matching . we found that regioselectivity of the heterogeneous nucleation on cubic pt seeds plays an important role on the formation of nanotripods ( si schemes s1 and s2 ) . considering the possible nucleation regions and steric effect , 6.5 nm cubic seeds exclusively resulted in the formation of uniform au - tripods with high yield and improved quality by sequentially applying a set of known nucleation reactions and epitaxial growth processes . the theoretical calculation coupled with experimental results on such anisotropic tripod nanostructures we successfully identified tripods with two possible configurations as high absorbance nanomaterials from various gold multipods using a numerical simulation analysis . the enhancement of the electric field on the surface of au - tripods is due to the occurrence of coupled plasmons at the edges of tripods and the junctions between gold nanocrystals on the pt cubes . optimizing the geometrical configurations of gold nanocrystals on the pt cube could improve the plasmon resonances of au - tripods . the measured absorption spectrum of au - tripods was identified by the calculated fdtd spectrum . due to an obvious increase in the cross section of au - tripods on a per - weight basis compared to that of the gold rods , au - tripods could generate more contrast on pa images within the nir region . among nanoparticle - based pa contrast agents , au - tripods exhibit superior optical properties in the nir region , and more importantly , they have exceptionally small sizes , which are distinctive advantages over traditional gold - based nps or carbon nanotubes for in vivo molecular imaging . we have successfully validated novel au - tripods as multimodality probes for in vivo molecular imaging ( pai and pet ) . the in vivo biodistribution of au - tripods favorable for living subject imaging was confirmed by cu radiolabeling and imaging their localization over time using pet . after intravenous administration , the au - tripods accumulated in liver and spleen , suggesting that the hepatic excretion is a major route of elimination of au - tripods . interestingly , the kidney retention of au - tripods over time revealed that the renal excretion could be an additional clearance route for au - tripods . considering the anisotropic shape of au - tripods [ the thickness of tripods is close to the renal clearance threshold ( less than 7 nm ) ] , it is possible for au - tripods to be cleared to some extent through the renal system . intravenous administration of rgd - au - tripods to u87 mg tumor - bearing mice showed remarkably higher contrast in tumors than competitive injection controls even at subnanomolar concentrations . quantitative biodistribution data revealed that 7.9% id / g of rgd - au - tripods accumulated in the u87 mg tumor after 24 h post - injection , but an obvious decrease in res uptake ( low liver and spleen accumulation , figure 4 ) of rgd - au - tripods was observed during the imaging . it is clear that their tumor uptake was higher than most of traditional gold - based nps for in vivo molecular imaging , probably due to their unique anisotropic shape and relatively small size . a pilot mouse toxicology study confirmed that no evidence of significant acute and systemic toxicity was observed in histopathological examination . by sequentially applying a set of nucleation reactions and epitaxial growth processes , we herein developed a control and stepwise strategy to build novel anisotropic au - tripods with predesigned shape , high yield , and excellent quality . these au - tripods exhibit superior optical and physical properties compared to their counterparts with regular architectures . we showed the ability to tune their shape - dependent plasmon resonances to the nir window without compromising their pharmacokinetic profile . our strategy opens up novel ways to enable the creation of sophisticated nanostructures with predetermined optical and physical properties for specific biomedical applications . moreover , our study suggests that highly selective and sensitive detection of cancer cells in a living subject is possible using molecular specific au - tripods as pai contrast agents . the functionalized rgd - tripods showed significantly enhanced photoacoustic contrast effect in both phantom and small animal imaging experiments . functional and molecular information of the tumor with high spatial resolution was further obtained by pai , which correlated well with the corresponding pet quantification . due to their excellent biocompatibility and stability in a biological environment , ease of functionalization , passive and activated targeting capabilities and potential hepatic and renal clearance , the au - tripods represent a new generation of a nanoplatform for biomedical research and personalized therapy . the integrin v3 targeting peptide cyclo ( arg - gly - asp - d - phe - cys ) ( crgdfc ) was purchased from peptides international , inc . ( louisville , ky ) . the p - scn - bn - nota was purchased from macrocyclics , inc . hydrogen tetrachloroaurate(iii ) hydrate ( haucl4 ) and platinum(ii ) acetylacetonate ( pt(acac)2 ) was ordered from strem chemicals , inc . n - hydroxysuccinimide ( nhs ) , n - hydroxysulfosuccinimide ( sulfo - nhs ) , succinimidyl 4-(n - maleimidomethyl ) cyclohexane-1-carboxylate ( sulfo - smcc ) , and 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide , hydrochloride ( edc ) were purchased from thermo fisher scientific . the dialysis membrane tubing ( mwco : 12,00014,000 , and 100,000 ) were purchased from spectrum laboratories . the size of various cubic pt nps was successfully tuned by adjustment of the reaction conditions in a precise manner . monodisperse pt seeds were synthesized by thermal decomposition of platinum precursor [ pt(acetylacetonate)2 , pt(acac)2 ] , in 1-octadecene solution containing a trace amount of iron pentacarbonyl [ fe(co)5 ] , which facilitates a fast nucleation and improves the homogeneous growth of platinum crystals . the shape and size of pt nps are dependent on the reaction temperature at which a trace amount of fe(co)5 was injected . multiple stepwise seed - mediated growth processes were applied to obtain monodisperse pt nanocubes . the gold precursor ( hydrogen tetrachloroaurate , haucl4 , 100 mg , 0.29 mmol ) was dissolved in 20 ml of 1-octadecene ( ode ) containing 2 ml of oleylamine under nitrogen protection . after stirring at room temperature for 5 min , twenty milligrams of 6.5 nm freshly synthesized pt nps ( dispersed in 1 ml of hexane ) was quickly injected into the above solution . the resultant mixture was then heated up to 110 c and kept at this temperature for one hour before it was cooled down to room temperature . the solution finally turned to gray - purplish color , indicating the formation of gold branched nanostructures . the particles were precipitated out by adding 30 ml of isopropanol and collected by a centrifuge ( 3000 rcf , 5 min ) . the resultant particles were redispersed in 5 ml of hexane and then precipitated out by adding ethanol . this purification step was repeated twice to remove the extra surfactant and ode . the final product ( au - tripods ) was dispersed in 10 ml of hexane in the presence of 0.01 ml of oleylamine for further use . surface pegylation of au - tripods is described in detail in the si sections a.4 , b.2 , c.5 , and c.6 . briefly , the au - tripods ( 10 mg ) were suspended in 3 ml of a chloroform solution of bidentate thiol - termined peg chains ( o-(3-aminopropyl)polyethylene glycol lipoate amide , lp - peg-3400-nh2 , 0.02 mmol , si section a.3 ) . after stirring at room temperature for 24 h , pegylated au - tripods were precipitated by adding 20 ml of hexane , collected by a brief centrifugation , and dried under the nitrogen gas flow . pegylated au - tripods were then dispersed in water , and the unbound peg and any other excess reagents were removed by dialysis against water or phosphate - buffered saline ( pbs ) ( 10 mm , ph 7.4 ) by dialysis tubing ( spectrum spectra / por dialysis membrane tubing , mwco = 12 kda ) . any impurity or precipitate was removed by a 0.22 m syringe filter . the final gold or platinum concentration of pegylated au - tripods was measured by inductively coupled plasma mass spectrometry ( icp - ms ) analysis . those nps were further characterized with transmission electron microscopy ( tem ) , uv vis absorption spectroscopy , and dynamic light scattering ( dls ) ( si sections b.2 , b.3 , b.5 , and sections c.4 , c.6 ) . conjugation of au - tripods with crgd ( rgd - au - tripods ) is described in detail in the si sections a.5 , c.6 , and c.7 . typically , the cross - linker solution , the water - soluble au - tripods ( 100 nm , 0.5 ml , 5 10 mol , see the determination of concentrations of tripods and rods in si section c.3 ) in 10 mm pbs ( ph = 7.2 ) were incubated with the cross - linker solution ( sulfo - smcc [ 0.5 mg , 1.5 mol ] , was freshly prepared in 15 l of dmso ) for 2 h at room temperature . after removal of excessive sulfo - smcc and byproducts using a pd-10 column ( ge healthcare , piscataway , nj ) , the resultant thiol - active au - tripods were concentrated to the final volume of 0.5 ml with a centrifugal - filter ( amicon centrifugal filter device , mwco = 30 kda ) and were incubated with the crgdfc stock solution ( 50 l of 5 mm in the degassed water , 0.25 mol , the final rgd concentration in the mixture was 0.5 mm ) with stirring . after the uncoupled rgd and byproducts were removed through pd-10 column , the resultant product , rgd - au - tripods , was concentrated by a centrifugal - filter ( amicon centrifugal filter device , mwco = 30 kda ) and stored at 4 c for one month without losing targeting activity . the final rgd - au - tripods was reconstituted in pbs and filtered through a 0.22 m filter for cell and animal experiments . the gold and platinum concentrations of rgd - au - tripods were measured by icp - ms analysis . the process to conjugate the tripods with both rgd and nota was similar to the conjugation of tripods and rgd , except that sulfo - smcc solution was mixed with nota aqueous solution in the first step before being added into the tripod solution . briefly , the sulfo - smcc ( 0.5 mg , 1.5 mol ) was dissolved in 15 l of dmso and mixed with 4.5 l of p - scn - bn - nota solution in the water ( 33 mm , 0.15 mol ) . the ratio of smcc to nota was optimized according to the specific activity of radioactive tracer - labeled nps ( si section c.7 ) . pet imaging was carried out on a micropet r4 rodent model scanner ( manufactured by cti concorde microsystems , knoxville , tn ) as previously reported . pet scans were performed at 1 , 2 , 4 , 24 , and 48 h post - injection ( p.i . ) . u87 mg tumor - bearing mice were divided into several injection groups ( 4 mice per each group ) to evaluate differences in specific targeting and biodistribution of au - tripods and rgd modified tripods ( si section a.5 and c.7 ) . for the tripod groups , each mice was injected with 3.7 mbq of cu - rgd - au - tripod ( 200 pmol / kg of mouse body weight , or 2 mg / kg of mouse body weight , normally 5 pmol per each mouse ) in 100200 l pbs via the tail vein . for the receptor - blocking experiment , u87 mg tumor - bearing mice were coinjected with 12 mg of c(rgdfc)/kg of mouse body weight and 100200 l of cu - rgd - au tripod ( 200 pmol per kg mouse body weight ) in pbs via the tail vein . prior to imaging , mice were anesthetized with isoflurane ( 5% for induction and 2% for maintenance in house oxygen at 2 l / min ) . mice ( 4 mice per group ) were placed in the prone position and near the center of the field of view ( fov ) of the scanner . the 5-min static scans were obtained at the predetermined time points after post - injection . all the pet images were then reconstructed by a two - dimensional ordered - subset expectation maximization ( osem ) algorithm with a spatial resolution of 1.661.85 mm . the custom - built photoacoustic instrument was described previously and is shown in si figure s2 . the photoacoustic characterizations of au - tripods were further determined by nexus 128 ( endra , mi ) . two groups of female nude mice ( n = 3 in each group , 68 weeks old ) were inoculated subcutaneously at their right hind side with 5 10 u87 mg cells in 50 l of pbs . the mouse was placed on its left side ( left lateral recumbent ) and partially embedded in the agarose gel covered with a water bath ( si figure s2 ) , and was then scanned under the photoacoustic system . before the injection , the photoacoustic and ultrasound images of the mice were taken . a photoacoustic image with lateral step size of 0.25 mm was acquired using the 5 mhz transducer at 670 , 700 , and 725 nm wavelength . following the photoacoustic scan , the u87 mg tumor - bearing mouse was then injected with 100200 l of rgd - au - tripods ( 200 pmol per kg mouse body weight ) in pbs through the tail vein using a butterfly catheter to avoid any position change during the injection ( 50 l of dead volume ) ( si figure s2b ) . for the receptor - blocking experiment , mice were coinjected with 21 mol of c(rgdfc)/kg of mouse body weight and 100200 l of rgd - au - tripods ( 200 pmol per kg mouse body weight ) in pbs through the tail vein . after injection , photoacoustic images ( 20 mm 20 mm ) were acquired at 670 , 700 , and 725 nm with step size of 0.25 mm and at 0.5 , 1 , 2 , and 4 h post - injection . following the photoacoustic scan , the photoacoustic and ultrasound images were analyzed , coregistered , and displayed using amide software . quantitative analysis of the photoacoustic signal from the tumor was done by drawing three - dimensional rois around the tumor on the basis of the ultrasound images . after 4 h post - injection , the mice were sacrificed , and tumors and tissues of interest were collected for tem ( si section b.1 ) , elemental analysis ( si section b.5 ) , and histology study ( si section b.6 ) .

anisotropic colloidal hybrid nanoparticles exhibit superior optical and physical properties compared to their counterparts with regular architectures . we herein developed a controlled , stepwise strategy to build novel , anisotropic , branched , gold nanoarchitectures ( au - tripods ) with predetermined composition and morphology for bioimaging . the resultant au - tripods with size less than 20 nm showed great promise as contrast agents for in vivo photoacoustic imaging ( pai ) . we further identified au - tripods with two possible configurations as high - absorbance nanomaterials from various gold multipods using a numerical simulation analysis . the pai signals were linearly correlated with their concentrations after subcutaneous injection . the in vivo biodistribution of au - tripods favorable for molecular imaging was confirmed using small animal positron emission tomography ( pet ) . intravenous administration of cyclic arg - gly - asp - d - phe - cys ( rgdfc ) peptide conjugated au - tripods ( rgd - au - tripods ) to u87 mg tumor - bearing mice showed pai contrasts in tumors almost 3-fold higher than for the blocking group . pai results correlated well with the corresponding pet images . quantitative biodistribution data revealed that 7.9% id / g of rgd - au - tripods had accumulated in the u87 mg tumor after 24 h post - injection . a pilot mouse toxicology study confirmed that no evidence of significant acute or systemic toxicity was observed in histopathological examination . our study suggests that au - tripods can be reliably synthesized through stringently controlled chemical synthesis and could serve as a new generation of platform with high selectivity and sensitivity for multimodality molecular imaging .

Introduction Results Discussion Conclusion Methods and Materials

as a relatively new molecular imaging technique , photoacoustic imaging ( pai ) pai takes advantage of individual strengths of both optical and acoustic imaging while largely overcoming the weaknesses associated with each modality , providing functional and molecular information of abnormalities with deep tissue penetration , high sensitivity , and excellent spatial resolution . as representative anisotropic nanogeometries , au - nanorods and -nanocages have shown great promise as pai contrasts ; however , they generally have relatively large particle sizes ( 50 nm ) , which could result in unfavorable in vivo behavior and severely limit their application for targeted cancer imaging . in order to build novel hybrid nanostructures with optimal structure and morphology for spr , we herein developed a synthetic strategy to construct a series of anisotropic gold - based nanomultipods , including dipods , tripods , and tetrapods ( figure 1 ) , with improved yield and excellent quality in a predictable , controlled , and stepwise manner . among them , the gold tripods ( au tripods ) have relatively small sizes with a narrow size distribution and display stringently controlled morphology and , more importantly , have well - defined absorptions in visible and nir regions . as a proof of concept , we further conjugated v3 integrins targeting the peptide , cyclic c(rgdfc ) peptide , to pegylated au - tripods ( rgd - au - tripods ) and used this novel nanoprobe as a pai contrast agent to image tumor angiogenesis . the tumor targeting efficacy and in vivo profile of pegylated au - tripods ( modified with different sizes of pegs and/or rgd ) labeled with radionuclide cu were evaluated in a subcutaneous v3-positive u87 mg glioblastoma xenograft model using small animal positron emission tomography ( pet ) . finally , pai was performed to investigate the targeting and imaging performance of rgd - au - tripods as photoacoustic contrast agents . there are two meta positions favorable for the nucleation of incoming au crystals when an intermediate has a bent shape , thereby resulting in the formation of tripod - a ; there are six nucleation positions equal to the incoming au crystals in the case of a linear intermediate , producing the other type of tripods ( tripod - t ) . on the basis of the linear relationship between a and photoacoustic signal amplitude and the known a of methylene blue ( si figure s15 ) , the photoacoustic signals from au - tripods with different concentrations were converted into a , and the a of au - tripods was calculated by dividing the a by the corresponding concentration of the au - tripods . the contributions of absorption cross sections of two types of au - tripods ( tripod - t and tripod - a ) as a function of the rotated angle around the x - axis were obtained using the fdtd simulation and are shown in c and d of figure 3 . it is worth noting that the electric fields on the surface of au - tripods ( especially au - tripod - t ) are 23 orders of magnitude higher when compared to fields around spherical gold nanoparticles at the same weight in the nir range . in order to stabilize the au - tripods in the aqueous solution and provide the capability for subsequent surface modification , we developed a facile , versatile pegylation strategy which can significantly increase in vivo circulation time of resultant nps and reduce their reticuloendothelial system ( res ) accumulation versus uncoated counterparts . in order to track the rgd - au - tripods in vivo by pet , the radioactive metal chelator , 2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid ( p - scn - bn - nota ) was further conjugated to the surface of rgd - au - tripods in a well - defined manner for cu radiolabeling . we also validated the targeting ability of rgd - au - tripods to v3-positive u87 mg cells in vitro by determining their cellular uptake using tem and inductively coupled plasma mass spectrometry ( icp - ms ) analysis ( si figures s28 and s29 ) . in order to obtain a better understanding of the in vivo behavior of au - tripods and study their potential toxicity , we carried out a pilot preclinical animal toxicity study to assess the potential toxicity of tripods to pave the way for their clinical translation . both hematology and serum biochemistry analyses , and histologic and microscopic examination revealed that no evidence of significant acute toxicity was observed and the tripods are likely highly biocompatible in small living subjects ( si figures s35s37 ) . importantly , the cu radiolabels on the tripods remained intact on the tripods even though they were incubated in the mouse serum over 24 h. the u87 mg tumor - bearing mice ( n = 4 ) were tail - vein injected with cu au - tripod , followed by small animal pet scans at different time points ( 1 , 2 , 4 , 24 , and 48 h ) . small animal pet images and pet quantification of intravenous injected tripods with different surface functionalization in mice bearing the u87 mg human glioblastroma tumor . decay - corrected whole - body coronal pet images of nude mice bearing human u87 mg tumors at 1 , 4 , 24 , and 48 h after injection of 3.7 mbq of cu - rgd - au - tripod , cu - rgd - au - tripod with a blocking dose of c(rgdfc ) ( 21 mol of c(rgdfc)/kg of mouse body weight ) , and cu au - tripod ( 200 pmol / kg of mouse body weight , or 2 mg / kg of mouse body weight ) . ( g i ) , comparison of tumor and major organ uptake of cu - rgd - au - tripod , cu - rgd - au - tripod with a blocking dose of c(rgdfc ) , and cu au - tripod for a time period up to 48 h after intravenous injection to u87 mg tumor - bearing mice ( n = 4 per group ) . the targeting ability and specificity of rgd - au - tripods for the v3 integrin were evaluated in the u87 mg tumor - bearing mice ( n = 4 ) . the representative coronal and transverse pet images of a mouse acquired at 1 , 4 , 24 , and 48 h after tail vein injection of cu - rgd - au - tripods , cu au - tripods , or cu - rgd - au - tripods coinjected with a blocking dose of c(rgdfc ) are shown in figure 4 . quantitative analysis showed that tumor uptake of cu - rgd - au - tripods gradually accumulated in the tumor between 1 and 24 h , reaching a plateau at about 24 h post - injection , and then leveled off in the next 24 h. the cu - rgd - au - tripods exhibited a significantly high tumor uptake of 7.9% id / g after 24 h post - injection , which was more than 3 times higher than that of plain cu au - tripods ( 2.6% id / g ) . in the control group , the tumor uptake was significantly blocked when a blocking dose of c(rgdfc ) was injected into tumor - bearing mice along with cu - rgd - au - tripods . the reduced tumor uptake was observed ( 3.8% id / g after 24 h post - injection ) , which is significantly lower than that of unblocked one ( p < 0.05 ) . interestingly , compared to plain tripods , cu - rgd - au - tripods displayed much longer blood circulation time ( t1/2 = 10.3 h for cu - rgd - au - tripods vs t1/2 = 6.4 h for cu au - tripods ) and less res uptake . cu - rgd - au - tripods were also found to be excreted by both hepatobiliary and renal routes , as indicated by the deposition of radioactivity in the kidney and the gastrointestinal tract ( b and e of figure 4 ) , which were also confirmed by the tissue histology following in vivo imaging ( si figure s40 ) . to confirm the photoacoustic detection of tripods in tumor cells , we incubated u87 mg cells with rgd - au - tripods for 2 h under standard conditions . quantitative analysis of the photoacoustic signals from the phantom revealed that the minimum detectable number of tumor cells exposed to rgd - au - tripods could be as low as 11.5 10 ( a and b of figure 5 , and figure s17 of si ) . we observed a linear correlation between the number of rgd - au - tripod - loaded cells and the corresponding photoacoustic signal . to test the tripod s pai sensitivity in living mice , we subcutaneously injected the right hind side of a mouse with 30 l of rgd - au - tripods mixed with matrigel ( n = 3 ) at increasing concentrations of 0.39 , 0.78 , 1.56 , 3.125 , 6.25 , and 12.5 nm . compared to the tissue background , about 200 pm of rgd - au - tripods extrapolated from the signal - concentration curve provided the equivalent photoacoustic signal as the tissue background . similar to pet imaging , the pai imaging ability of rgd - au - tripods to v3 integrin - positive tumor was evaluated in the u87 mg tumor - bearing mice ( n = 3 ) ( see figure 6 ) . the u87 mg tumor - bearing mice were then injected with 100200 l of rgd - au - tripods ( 200 pmol per kg mouse body weight ) in pbs through the tail vein . for the receptor - blocking experiment , mice were coinjected with 21 mol of c(rgdfc)/kg of mouse body weight and 100200 l of rgd - au - tripods ( 200 pmol per kg mouse body weight ) in pbs through the tail vein . mice injected with rgd - au - tripods showed significantly higher photoacoustic signal in the tumor compared with the blocking group coinjected with rgd after 2 h post - injection . quantitative analysis showed that tumor uptake of rgd - au - tripods gradually accumulated in the tumor between 1 and 4 h , reaching a plateau at about 2 h post - injection , and then leveled off in the next 2 h. the mice injected with rgd - au - tripods at 2 h post - injection showed more than 3 times higher photoacoustic signal in the tumor than the mice coinjected with rgd and rgd - au - tripods . the tumor tissue histology combined with silver staining further confirmed the specific targeting ability of rgd - au - tripods toward u87 mg tumors ( si figure s40 ) . ( a ) the coronal , sagittal , and transverse views of 3d volume rendering of photoacoustic images and ultrasound images of nude mice bearing u87 mg tumors were obtained before injection or at 1 , 4 , 24 , and 48 h after intravenous injection of rgd - au - tripod ( 200 pmol / kg of mouse body weight , or 2 mg / kg of mouse body weight ) . considering the possible nucleation regions and steric effect , 6.5 nm cubic seeds exclusively resulted in the formation of uniform au - tripods with high yield and improved quality by sequentially applying a set of known nucleation reactions and epitaxial growth processes . the theoretical calculation coupled with experimental results on such anisotropic tripod nanostructures we successfully identified tripods with two possible configurations as high absorbance nanomaterials from various gold multipods using a numerical simulation analysis . due to an obvious increase in the cross section of au - tripods on a per - weight basis compared to that of the gold rods , au - tripods could generate more contrast on pa images within the nir region . among nanoparticle - based pa contrast agents , au - tripods exhibit superior optical properties in the nir region , and more importantly , they have exceptionally small sizes , which are distinctive advantages over traditional gold - based nps or carbon nanotubes for in vivo molecular imaging . we have successfully validated novel au - tripods as multimodality probes for in vivo molecular imaging ( pai and pet ) . the in vivo biodistribution of au - tripods favorable for living subject imaging was confirmed by cu radiolabeling and imaging their localization over time using pet . after intravenous administration , the au - tripods accumulated in liver and spleen , suggesting that the hepatic excretion is a major route of elimination of au - tripods . intravenous administration of rgd - au - tripods to u87 mg tumor - bearing mice showed remarkably higher contrast in tumors than competitive injection controls even at subnanomolar concentrations . quantitative biodistribution data revealed that 7.9% id / g of rgd - au - tripods accumulated in the u87 mg tumor after 24 h post - injection , but an obvious decrease in res uptake ( low liver and spleen accumulation , figure 4 ) of rgd - au - tripods was observed during the imaging . it is clear that their tumor uptake was higher than most of traditional gold - based nps for in vivo molecular imaging , probably due to their unique anisotropic shape and relatively small size . a pilot mouse toxicology study confirmed that no evidence of significant acute and systemic toxicity was observed in histopathological examination . by sequentially applying a set of nucleation reactions and epitaxial growth processes , we herein developed a control and stepwise strategy to build novel anisotropic au - tripods with predesigned shape , high yield , and excellent quality . these au - tripods exhibit superior optical and physical properties compared to their counterparts with regular architectures . moreover , our study suggests that highly selective and sensitive detection of cancer cells in a living subject is possible using molecular specific au - tripods as pai contrast agents . functional and molecular information of the tumor with high spatial resolution was further obtained by pai , which correlated well with the corresponding pet quantification . due to their excellent biocompatibility and stability in a biological environment , ease of functionalization , passive and activated targeting capabilities and potential hepatic and renal clearance , the au - tripods represent a new generation of a nanoplatform for biomedical research and personalized therapy . the integrin v3 targeting peptide cyclo ( arg - gly - asp - d - phe - cys ) ( crgdfc ) was purchased from peptides international , inc . the final product ( au - tripods ) was dispersed in 10 ml of hexane in the presence of 0.01 ml of oleylamine for further use . conjugation of au - tripods with crgd ( rgd - au - tripods ) is described in detail in the si sections a.5 , c.6 , and c.7 . typically , the cross - linker solution , the water - soluble au - tripods ( 100 nm , 0.5 ml , 5 10 mol , see the determination of concentrations of tripods and rods in si section c.3 ) in 10 mm pbs ( ph = 7.2 ) were incubated with the cross - linker solution ( sulfo - smcc [ 0.5 mg , 1.5 mol ] , was freshly prepared in 15 l of dmso ) for 2 h at room temperature . after removal of excessive sulfo - smcc and byproducts using a pd-10 column ( ge healthcare , piscataway , nj ) , the resultant thiol - active au - tripods were concentrated to the final volume of 0.5 ml with a centrifugal - filter ( amicon centrifugal filter device , mwco = 30 kda ) and were incubated with the crgdfc stock solution ( 50 l of 5 mm in the degassed water , 0.25 mol , the final rgd concentration in the mixture was 0.5 mm ) with stirring . after the uncoupled rgd and byproducts were removed through pd-10 column , the resultant product , rgd - au - tripods , was concentrated by a centrifugal - filter ( amicon centrifugal filter device , mwco = 30 kda ) and stored at 4 c for one month without losing targeting activity . the gold and platinum concentrations of rgd - au - tripods were measured by icp - ms analysis . u87 mg tumor - bearing mice were divided into several injection groups ( 4 mice per each group ) to evaluate differences in specific targeting and biodistribution of au - tripods and rgd modified tripods ( si section a.5 and c.7 ) . for the receptor - blocking experiment , u87 mg tumor - bearing mice were coinjected with 12 mg of c(rgdfc)/kg of mouse body weight and 100200 l of cu - rgd - au tripod ( 200 pmol per kg mouse body weight ) in pbs via the tail vein . following the photoacoustic scan , the u87 mg tumor - bearing mouse was then injected with 100200 l of rgd - au - tripods ( 200 pmol per kg mouse body weight ) in pbs through the tail vein using a butterfly catheter to avoid any position change during the injection ( 50 l of dead volume ) ( si figure s2b ) . for the receptor - blocking experiment , mice were coinjected with 21 mol of c(rgdfc)/kg of mouse body weight and 100200 l of rgd - au - tripods ( 200 pmol per kg mouse body weight ) in pbs through the tail vein .

the online version of this article ( doi:10.1007/s00267 - 011 - 9751-z ) contains supplementary material , which is available to authorized users . habitat fragmentation by transport networks and consequential secondary development has become one of the most serious global threats to biological diversity ( eea 2002 ; iuell and others 2003 ; laurance and others 2009 ; bentez - lpez and others 2010 ) . with more than 100 million km of roads worldwide ( cia 2008 ) , the road network plays a main role in shaping the environment . road effects have been widely studied and include biodiversity decline , environmental degradation , alteration of ecological processes and ecosystem services , and increases in both extinction and outbreak probabilities ( forman and alexander 1998 ; trombulak and frissell 2000 ; forman and others 2003 ; fahrig and rytwinski 2009 ) . by acting as a barrier , roads isolate populations , reduce the overall landscape connectivity and restrict ( or even block off ) gene flow for a wide variety of taxa ( keller and others 2004 ; epps and others 2005 ; riley and others 2006 ; balkenhol and waits 2009 ) . in a long term , this loss of genetic diversity and connectivity increases the extinction risk of populations and , reduces their ability to adapt to future global changes . in general , these effects are synergistic , extremely complex , cumulative and time - lagged ( forman and alexander 1998 ; findlay and bourdages 2000 ; mcgarigal and others 2001 ; forman and others 2003 ) . the spatial influence of these effects ( road - effect zone ) ranges from a few meters to kilometres ( e.g. , forman and deblinger 2000 ) . when taking into account this road area - of - influence , about one fifth of the land in usa and the netherlands is ecologically affected by the road system ( reijnen and others 1995 ; forman 2000 ) . estimates indicate that only 18% of the usa lands are more than 1 km away from the closest road ( riitters and wickham 2003 ) . not only major roads , but also minor and unpaved roads may have a considerable impact on the environment ( e.g. , van langevelde and others 2009 ) natural areas are being increasingly fragmented by a rapidly expanding transport network , together with urban sprawl ( eea 2006 , 2010 ) . in the case of usa forests , estimations yield about 11% of all forest located within 85 m of a road ( riitters and wickham 2003 ) . the secondary impacts of roads account for an even more serious threat to biodiversity and ecosystem resilience . land use and transport networks are interdependent in complex ways ( wilkie and others 2000 ; eea 2006 ; mller and others 2010 ) . roads facilitate human access into formerly remote areas , accelerating land use change , habitat degradation and biodiversity loss , due to an increase in hunting , poaching , fishing , tourism , logging , mining , fires and urbanization ( trombulak and frissell 2000 ; wilkie and others 2000 ; hawbaker and others 2006 ) . urban development and sprawl are strongly linked to transport networks , also in rural and natural environments ( wilkie and others 2000 ; eea 2006 ; mller and others 2010 ) . thus , the density of transport infrastructures is a good indicator of the intensity of human activities and their impacts on biological diversity , and can be taken as a proxy for general disturbance levels ( e.g. , wilkie and others 2000 ; sanderson and others 2002 ; hawbaker and others 2006 ; theobald 2008 ; laurance and others 2009 ) . anthropogenic disturbances , in general , cause stress and reduce resilience and adaptive capacity of populations and species . this is of special concern in the context of climate change , which is increasing local extinction rates and forcing latitudinal and elevational shifts in species ranges ( walther and others 2002 ; parmesan 2006 ) . europe , as the cradle of industrialization and vehicular transport , is probably the continent most highly fragmented by transport infrastructures . especially the eu-12 countries ( austria , belgium , denmark , finland , france , germany , ireland , italy , luxembourg , netherlands , sweden , united kingdom ) , historically highly developed and crowded , constitute the epicenter of habitat conversion and fragmentation ( pullin and others 2009 ) . it would be informative , for example , to differentiate between urban and rural roads , as well as among road types ( motorways , highways , minor roads ) . however , such specific data are available only regionally or even locally . taking into account these constraints ( tent - t , a subset of the continent s overall road network targeted to support the economical integration of the eu ) in 2005 was 98,500 km , of which 78% corresponded to roads in eu-15 countries ( eu-12 plus greece , portugal , spain , data from tina 2008 ) . in a 10-year period ( 19952005 ) the length of motorways has increased by more than 13,000 km in the eu countries ( steer davies gleave 2009 ) . while eu plans imply slight increases of its total length , planned upgradings would increase the proportion of motorways in the network from 49% ( 2005 ) to 63% ( 2020 ) . the land dedicated to transport networks continues to increase in the whole eu . in the period 19901998 approximately 10 ha of land were taken for new motorway construction every day in the eu-15 ( eea 2002 ) . for example , in the netherlands , on average 1 km of land is crossed by 3 km of asphalt road ( eea 2002 ) ; and the average size of polygons enclosed by the network of all roads is 1.14 km ( van langevelde and others 2009 ) . in belgium , transport infrastructure already occupies more than 4% of the country the environmental consequences of this process may be different in the already highly fragmented eu-15 and in the new member states ( accessions from 2004 ; bulgaria , cyprus , czech republic , estonia , hungary , latvia , lithuania , malta , poland , romania , slovakia , slovenia , eu-27 ) with a lower density of roads and generally harbouring relatively well - preserved biodiversity and ecosystems , but where transport infrastructures are developing quickly after their accession . fragmentation by transport infrastructures in hungary and in the czech and slovak republics are already more severe than the eu-15 average . in the 1990s , total motorway length doubled in the new members ( 2300 km built ) , while in the eu-15 it increased by almost one third ( 12000 km built , eea 2002 ) . major transport infrastructures in the new members ( 174 km , eea 2002 , data from 1998 ) is still above the average of the eu-15 ( 121 km ) . however , differences between individual eu member states are more pronounced ; finland , and scandinavian countries in general , being the least fragmented in the continent , followed by countries in the carpathian region . mountainous countries , like italy or austria , still maintain relatively large unfragmented patches ( eea 2002 ) . thus , it seems reasonable to hypothesize that the few areas of low or no road fragmentation still existing in europe might be an important focus of future conservation efforts . in this paper , the term roadless areas refers to relatively large areas without any roads , whereas areas containing only roads with low - traffic intensity , below an established limit , are termed low - traffic areas ( see definition below ) . our main goal is to bring attention to the conservation value of roadless and low - traffic areas in europe , specifically in the eu . we aim at ( 1 ) describing the benefits of roadless and low - traffic areas for biodiversity conservation ; ( 2 ) exploring how these areas are valued and considered in european legislation ; ( 3 ) assessing how low - traffic areas are protected as natura 2000 sites in germany , as a case study , comparing the western and eastern parts of the country ; and , ( 4 ) discussing the potential role of roadless and low - traffic areas to strengthen the effectiveness of the natura 2000 network , especially in the context of climate change and the unaccomplished goal of halting biodiversity loss by 2010 ( eu 2001 ; ec 2010 ) , as well as their integration into legal instruments and transport policies . roadless areas and , to a lesser extent , low - traffic areas , represent relatively undisturbed natural habitats and functioning ecosystems . as at low traffic intensity , road impacts , such as the barrier effect for fauna , wildlife disturbance or pollution , are dampened ( iuell and others 2003 ; jaeger and others 2006 ; theobald 2008 ; charry and jones 2009 ) , it is sensible to assume that low - traffic areas may also represent sites of high conservation value , especially in europe , a human - dominated landscape . under this scenario , the ecological benefits described for roadless areas , which have been the focus of most research , may well apply to low - traffic areas as well . scientific evidence shows that roadless areas are critical in maintaining biodiversity , ecosystem processes , connectivity and overall ecosystem integrity . large , well - connected patches increase landscape connectivity and complement the network of protected areas ( e.g. , develice and martin 2001 ; loucks and others 2003 ; crist and others 2005 ) . thus , roadless areas sustain important elements of ecosystem integrity , such as the ability of species to move and natural processes to function . they largely contribute to the preservation of native biodiversity and contain more species and individuals , species with large spatial requirements ( e.g. , top carnivores ) , and species sensitive to human disturbance ( haskell 2000 ; watkins and others 2003 ; angelstam and others 2004 ; blake and others 2008 ; chen and roberts 2008 ) . they have the potential to ensure sufficient habitat for viable populations of species of conservation concern , as well as to increase the representation of rare ecological communities ( strittholt and dellasala 2001 ; loucks and others 2003 ; crist and others 2005 ) . they serve as a barrier against pests , diseases ( of wildlife , livestock and humans , e.g. , the lyme disease ) and invasive species ( strittholt and dellasala 2001 ; allan and others 2003 ; gelbard and harrison 2003 ; holdsworth and others 2007 ; von der lippe and kowarik 2007 ) . it is in these large areas of unfragmented land that ecosystem services , vital for human societies , are rendered ( millennium ecosystem assessment 2005 ) . roadless and low - traffic areas are of special importance in the context of climate change because they are more resilient than areas more fragmented by roads , and because they have a vast buffering capacity ( mcgarigal and others 2001 ) . their ecosystem dynamics are still internally driven , as opposite to the dynamics of fragmented patches , which are predominantly driven by external forces ( saunders and others 1991 ) . ecosystems already fragmented and stressed by human activities will be more vulnerable to climatic threats , while large intact areas better resist and recover from climate change impacts ( markham 1996 ; laurance and williamson 2001 ; noss 2001 ; opdam and wascher 2004 ; ferguson and others 2008 ) . these relatively undisturbed habitat patches can facilitate movements of organisms in the case of climate - forced range shifting . in general , they may represent havens for many species that are displaced from former habitats ( noss 2001 ; lovejoy 2006 ) . by slowing the rates of changes , moderating local climate , and being more diverse and resilient , roadless and low - traffic areas may contribute to mitigate the effects of species phenology changes and trophic mismatches caused by climate change ( parmesan 2006 ) , and to facilitate species adaptation . especially when comprising forest ecosystems or peatlands , roadless and low - traffic areas can play an important role in carbon fixation . undisturbed habitats may be better players in carbon sequestration than their fragmented and degraded counterparts ( harmon and others 1990 ; laurance and williamson 2001 ; ferguson and others 2008 ; luyssaert and others 2008 ) . it should be a relevant hypothesis to test that relatively carbon - rich ecosystem types in roadless and low - traffic areas store more carbon for maintaining a less stressed and thus more functional status . in the case of forests , in areas with poor access ( e.g. , in remote mountain areas ) roadless and low - traffic areas also provide protection against the impacts of storm events , like flooding or landslides , and wildfires ( lower fire risk and higher resilience ; usda forest service 2000 , 2001 ; dellasala and frost 2001 ; laurance and williamson 2001 ; ferguson and others 2008 ) . these relatively intact areas contribute to floodplain protection and drought abatement , as well as to maintain local climates stables and buffering weather extremes . the social and economic benefits of roadless and low - traffic areas , such as recreation , have also been well documented ( noss 1991 ; loomis and richardson 2000 ; krieger 2001 ) . roadless and low - traffic areas , as lands with a relatively low human footprint and good conservation status , have been considered a priority in regional conservation planning in several countries outside europe . for example , in bolivia , various conservation planning exercises on regional and national scales followed a functional approach and integrated roadless areas as surrogates for functional conservation targets , such as ecosystem processes and emergent features of biodiversity , especially required in the face of environmental change ( viability , resilience and adaptive capacity ; ibisch and others 2005 ) . however , the most ground - breaking initiative and important precedent has been the u.s . roadless area conservation rule of 2001 . it stated that 237,000 km within the u.s . national forest system ( 2% of us continental land ) the two inventories of roadless areas , rare i and rare ii , included unfragmented patches larger than 2024 ha ( an important part of which was designated as wilderness ) and 405 ha , respectively ( see usda forest service 2001 ; strittholt and dellasala 2001 ; turner 2006 , 2009 for details ) . in europe , initiatives specifically restricting road development in natural areas and giving special attention to the protection of roadless and low - traffic areas have hardly been launched . the centerpiece of the eu nature conservation policy is the natura 2000 network , which consists of special protection areas and special areas of conservation delineated according to the provisions of the birds and habitats directives , respectively ( 79/409/eec and 92/43/eec , see pullin and others 2009 ) . although both european directives oblige the member states to take the appropriate measures to maintain the integrity of natura 2000 sites and to guarantee the long - term persistence of species and ecosystems , in practice they face enormous difficulties in avoiding habitat fragmentation . very illustrative are recent conflicts like the planned construction of the via baltica express - way through the biebrza marshes , the unique rospuda mire and two large natural forests in northeastern poland ( eea 2006 ) . moreover , a high proportion of natura 2000 sites is already in close proximity to major transport infrastructures and/or will be potentially affected by the future development of the european transport network ( fisher and waliczky 2001 ; eea 2002 ) . we conducted an exploration of european legal instruments , ranging from selected national laws to eu nature conservation directives and european conventions ( table 1 , see supplementary material ( appendix ) for detailed description of the laws ) . only one of the explored laws considered roadless or low - traffic areas as a conservation target , although , paradoxically , the majority of them aimed at protecting other targets which are inherently and intimately related to fragmentation like connectivity , ecosystem processes or integrity . one important finding is an apparent conceptual shift from mere species and habitat protection ( e.g. , bern convention 1979 ) to more holistic approaches of ecosystem conservation , including processes , functions and aspects of integrity . we especially consider the carpathian convention ( 2003 ) that explicitly addresses regulations of traffic impacts and development , and encourages the parties to develop sustainable transport policies . traffic and energy infrastructure and similar projects shall be integrated so that fragmentation and consumption of the landscape as well as ecological impairment is avoided or reduced to a minimum ( federal nature conservation act from 29 july 2009 ; table 1 ) . in spite of the apparent trend that european nature legislation is starting to consider minimising fragmentation by transport infrastructures , it is unfortunate that neither the eu nor the large majority of national laws recognise the significance of areas with low levels of fragmentation by roads in their conservation policies.table 1conservation targets and entities explicitly considered in selected european legal instruments , including conventions , european directives and national lawscountry or regionroadless and low - traffic areasecosystem servicesnatural resourcesecosystem integrityecosystem functioningecological processesconnectivitybiodiversitylandscapenatural ecosystemshabitatsnative species ( flora&fauna)natural heritagebern convention regionxx alpine convention region xxxxxxxxxxx carpathian convention region xxxxxxxxxxxxeuropean unionxxx xxxxxxxgermanyxxx 1 and supplementary material ( appendix ) for details of the countries included in each region and the respective laws . symbol x indicates inclusion of targets and symbol indicates exclusion ; ( ) except scotland , whose law includes those conservation targets conservation targets and entities explicitly considered in selected european legal instruments , including conventions , european directives and national laws see fig . 1 and supplementary material ( appendix ) for details of the countries included in each region and the respective laws . x indicates inclusion of targets and symbol indicates exclusion ; ( ) except scotland , whose law includes those conservation targets germany is one of the largest european countries , located in the center of the continent , including a wide variety of biogeographical regions . after world war ii , germany was divided into the federal republic of germany ( western germany ) and the german democratic republic ( eastern germany ) ; in 1990 they were unified again . differences in natural conditions , history , political regimes , social models and economic development between eastern and western germany illustrate well the differences between the so - called western and eastern europe , once separated by the iron curtain . germany lies in the transition zone between these two parts of europe ( fig . it reflects somehow different situations between the highly developed and industrialized countries in eu-15 and the new member states , more rural and less fragmented by transport infrastructures . these two german regions also show strong differences in population density ; the old federal states ( former federal republic of germany ) have almost twice the population density of the new federal states ( former german democratic republic).fig . 1map of europe indicating the members of the european union ( with a distinction of the new member states that have acceded since 2004 ) and the contracting parties under the alpine and carpathian convention map of europe indicating the members of the european union ( with a distinction of the new member states that have acceded since 2004 ) and the contracting parties under the alpine and carpathian convention germany is the first european country where data on the distribution and size of low - traffic areas have become available . the german federal agency for nature conservation conducted a first inventory of large areas not cut by major transport infrastructures ( bfn 2008 ) , whose data were used for the present study ( federal agency for nature conservation , technical data on natura 2000 from 2008 , unpublished ) . these low - traffic areas were defined as larger than 100 km and not dissected by roads with more than 1000 vehicles per day , by railway lines ( twin - track and single - track electrified lines ) or by human settlements , airports or channels ( with the status of a category iv federal waterway or above ) . these criteria are generally applied in europe ( e.g. , andel and others 2005 ; jaeger and others 2007 ; bfn 2008 ) . although further research to identify threshold values for traffic volumes are needed , the value of 1000 vehicles per day seems to be an acceptable synthesis of the current ecological evidence and models and may serve as a preliminary basis for further discussions . below this threshold many , though certainly not all , populations of conservation - relevant species dissected by a road are thought to remain viable by several authors ( e.g. , hels and buchwald 2001 ; iuell and others 2003 ; seiler 2003 , 2005 ; jaeger and others 2006 ; charry and jones 2009 ) . we superimposed the gis shape files of these low - traffic areas with those of natura 2000 sites in germany ( merging special areas of conservation and special protection areas , federal agency for nature conservation , technical data on moderately fragmented areas with low traffic intensity from 2006 , unpublished ) to analyze their spatial relationship . particularly , our aim was to determine to what extent low - traffic areas are protected within the natura 2000 network and to what extent natura 2000 sites lie outside these areas . as the ecological coherence of the natura 2000 network is one of the main goals of the habitats directive , we were also interested in determining the degree of fragmentation of natura 2000 sites . for this purpose , we counted the number of spatially isolated subareas each natura 2000 site is composed of and analyzed the class size distribution of subareas for special areas of conservation and special protection areas separately . although the natura 2000 network covers an important proportion of the country ( 16% ; table 2 ; fig . 2 ) , most low - traffic areas ( 75% ) lie outside the network and thus remain without protection . this proportion is higher for the old federal states ( western germany ) , where only one fifth of low - traffic patches are protected . the new federal states are much less fragmented , and about 45% of its surface consists of low - traffic areas , as against 18% in the old federal states . more than half of the natura 2000 sites in germany lie outside low - traffic areas , especially in the old federal states ( 72% of the sites ; table 2 ) . natura 2000 sites are highly fragmented , and often they consist of several subareas , many of which have a very small size . almost 64% of these subareas classified as special areas of conservation are less than 50 ha ( fig . 3 ) . in the case of the special protection areas these figures are better ( 27% ) . in general , special areas of conservation comprise smaller subareas ; only 4% of them cover more than 1000 ha , against 26% in the case of special protection areas ( fig . 3).table 2representation of low - traffic areas and natura 2000 sites in germany , with a distinction between the old and the new federal statesold federal statesnew federal stateswhole germanysurface248,884109,025357,909low - traffic areas45,161 ( 18%)48,843 ( 45%)94,004 ( 26%)natura 2000 sites33,662 ( 14%)23,753 ( 22%)57,415 ( 16%)low - traffic areas not covered by natura 2000 sites35,677 ( 79%)34,398 ( 70%)70,075 ( 75%)natura 2000 sites lying outside low - traffic areas24,178 ( 72%)9,308 ( 39%)33,486 ( 58%)the surface ( km ) and percentage of low - traffic areas , natura 2000 sites and their overlap are also indicated . low - traffic areas are defined as larger than 100 km and crossed only by small roads with less than 1000 vehicles per day ( data from 2007 , bfn 2008)fig . 3size class distribution of the subareas forming natura 2000 sites in germany : special protection areas ( birds directive ) and special areas of conservation ( habitats directive ) representation of low - traffic areas and natura 2000 sites in germany , with a distinction between the old and the new federal states the surface ( km ) and percentage of low - traffic areas , natura 2000 sites and their overlap are also indicated . low - traffic areas are defined as larger than 100 km and crossed only by small roads with less than 1000 vehicles per day ( data from 2007 , bfn 2008 ) natura 2000 network and low - traffic areas coverage in germany size class distribution of the subareas forming natura 2000 sites in germany : special protection areas ( birds directive ) and special areas of conservation ( habitats directive ) biodiversity continues to decline in europe in spite of considerable conservation efforts carried out by administrations as well as non - governmental organisations ( ec 2010 ) . the unaccomplished target of halting biodiversity loss by 2010 ( eu 2001 ; ec 2010 ) calls for additional conservation measures to be put into practice as soon . fruit of this concern are recent initiatives of the european commission on defining targets beyond 2010 , developing green infrastructures or scaling up efforts to protect wilderness ( ep 2008 , 2009 ; ec 2011a , b ) . the need to strengthen the natura 2000 network and adapt it to the rising challenges of climate change has also been highlighted . in this context , ecosystem resilience and landscape connectivity are key goals that should be reinforced ( ep 2010a , b ) . the macis report ( berry and others 2008 ) states that to prevent and minimise future impacts of climate change on biological diversity in the eu , the minimization of fragmentation and the creation of connectors between protected areas is of extreme importance . therefore , maintaining unfragmented large patches of natural habitats , i.e. , roadless and low - traffic areas , seems a prudent strategy under any climate change scenario . the capacity of an ecosystem to preserve its integrity and biodiversity increases in large habitats that are well connected and which are far from the influence of external disturbances ( e.g. , fahrig and merriam 1985 ) . we feel that there is enough evidence of the benefits of roadless and low - traffic areas for nature conservation , especially in the context of global change . they represent sites with low human footprint , a high level of ecological integrity , and thus , intact ecosystem functioning and ongoing ecological processes , which translates into higher resilience and adaptive capacity . even in densely populated and intensively used landscapes , like germany , the remaining roadless and low - traffic areas may be of enormous value in supporting resilience and adaptive capacity of the biodiversity . in many areas of europe , it is probably too late to conserve roadless areas sensu stricto to a substantial extent . in this context , the last remnants are of special value , much more if they are primeval or close to a natural state . although the importance of keeping unfragmented large patches of natural habitats is increasingly recognised ( e.g. , jaeger and others 2006 ; charry and jones 2009 ; bentez - lpez and others 2010 ) , more research on the ecological benefits of both roadless and low - traffic areas in europe is deemed necessary . such research efforts should be accompanied by improving the quality and availability of road data ( e.g. , across road types unpaved roads included or differentiating between urban and rural roads ) and securing data coverage for the entire continent . conservation policies should focus not only on tangible elements , like species or habitats , but also include surrogates ( such as roadless areas ) of more abstract conservation targets , like ecosystem functioning , ecological processes or ecosystem services . the identification and inventory of the roadless and low - traffic areas in europe a detailed inventory of these areas , based on sound and concrete criteria , and more detailed estimations of the level of fragmentation by roads in europe shall form the basis for a proper assessment of the magnitude of the problem . given the considerable impacts of minor and unpaved roads in natural ecosystems ( e.g. , reed and others 1996 ; riitters and wickham 2003 ; van langevelde and others 2009 ) , fragmentation and impact assessments should also consider them . as an example , in sweden , classically considered as one of the least fragmented countries in europe ( 400 km is the average size of non - fragmented land according to eea 2002 ) , the figures may look quite different when forest roads are taken into account . during the last decade , the rate of road construction in swedish forests has been approximately 1700 km per year , as a long - term goal of the forestry sector is to have no more than 500 m or 1000 m to the nearest road in southern and northern sweden , respectively ( swedish forest agency 2008 ) . data on the habitat types covered by roadless and low - traffic areas and their overlap with the natura 2000 network should be compulsory in europe . stronger scientific evidence on traffic and area thresholds following an ecosystem approach is needed ; in general , road research needs to effectively address questions of direct management relevance and design studies that have high inferential strength , e.g. evaluating impacts before and after road construction ( gontier and others 2006 ; roedenbeck and others 2007 ) . as a second step , inventoried roadless and low - traffic areas should be considered in european legal instruments in a more explicit way . fortunately , european legislation has just started to take them into account . in this sense , the recent carpathian convention and the german federal conservation act are among the most progressive legal instruments in europe ( see table 1 ) . the urgent need to protect large , undisturbed habitat patches in europe , the european parliament adopted the report on wilderness in europe ( ep 2008 , 2009 ) that calls on the commission for better protection of wild areas , as a means for climate protection and maintenance of ecosystem services and biodiversity . in this context , a another possible step would be to include roadless and low - traffic areas in the habitats directive , either within a new annex of functional targets complementing and facilitating the adaptation of the natura 2000 network to climate change , or even as a new category of site . whether a special protection category or designation of these areas is needed or not , large natural areas without roads are protected de facto . mirroring the us legislation , the main goal would be to protect roadless and low - traffic areas from further road development , namely building new roads , increase of traffic volume on existing small roads and use of off - road vehicles . inclusion of roadless and low - traffic areas in the national and eu biodiversity strategies would also be desirable . only after being inventoried and receiving a legal status , roadless and low - traffic areas could be properly integrated into transport and spatial planning . the environmental impact assessment ( eia , 85/337/eec ) and the strategic environmental assessment directives ( sea , 2001/42/eec ) are essential instruments to assess the impacts from transport infrastructures and for the integration of ecological issues into spatial planning ; however the quality of ecological assessments is still limited ( e.g. , inappropriate criteria and methods , local approach , lack of integration between ecological and landscape assessments , gontier and others 2006 ; joumard and nicolas 2010 ) . the consideration of biodiversity in environmental assessments requires a holistic approach , i.e. , to scale - up to the ecosystem level and to link local and global aspects ( gontier and others 2006 ; joumard and nicolas 2010 ) . a concrete step towards the accomplishment of this vision will be to consider roadless and low - traffic areas in environmental assessments . bearing in mind the strong meaning of the term ( e.g. , joumard and nicolas 2010 ) , a sustainable and conservation - sound planning of the european transport network should take into account the still existing roadless and low - traffic areas . especially in the new member states , containing highly valuable areas but where transport networks are developing at a brisk pace , more sustainable transport policies are necessary . research shows that , whenever possible , the design of new routes should avoid dissecting remote and roadless areas ( jaeger and others 2006 ; forman 2007 ; charry and jones 2009 ; bentez - lpez and others 2010 ) . studies on road animal mortality , population persistence and road configuration ( jaeger and others 2006 ; charry and jones 2009 ) support the bundling traffic concept , concluding that ( 1 ) the road network should leave areas as large as possible free from disturbances due to traffic , ( 2 ) traffic should be concentrated on highly travelled roads , and ( 3 ) when traffic can not be combined on one road , it is better to bundle roads close together than to distribute them evenly across the landscape . thus , in natural areas with low level of fragmentation and human footprint ( e.g. , crossed by small roads ) , the general recommendation is to prevent increases in traffic volume ( charry and jones 2009 ) . when avoidance of habitat fragmentation is not possible , mitigation measures and strategies to maintain landscape connectivity , like ecological corridors , should be designed ( iuell and others 2003 ) . so far , scientific evidence of their effectiveness from a genetic point of view is still needed ( corlatti and others 2009 ) . in minor roads , traffic calming is another type of intervention used to mitigate negative impacts by reducing traffic volumes and speeds , and which has been shown to increase the persistence on animal populations in areas with a dense road network ( van langevelde and jaarsma 2009 ) . road closure , especially in remote areas , has also been suggested as a measure to decrease fragmentation ( berry and others 2008 ; charry and jones 2009 ) . integrated solutions are obliged to include urban planning . given the magnitude of urban sprawl in europe , also affecting natural habitats ( eea 2006 ) , an equally important goal should be to impede secondary effects along existing roads in roadless and low - traffic areas . this is of high concern in countries lacking spatial plans in most of their surface ( e.g. , poland ) and where roads are inevitably followed by urban development . the implementation of sustainable development schemes at large spatial scales , linking long and short terms , as well as local and global issues , would prevent the degradation of the integrity of roadless and low - traffic areas and thus , of their contribution to ecosystem and landscape resilience . clearly , in the preservation of roadless areas and sustainable development of low - traffic areas , synergies of nature conservation and other societal goals are manifold . just to name a few examples , traffic calming also benefits health and well - being ( less accidents , noise and pollution ) , and water provision to society generally improves in volume and quality with the functionality of roadless and low - traffic areas ( e.g. , van langevelde and jaarsma 2009 ) . such synergies make roadless areas and low - traffic areas worth considering by a variety of stakeholders beyond the conservation community . with broader societal support , roads have brought benefits to human societies for centuries . in the current situation of road encroachment , biodiversity crisis and global and climate change , roadless and low - traffic areas may far exceed roaded areas in the benefits provided . we call for a pan - european conservation strategy defining relatively unfragmented and low- footprint areas ( roadless and low - traffic areas ) regardless of their biotic characteristics as conservation targets . further scientific evidence on their ecological benefits and further research to answer key questions in road ecology regarding low - traffic and roadless areas under the different conditions in european countries is urgently needed . conservation scientists and administrations should join forces to halt the loss of biodiversity in europe , to keep healthy and resilient ecosystems and to preserve the services they provide . preserving the below is the link to the electronic supplementary material . supplementary material 1 ( docx 19 kb )

with increasing road encroachment , habitat fragmentation by transport infrastructures has been a serious threat for european biodiversity . areas with no roads or little traffic ( roadless and low - traffic areas ) represent relatively undisturbed natural habitats and functioning ecosystems . they provide many benefits for biodiversity and human societies ( e.g. , landscape connectivity , barrier against pests and invasions , ecosystem services ) . roadless and low - traffic areas , with a lower level of anthropogenic disturbances , are of special relevance in europe because of their rarity and , in the context of climate change , because of their contribution to higher resilience and buffering capacity within landscape ecosystems . an analysis of european legal instruments illustrates that , although most laws aimed at protecting targets which are inherent to fragmentation , like connectivity , ecosystem processes or integrity , roadless areas are widely neglected as a legal target . a case study in germany underlines this finding . although the natura 2000 network covers a significant proportion of the country ( 16% ) , natura 2000 sites are highly fragmented and most low - traffic areas ( 75% ) lie unprotected outside this network . this proportion is even higher for the old federal states ( western germany ) , where only 20% of the low - traffic areas are protected . we propose that the few remaining roadless and low - traffic areas in europe should be an important focus of conservation efforts ; they should be urgently inventoried , included more explicitly in the law and accounted for in transport and urban planning . considering them as complementary conservation targets would represent a concrete step towards the strengthening and adaptation of the natura 2000 network to climate change.electronic supplementary materialthe online version of this article ( doi:10.1007/s00267 - 011 - 9751-z ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction Importance of Roadless and Low-Traffic Areas in Biodiversity Conservation Roadless and Low-Traffic Areas in the European Legislation Roadless and Low-Traffic Areas and Natura 2000: Germany as a Case Study Discussion: Roadless and Low-Traffic Areas as a Key Element of European Conservation Policy Electronic supplementary material

the online version of this article ( doi:10.1007/s00267 - 011 - 9751-z ) contains supplementary material , which is available to authorized users . habitat fragmentation by transport networks and consequential secondary development has become one of the most serious global threats to biological diversity ( eea 2002 ; iuell and others 2003 ; laurance and others 2009 ; bentez - lpez and others 2010 ) . thus , the density of transport infrastructures is a good indicator of the intensity of human activities and their impacts on biological diversity , and can be taken as a proxy for general disturbance levels ( e.g. anthropogenic disturbances , in general , cause stress and reduce resilience and adaptive capacity of populations and species . this is of special concern in the context of climate change , which is increasing local extinction rates and forcing latitudinal and elevational shifts in species ranges ( walther and others 2002 ; parmesan 2006 ) . in belgium , transport infrastructure already occupies more than 4% of the country the environmental consequences of this process may be different in the already highly fragmented eu-15 and in the new member states ( accessions from 2004 ; bulgaria , cyprus , czech republic , estonia , hungary , latvia , lithuania , malta , poland , romania , slovakia , slovenia , eu-27 ) with a lower density of roads and generally harbouring relatively well - preserved biodiversity and ecosystems , but where transport infrastructures are developing quickly after their accession . fragmentation by transport infrastructures in hungary and in the czech and slovak republics are already more severe than the eu-15 average . thus , it seems reasonable to hypothesize that the few areas of low or no road fragmentation still existing in europe might be an important focus of future conservation efforts . in this paper , the term roadless areas refers to relatively large areas without any roads , whereas areas containing only roads with low - traffic intensity , below an established limit , are termed low - traffic areas ( see definition below ) . our main goal is to bring attention to the conservation value of roadless and low - traffic areas in europe , specifically in the eu . we aim at ( 1 ) describing the benefits of roadless and low - traffic areas for biodiversity conservation ; ( 2 ) exploring how these areas are valued and considered in european legislation ; ( 3 ) assessing how low - traffic areas are protected as natura 2000 sites in germany , as a case study , comparing the western and eastern parts of the country ; and , ( 4 ) discussing the potential role of roadless and low - traffic areas to strengthen the effectiveness of the natura 2000 network , especially in the context of climate change and the unaccomplished goal of halting biodiversity loss by 2010 ( eu 2001 ; ec 2010 ) , as well as their integration into legal instruments and transport policies . roadless areas and , to a lesser extent , low - traffic areas , represent relatively undisturbed natural habitats and functioning ecosystems . as at low traffic intensity , road impacts , such as the barrier effect for fauna , wildlife disturbance or pollution , are dampened ( iuell and others 2003 ; jaeger and others 2006 ; theobald 2008 ; charry and jones 2009 ) , it is sensible to assume that low - traffic areas may also represent sites of high conservation value , especially in europe , a human - dominated landscape . under this scenario , the ecological benefits described for roadless areas , which have been the focus of most research , may well apply to low - traffic areas as well . scientific evidence shows that roadless areas are critical in maintaining biodiversity , ecosystem processes , connectivity and overall ecosystem integrity . large , well - connected patches increase landscape connectivity and complement the network of protected areas ( e.g. they largely contribute to the preservation of native biodiversity and contain more species and individuals , species with large spatial requirements ( e.g. they serve as a barrier against pests , diseases ( of wildlife , livestock and humans , e.g. roadless and low - traffic areas are of special importance in the context of climate change because they are more resilient than areas more fragmented by roads , and because they have a vast buffering capacity ( mcgarigal and others 2001 ) . these relatively undisturbed habitat patches can facilitate movements of organisms in the case of climate - forced range shifting . by slowing the rates of changes , moderating local climate , and being more diverse and resilient , roadless and low - traffic areas may contribute to mitigate the effects of species phenology changes and trophic mismatches caused by climate change ( parmesan 2006 ) , and to facilitate species adaptation . especially when comprising forest ecosystems or peatlands , roadless and low - traffic areas can play an important role in carbon fixation . it should be a relevant hypothesis to test that relatively carbon - rich ecosystem types in roadless and low - traffic areas store more carbon for maintaining a less stressed and thus more functional status . in the case of forests , in areas with poor access ( e.g. , in remote mountain areas ) roadless and low - traffic areas also provide protection against the impacts of storm events , like flooding or landslides , and wildfires ( lower fire risk and higher resilience ; usda forest service 2000 , 2001 ; dellasala and frost 2001 ; laurance and williamson 2001 ; ferguson and others 2008 ) . the social and economic benefits of roadless and low - traffic areas , such as recreation , have also been well documented ( noss 1991 ; loomis and richardson 2000 ; krieger 2001 ) . roadless and low - traffic areas , as lands with a relatively low human footprint and good conservation status , have been considered a priority in regional conservation planning in several countries outside europe . for example , in bolivia , various conservation planning exercises on regional and national scales followed a functional approach and integrated roadless areas as surrogates for functional conservation targets , such as ecosystem processes and emergent features of biodiversity , especially required in the face of environmental change ( viability , resilience and adaptive capacity ; ibisch and others 2005 ) . national forest system ( 2% of us continental land ) the two inventories of roadless areas , rare i and rare ii , included unfragmented patches larger than 2024 ha ( an important part of which was designated as wilderness ) and 405 ha , respectively ( see usda forest service 2001 ; strittholt and dellasala 2001 ; turner 2006 , 2009 for details ) . in europe , initiatives specifically restricting road development in natural areas and giving special attention to the protection of roadless and low - traffic areas have hardly been launched . the centerpiece of the eu nature conservation policy is the natura 2000 network , which consists of special protection areas and special areas of conservation delineated according to the provisions of the birds and habitats directives , respectively ( 79/409/eec and 92/43/eec , see pullin and others 2009 ) . although both european directives oblige the member states to take the appropriate measures to maintain the integrity of natura 2000 sites and to guarantee the long - term persistence of species and ecosystems , in practice they face enormous difficulties in avoiding habitat fragmentation . moreover , a high proportion of natura 2000 sites is already in close proximity to major transport infrastructures and/or will be potentially affected by the future development of the european transport network ( fisher and waliczky 2001 ; eea 2002 ) . we conducted an exploration of european legal instruments , ranging from selected national laws to eu nature conservation directives and european conventions ( table 1 , see supplementary material ( appendix ) for detailed description of the laws ) . only one of the explored laws considered roadless or low - traffic areas as a conservation target , although , paradoxically , the majority of them aimed at protecting other targets which are inherently and intimately related to fragmentation like connectivity , ecosystem processes or integrity . in spite of the apparent trend that european nature legislation is starting to consider minimising fragmentation by transport infrastructures , it is unfortunate that neither the eu nor the large majority of national laws recognise the significance of areas with low levels of fragmentation by roads in their conservation policies.table 1conservation targets and entities explicitly considered in selected european legal instruments , including conventions , european directives and national lawscountry or regionroadless and low - traffic areasecosystem servicesnatural resourcesecosystem integrityecosystem functioningecological processesconnectivitybiodiversitylandscapenatural ecosystemshabitatsnative species ( flora&fauna)natural heritagebern convention regionxx alpine convention region xxxxxxxxxxx carpathian convention region xxxxxxxxxxxxeuropean unionxxx xxxxxxxgermanyxxx 1 and supplementary material ( appendix ) for details of the countries included in each region and the respective laws . after world war ii , germany was divided into the federal republic of germany ( western germany ) and the german democratic republic ( eastern germany ) ; in 1990 they were unified again . these two german regions also show strong differences in population density ; the old federal states ( former federal republic of germany ) have almost twice the population density of the new federal states ( former german democratic republic).fig . 1map of europe indicating the members of the european union ( with a distinction of the new member states that have acceded since 2004 ) and the contracting parties under the alpine and carpathian convention map of europe indicating the members of the european union ( with a distinction of the new member states that have acceded since 2004 ) and the contracting parties under the alpine and carpathian convention germany is the first european country where data on the distribution and size of low - traffic areas have become available . the german federal agency for nature conservation conducted a first inventory of large areas not cut by major transport infrastructures ( bfn 2008 ) , whose data were used for the present study ( federal agency for nature conservation , technical data on natura 2000 from 2008 , unpublished ) . we superimposed the gis shape files of these low - traffic areas with those of natura 2000 sites in germany ( merging special areas of conservation and special protection areas , federal agency for nature conservation , technical data on moderately fragmented areas with low traffic intensity from 2006 , unpublished ) to analyze their spatial relationship . particularly , our aim was to determine to what extent low - traffic areas are protected within the natura 2000 network and to what extent natura 2000 sites lie outside these areas . as the ecological coherence of the natura 2000 network is one of the main goals of the habitats directive , we were also interested in determining the degree of fragmentation of natura 2000 sites . although the natura 2000 network covers an important proportion of the country ( 16% ; table 2 ; fig . 2 ) , most low - traffic areas ( 75% ) lie outside the network and thus remain without protection . this proportion is higher for the old federal states ( western germany ) , where only one fifth of low - traffic patches are protected . the new federal states are much less fragmented , and about 45% of its surface consists of low - traffic areas , as against 18% in the old federal states . more than half of the natura 2000 sites in germany lie outside low - traffic areas , especially in the old federal states ( 72% of the sites ; table 2 ) . natura 2000 sites are highly fragmented , and often they consist of several subareas , many of which have a very small size . in general , special areas of conservation comprise smaller subareas ; only 4% of them cover more than 1000 ha , against 26% in the case of special protection areas ( fig . 3).table 2representation of low - traffic areas and natura 2000 sites in germany , with a distinction between the old and the new federal statesold federal statesnew federal stateswhole germanysurface248,884109,025357,909low - traffic areas45,161 ( 18%)48,843 ( 45%)94,004 ( 26%)natura 2000 sites33,662 ( 14%)23,753 ( 22%)57,415 ( 16%)low - traffic areas not covered by natura 2000 sites35,677 ( 79%)34,398 ( 70%)70,075 ( 75%)natura 2000 sites lying outside low - traffic areas24,178 ( 72%)9,308 ( 39%)33,486 ( 58%)the surface ( km ) and percentage of low - traffic areas , natura 2000 sites and their overlap are also indicated . low - traffic areas are defined as larger than 100 km and crossed only by small roads with less than 1000 vehicles per day ( data from 2007 , bfn 2008)fig . 3size class distribution of the subareas forming natura 2000 sites in germany : special protection areas ( birds directive ) and special areas of conservation ( habitats directive ) representation of low - traffic areas and natura 2000 sites in germany , with a distinction between the old and the new federal states the surface ( km ) and percentage of low - traffic areas , natura 2000 sites and their overlap are also indicated . low - traffic areas are defined as larger than 100 km and crossed only by small roads with less than 1000 vehicles per day ( data from 2007 , bfn 2008 ) natura 2000 network and low - traffic areas coverage in germany size class distribution of the subareas forming natura 2000 sites in germany : special protection areas ( birds directive ) and special areas of conservation ( habitats directive ) biodiversity continues to decline in europe in spite of considerable conservation efforts carried out by administrations as well as non - governmental organisations ( ec 2010 ) . the need to strengthen the natura 2000 network and adapt it to the rising challenges of climate change has also been highlighted . in this context , ecosystem resilience and landscape connectivity are key goals that should be reinforced ( ep 2010a , b ) . the macis report ( berry and others 2008 ) states that to prevent and minimise future impacts of climate change on biological diversity in the eu , the minimization of fragmentation and the creation of connectors between protected areas is of extreme importance . , roadless and low - traffic areas , seems a prudent strategy under any climate change scenario . we feel that there is enough evidence of the benefits of roadless and low - traffic areas for nature conservation , especially in the context of global change . they represent sites with low human footprint , a high level of ecological integrity , and thus , intact ecosystem functioning and ongoing ecological processes , which translates into higher resilience and adaptive capacity . even in densely populated and intensively used landscapes , like germany , the remaining roadless and low - traffic areas may be of enormous value in supporting resilience and adaptive capacity of the biodiversity . although the importance of keeping unfragmented large patches of natural habitats is increasingly recognised ( e.g. , jaeger and others 2006 ; charry and jones 2009 ; bentez - lpez and others 2010 ) , more research on the ecological benefits of both roadless and low - traffic areas in europe is deemed necessary . conservation policies should focus not only on tangible elements , like species or habitats , but also include surrogates ( such as roadless areas ) of more abstract conservation targets , like ecosystem functioning , ecological processes or ecosystem services . the identification and inventory of the roadless and low - traffic areas in europe a detailed inventory of these areas , based on sound and concrete criteria , and more detailed estimations of the level of fragmentation by roads in europe shall form the basis for a proper assessment of the magnitude of the problem . as an example , in sweden , classically considered as one of the least fragmented countries in europe ( 400 km is the average size of non - fragmented land according to eea 2002 ) , the figures may look quite different when forest roads are taken into account . data on the habitat types covered by roadless and low - traffic areas and their overlap with the natura 2000 network should be compulsory in europe . as a second step , inventoried roadless and low - traffic areas should be considered in european legal instruments in a more explicit way . the urgent need to protect large , undisturbed habitat patches in europe , the european parliament adopted the report on wilderness in europe ( ep 2008 , 2009 ) that calls on the commission for better protection of wild areas , as a means for climate protection and maintenance of ecosystem services and biodiversity . in this context , a another possible step would be to include roadless and low - traffic areas in the habitats directive , either within a new annex of functional targets complementing and facilitating the adaptation of the natura 2000 network to climate change , or even as a new category of site . mirroring the us legislation , the main goal would be to protect roadless and low - traffic areas from further road development , namely building new roads , increase of traffic volume on existing small roads and use of off - road vehicles . inclusion of roadless and low - traffic areas in the national and eu biodiversity strategies would also be desirable . only after being inventoried and receiving a legal status , roadless and low - traffic areas could be properly integrated into transport and spatial planning . the environmental impact assessment ( eia , 85/337/eec ) and the strategic environmental assessment directives ( sea , 2001/42/eec ) are essential instruments to assess the impacts from transport infrastructures and for the integration of ecological issues into spatial planning ; however the quality of ecological assessments is still limited ( e.g. a concrete step towards the accomplishment of this vision will be to consider roadless and low - traffic areas in environmental assessments . , joumard and nicolas 2010 ) , a sustainable and conservation - sound planning of the european transport network should take into account the still existing roadless and low - traffic areas . thus , in natural areas with low level of fragmentation and human footprint ( e.g. when avoidance of habitat fragmentation is not possible , mitigation measures and strategies to maintain landscape connectivity , like ecological corridors , should be designed ( iuell and others 2003 ) . given the magnitude of urban sprawl in europe , also affecting natural habitats ( eea 2006 ) , an equally important goal should be to impede secondary effects along existing roads in roadless and low - traffic areas . the implementation of sustainable development schemes at large spatial scales , linking long and short terms , as well as local and global issues , would prevent the degradation of the integrity of roadless and low - traffic areas and thus , of their contribution to ecosystem and landscape resilience . clearly , in the preservation of roadless areas and sustainable development of low - traffic areas , synergies of nature conservation and other societal goals are manifold . just to name a few examples , traffic calming also benefits health and well - being ( less accidents , noise and pollution ) , and water provision to society generally improves in volume and quality with the functionality of roadless and low - traffic areas ( e.g. such synergies make roadless areas and low - traffic areas worth considering by a variety of stakeholders beyond the conservation community . in the current situation of road encroachment , biodiversity crisis and global and climate change , roadless and low - traffic areas may far exceed roaded areas in the benefits provided . we call for a pan - european conservation strategy defining relatively unfragmented and low- footprint areas ( roadless and low - traffic areas ) regardless of their biotic characteristics as conservation targets .

the neuroprotective effect of stem cells for the treatment of cns injury has been shown in several preclinical studies . potential mechanisms currently under investigation include engraftment and transdifferentiation , modulation of the inflammatory milieu , and modulation of the systemic immunologic / inflammatory response . lundberg et al administered human mesenchymal stem cells in the ipsilateral internal carotid artery of rats which had been subjected to experimental tbi . kuh et al implanted human umbilical cord blood - derived progenitor cells ( hucbcs ) into the injury site after spinal cord contusion in a rodent model . the transplanted hucbcs were differentiated into various neural cells , which were confirmed by double immunofluorescence staining of bromodeoxyuridine ( brdu ) and glial fibrillary acidic protein ( gfap ) and microtubule - associated protein-2 ( map-2 ) staining . locomotor testing showed functional improvement for all time points tested up to 8 weeks after spinal cord injury . salazar et al transplanted human neural stem cells into immunodeficient nod - scid mice 30 days post spinal cord contusion injury . the transplanted mice demonstrated significantly improved locomotor recovery compared with vehicle controls using open field locomotor testing and cat walk gait analysis . the transplanted neural stem cells exhibited long - term engraftment , migration , limited proliferation , and differentiation predominantly to oligodendrocytes and neurons . also , differentiated nscs integrated with the host as was demonstrated by colocalization of human cytoplasm with discrete staining for the paranodal marker contactin - associated protein . dramatic cerebral responses following tbi comprise inflammation , cell death , and modulation of trophic factor release . walker et al directly implanted mscs into the brains of rats which had been subjected to tbi . brain supernatant analysis showed an increase in interleukin ( il)-6 , which has both direct and indirect neurotrophic effects on neurons . glazova et al implanted neuronal phenotype es cells in mice after experimentally induced spinal cord injury . transplantation of the es cells activated both brainderived neurotrophic factor il-6 signaling pathways in the host tissue , leading to activation of camp / pka , phosporylation of cofilin and synapsin i , and promoting regenerative growth and neuronal survival . given the results of these preclinical studies , modulation of the proinflammatory environment could afford neuroprotection . models of tbi invariably show activation of microglial cells , although it is unclear whether such activation promotes neuronal survival , or exacerbates neuronal damage . also , adaptive immune responses play a role . for example cd4 + t cells are observed in the substantia nigra in tbi patients , and in a model of spinal cord injury , t cells isolated from diseased animals induced transient hind limb paralysis and spinal cord inflammation when injected into nave recipients . although innate responses are considered protective , there is a delicate balance between the innate immune system and the adaptive immune system in mediating either pathogenic or repair processes under these conditions . walker et al were able to show that the intravenous injection of multipotcnt adult progenitor cells after experimental tbi in rodents preserved splenic mass and increased the num - ber and proliferative rate of cd4 + t cells as well as the production of il-4 and il-10 in stimulated splenocyt.es . hence , the colocalization of transplanted mapc and resident cd4 + splenocyt.es seems to be associated with a global increase in il-4 and il-10 production and stabilization of the cerebral microvasculature tight junction proteins . nemeth et al administered bone marrow stromal cells to mice before or shortly after inducing sepsis by cecal ligation and puncture , and found monocytes and/or macrophages from septic lungs made more il-10 when prepared from mice treated with bone mesenchymal stem cells ( bmscs ) versus untreated mice , leading to reduced mortality and improved organ function . despite the promising preclinical results described above , there are problems to consider when trying to translate these studies into a clinical setting . intravenous infusion of mscs in rats which had been subjected to tbi failed to result in significant acute or prolonged cerebral engraftment of cells or to modify the recovery of motor or cognitive function . also , the transplantation of neuronal stem cells into the ipsilateral or contralateral corpus callosum of rats at 48 hours after severe experimental tbi failed to lead to proliferation of the implanted cells , regardless of the site of implantation . cao et al found pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord to be restricted to a glial lineage . zheng et al implanted neural stem cells derived from wistar rats into traumatized sprague - dawley rats and studied the local lymphocyte infiltration . the histological examination and immunohistochemistry revealed significant lymphocyte infiltration in the contusion , suggesting that immunosuppressive treatment is necessary following nsc transplantation . considering these problems , this pathway may not be feasible for a clinical translation at this point in time . encapsulated cell biodelivery has been put forward as a novel clinical strategy for cell therapy in the cns . it appears that semipermeable hollow fibers , as well as spherical polymeric microcapsules , protect cells transplanted into the brain from the immunological graft - versus - host response . as the capsules permit the free passage of nutrients , oxygen , and , indeed , smaller molecules , the cells are maintained within the capsules , and can produce and deliver therapeutic peptides to the brain . encapsulated cells have already been used for the therapy of diabetes mellitus , amyotropic lateral sclerosis , chronic pain , huntington 's disease , and for the treatment of malignant brain tumors . our group conducted a preclinical study testing the effect of encapsulated native mscs and encapsulated glucagon -like pcptide-1 ( glp-1 ) transfected mscs in experimental traumatic brain injur } ' ( controlled cortical impact- cci ) . glp-1 is an endogenous insulin - stimulating peptide that is secreted from the gastrointestinal tract in response to food intake . glp-1 has been shown to improve learning and memory in glp-1 receptor - deficient mice . the blood - to - brain delivery of native glp-1 is , however , affected because glp-1 rapidly degrades , with a plasma half - life of between 1 and 2 min . hence , the cells were used as a bioreactor which constantly releases glp-1 , while simultaneously bypassing the blood - brain barrier . a human bone this cell line was immortalized by transduction with the human telomerase reverse transcriptase ( htert ) gene . following transfection with a plasmid vector encoding a glp-1 fusion gene , the cells produced 8.7 kda of dimeric glp-1 . animals were randomized into five groups : controls ( no cci ) ; cci - only ; cci + native human bone - marrow derived mesenchymal stem cells ( hm'sc ) ; cci + glp1 producing hmsc ; and cci + empty capsules . twenty capsules were implanted into the right lateral ventricle immediately before cci . even though this technique does not mimic the clinical setting , it was necessary in order to ensure implantation of the encapsulated cells into the ventricle , since the standard stereotactic coordinates become invalid after the cci due to contusion - related brain tissue shifting . on day 14 , cisternal cerebrospinal fluid ( csf ) was sampled for measurement of glp-1 concentration , and brains were immuno - histochemically assessed using specific antibody staining for neun , m ap-2 and glial fibrillary acidic protein ( gfap ) . in order to determine the viability and the glp production of the glp-1 secreting hmscs , nine healthy animals were implanted with 20 capsules using the same stereotactic technique . capsules were retrieved , and viability and glp-1 production rate was assessed after 2,7 , and 14 days of cerebral transplantation . one third of the retrieved capsules were stained with propidium iodide ( staining of nonvital cells ) and sybr green ( staining of vital cells ) , and then visualized using fluorescence microscopy . in both of the stem cell - treated cci groups , hippocampal cell loss was reduced , along with an attenuation of cortical neuronal and glial abnormalities , as measured by map-2 and gfap expression . anti - neun staining demonstrated a major reduction of positively stained neurons in the hilus of the dentate gyrus in the cci - only and cci with empty capsule groups . this neuronal loss was not observed in cci animals implanted with native hmscs and with glp-1 - producing hmscs . similarly , both anti - gfap and anti - map-2 staining illustrated that the staining pattern in the animals with native and glp-1 producing stem cells were very similar to those of the healthy controls , whereas in the cci - only and cci with empty capsules groups , increased immunostaining was observed , indicating reactive neuronal and glial changes . however , the effects were more pronounced in animals treated with glp-1 secreting hmscs . in the cci animals with glp-1 producing hmscs , the csf concentration of glp-1 at day 14 was 17.3 + 3.4 pm.this concentration was significantly higher than that in the remaining groups : 3.1 1 .6 pm ( cci + capsules without cells ) , 3.32.9pm ( cci + native hmsc ) and 2.40.7pm ( cci - only ) . no measurable glp-1 concentrations ( detection limit : 2 pm ) were found in the healthy control group . following a temporary cerebral implantation in healthyrats , the mean in vitro glp-1 production rate of the hmcs explanted at day 2 was 3.680.49 fmol / capsule / h . on day 7 the rate was 2.850.45 fmol / capsule / h , and on day 14 it was 3.530.55 fmol / capsule / h . the production rate of non - implanted capsules was 7.03 fmol / capsule / h . the in vitro production rate of the encapsulated glp-1 stem cells , retrieved after temporary implantation in healthy rats , was maintained at about half the rate of the nonimplantcd glp-1 secreting stem cells . independently of the duration of implantation , propidium iodide and sybr green fluorescence microscopy revealed that more than 95% of the stem cells were viable in the explanted capsules . in a second study , we tested the encapsulated cells described above in a double transgenic mouse model of alzheimer 's disease ( ad ) after intraventricular implantation at 3 months of age . mice earning mutations in the amyloid precursor protein and presenilin-1 and -2 genes develop ad - like deposits composed of a - beta at an early age . since a - beta deposits as well as inflammation of the cns arc visible at 3 months starting in the frontal cortex , stem cell implantation was performed at this age to test whether early treatment may prevent the onset of a - beta deposition and associated inflammation . abeta 40/42 deposition , and glial ( gfap ) and microglial ( cd11b ) immunoreactivity were investigated 2 months after transplantation of either native msc or msc transfected with glp-1 and compared with untreated controls . cdllb immunostaining in the frontal lobes was significantly decreased in the glp-1 hmsc group compared with the untreated controls . also , the plaque - associated gfap immunoreactivity was only observed in one animal in the glp-1 msc group . a - bcta 40 whole brain ( enzyme - linked immunosorbent assay , elisa ) was decreased in both hmsc groups : 86.06 + /- 11.1 pg / ml . according to these experimental findings , encapsulated native hmscs possess anti - inflammatory and neuroprotective properties , which seem to be enhanced by genetical engineering of the cells to secrete glp-1 . tticrcf ore , glp-1 -secreting hmsc capsules may have a therapeutic potential in acute but also chronic neurological diseases . translating our experimental findings , intraccrcbal hemorrhage ( ich ) was chosen as disease model to investigate the safety of encapsulated mesenchymal cell biodelivery of glp-1 in a phase i / ii trial which is currently ongoing . microencapsulated allogenic hmscs are transplanted into the brain tissue cavity after neurosurgical evacuation of the hematoma . the objective of this approach is to improve the outcome after surgery for ich ; the local , neuroprotective , and anti - inflammatory cell therapy is targeting the secondary neuronal injury in the perihematomal area occuring in the first weeks after the bleeding . in the clinical trial , each microcapsule contains about 3000 glp-1 hmsc capsules , and approximately 7.8 x 106 cells are implanted . since approval agencies are concerned about possible long - term side effects due to stem cell transplantation , the cells are not implanted into the brain directly , but filled into a 1.5 x 1.5 cm - sized bag that is manually sutured from a polypropylene mesh with pores of up to 300 urn . a 5-cm tether for fixation of the implant to the skull surface after surgical hematoma evacuation , this mesh bag is implanted into the hematoma cavity , and it is removed 2 weeks after implantation by a second surgery . safety assessments include mri examinations , physical and neurological examinations , nih stroke scale ( nihss ) , barthel index ( iii ) , clinical laboratory profile , and any adverse events . different risk levels are defined that would eventually lead to the explantation of the containment including the study medication ( eg , systemic infection , local inflammatory reaction , anaphylactic reaction , seizures , unexpected neurological deterioration or other unexpected adverse events ) . the interim evaluation of the first 11 patients revealed neither side effects from the surgical interventions nor implant - related side effects . also , up to 30% of the transplanted mscs survived the 2-weck implantation period and were still secretorily active after explantation . the trial is still recruiting ; a thorough assessment of the application safety of the novel therapy , including a comprehensive analysis of neurological , radiological , and laboratory parameters will be possible after completion of the trial including a total of 20 cases . according to the existing preclinical studies and the preliminary results of the ongoing clinical trial in ich patients , glp-1-secreting hmsc capsules might be an effective treatment for tbi patients as well . presumably , the neuroprotective and anti - inflammatory properties of the cell capsules are most effective in the acute stage after tbi preventing ongoing secondary brain injury . however , additional preclinical studies are required to ascertain that the transplantation of cell capsules does not increase the risk of edema or may cause increased icp . however , the preliminary radiological ( mri ) results in the ich patients suggest that the cell capsules may even decrease cerebral edema . currently the therapeutic value of intracerebral injection of cell capsules into a traumatic lesion , ie , cerebral contusion , or into the cerebral ventricles is not established . the intraventricular application has been shown to be effective in our rodent tbi model ; however , it is controversial as to whether this application route is also effective in humans . while the cerebroventricular administration of trophic factors has influenced the pathology of neurodegenerative disorders , the rapid clearance of csf into the venous circulation has been recognized as a substantial limitation to the pharmakokinetics of this drug delivery route . the only reported clinical study investigating intraventricular , hollow fiber encapsulated cell biodelivery revealed only minimally increased csf concentration of the delivered factor . however , microencapsulation , as used in our clinical study , allows for the transplantation of a significantly higher number of cells , ie , millions compared with only hundreds of thousands in the hollow fiber encapsulation . thus , the higher release rates that can be achieved with the microencapsulation technique might compensate for the rapid csf clearance , and thereby build up pharmacologically active csf factor concentrations . also , it is not clarified , whether an enclosure , similar to the mesh bag used in the ich trial , is necessary for intraventricular or intracerebral implantation . it might be safe and effective to inject the cell capsules without such containment . however , to validate this application , additional preclinical work addressing mainly acute and chronic safety issues is required . despite the promising preclinical results described above , there are problems to consider when trying to translate these studies into a clinical setting . intravenous infusion of mscs in rats which had been subjected to tbi failed to result in significant acute or prolonged cerebral engraftment of cells or to modify the recovery of motor or cognitive function . also , the transplantation of neuronal stem cells into the ipsilateral or contralateral corpus callosum of rats at 48 hours after severe experimental tbi failed to lead to proliferation of the implanted cells , regardless of the site of implantation . cao et al found pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord to be restricted to a glial lineage . zheng et al implanted neural stem cells derived from wistar rats into traumatized sprague - dawley rats and studied the local lymphocyte infiltration . the histological examination and immunohistochemistry revealed significant lymphocyte infiltration in the contusion , suggesting that immunosuppressive treatment is necessary following nsc transplantation . considering these problems , this pathway may not be feasible for a clinical translation at this point in time . encapsulated cell biodelivery has been put forward as a novel clinical strategy for cell therapy in the cns . it appears that semipermeable hollow fibers , as well as spherical polymeric microcapsules , protect cells transplanted into the brain from the immunological graft - versus - host response . as the capsules permit the free passage of nutrients , oxygen , and , indeed , smaller molecules , the cells are maintained within the capsules , and can produce and deliver therapeutic peptides to the brain . encapsulated cells have already been used for the therapy of diabetes mellitus , amyotropic lateral sclerosis , chronic pain , huntington 's disease , and for the treatment of malignant brain tumors . our group conducted a preclinical study testing the effect of encapsulated native mscs and encapsulated glucagon -like pcptide-1 ( glp-1 ) transfected mscs in experimental traumatic brain injur } ' ( controlled cortical impact- cci ) . glp-1 is an endogenous insulin - stimulating peptide that is secreted from the gastrointestinal tract in response to food intake . glp-1 has been shown to improve learning and memory in glp-1 receptor - deficient mice . the blood - to - brain delivery of native glp-1 is , however , affected because glp-1 rapidly degrades , with a plasma half - life of between 1 and 2 min . hence , the cells were used as a bioreactor which constantly releases glp-1 , while simultaneously bypassing the blood - brain barrier . a human bone this cell line was immortalized by transduction with the human telomerase reverse transcriptase ( htert ) gene . following transfection with a plasmid vector encoding a glp-1 fusion gene , the cells produced 8.7 kda of dimeric glp-1 . animals were randomized into five groups : controls ( no cci ) ; cci - only ; cci + native human bone - marrow derived mesenchymal stem cells ( hm'sc ) ; cci + glp1 producing hmsc ; and cci + empty capsules . twenty capsules were implanted into the right lateral ventricle immediately before cci . even though this technique does not mimic the clinical setting , it was necessary in order to ensure implantation of the encapsulated cells into the ventricle , since the standard stereotactic coordinates become invalid after the cci due to contusion - related brain tissue shifting . on day 14 , cisternal cerebrospinal fluid ( csf ) was sampled for measurement of glp-1 concentration , and brains were immuno - histochemically assessed using specific antibody staining for neun , m ap-2 and glial fibrillary acidic protein ( gfap ) . in order to determine the viability and the glp production of the glp-1 secreting hmscs capsules were retrieved , and viability and glp-1 production rate was assessed after 2,7 , and 14 days of cerebral transplantation . one third of the retrieved capsules were stained with propidium iodide ( staining of nonvital cells ) and sybr green ( staining of vital cells ) , and then visualized using fluorescence microscopy . the remaining capsules were recultured to measure the glp-1 production rate . in both of the stem cell - treated cci groups , hippocampal cell loss was reduced , along with an attenuation of cortical neuronal and glial abnormalities , as measured by map-2 and gfap expression . anti - neun staining demonstrated a major reduction of positively stained neurons in the hilus of the dentate gyrus in the cci - only and cci with empty capsule groups . this neuronal loss was not observed in cci animals implanted with native hmscs and with glp-1 - producing hmscs . similarly , both anti - gfap and anti - map-2 staining illustrated that the staining pattern in the animals with native and glp-1 producing stem cells were very similar to those of the healthy controls , whereas in the cci - only and cci with empty capsules groups , increased immunostaining was observed , indicating reactive neuronal and glial changes . however , the effects were more pronounced in animals treated with glp-1 secreting hmscs . in the cci animals with glp-1 producing hmscs , the csf concentration of glp-1 at day 14 was 17.3 + 3.4 pm.this concentration was significantly higher than that in the remaining groups : 3.1 1 .6 pm ( cci + capsules without cells ) , 3.32.9pm ( cci + native hmsc ) and 2.40.7pm ( cci - only ) . no measurable glp-1 concentrations ( detection limit : 2 pm ) were found in the healthy control group . following a temporary cerebral implantation in healthyrats , the mean in vitro glp-1 production rate of the hmcs explanted at day 2 was 3.680.49 fmol / capsule / h . on day 7 the rate was 2.850.45 fmol / capsule / h , and on day 14 it was 3.530.55 fmol / capsule / h . the production rate of non - implanted capsules was 7.03 fmol / capsule / h . thus , the in vitro production rate of the encapsulated glp-1 stem cells , retrieved after temporary implantation in healthy rats , was maintained at about half the rate of the nonimplantcd glp-1 secreting stem cells . independently of the duration of implantation , propidium iodide and sybr green fluorescence microscopy revealed that more than 95% of the stem cells were viable in the explanted capsules . in a second study , we tested the encapsulated cells described above in a double transgenic mouse model of alzheimer 's disease ( ad ) after intraventricular implantation at 3 months of age . mice earning mutations in the amyloid precursor protein and presenilin-1 and -2 genes develop ad - like deposits composed of a - beta at an early age . since a - beta deposits as well as inflammation of the cns arc visible at 3 months starting in the frontal cortex , stem cell implantation was performed at this age to test whether early treatment may prevent the onset of a - beta deposition and associated inflammation . abeta 40/42 deposition , and glial ( gfap ) and microglial ( cd11b ) immunoreactivity were investigated 2 months after transplantation of either native msc or msc transfected with glp-1 and compared with untreated controls . cdllb immunostaining in the frontal lobes was significantly decreased in the glp-1 hmsc group compared with the untreated controls . also , the plaque - associated gfap immunoreactivity was only observed in one animal in the glp-1 msc group . a - bcta 40 whole brain ( enzyme - linked immunosorbent assay , elisa ) was decreased in both hmsc groups : 86.06 + /- 11.1 pg / ml . according to these experimental findings , encapsulated native hmscs possess anti - inflammatory and neuroprotective properties , which seem to be enhanced by genetical engineering of the cells to secrete glp-1 . tticrcf ore , glp-1 -secreting hmsc capsules may have a therapeutic potential in acute but also chronic neurological diseases . translating our experimental findings , intraccrcbal hemorrhage ( ich ) was chosen as disease model to investigate the safety of encapsulated mesenchymal cell biodelivery of glp-1 in a phase i / ii trial which is currently ongoing . microencapsulated allogenic hmscs are transplanted into the brain tissue cavity after neurosurgical evacuation of the hematoma . the objective of this approach is to improve the outcome after surgery for ich ; the local , neuroprotective , and anti - inflammatory cell therapy is targeting the secondary neuronal injury in the perihematomal area occuring in the first weeks after the bleeding . in the clinical trial , each microcapsule contains about 3000 glp-1 hmsc capsules , and approximately 7.8 x 106 cells are implanted . since approval agencies are concerned about possible long - term side effects due to stem cell transplantation , the cells are not implanted into the brain directly , but filled into a 1.5 x 1.5 cm - sized bag that is manually sutured from a polypropylene mesh with pores of up to 300 urn . a 5-cm tether for fixation of the implant to the skull surface after surgical hematoma evacuation , this mesh bag is implanted into the hematoma cavity , and it is removed 2 weeks after implantation by a second surgery . safety assessments include mri examinations , physical and neurological examinations , nih stroke scale ( nihss ) , barthel index ( iii ) , clinical laboratory profile , and any adverse events . different risk levels are defined that would eventually lead to the explantation of the containment including the study medication ( eg , systemic infection , local inflammatory reaction , anaphylactic reaction , seizures , unexpected neurological deterioration or other unexpected adverse events ) . the interim evaluation of the first 11 patients revealed neither side effects from the surgical interventions nor implant - related side effects . also , up to 30% of the transplanted mscs survived the 2-weck implantation period and were still secretorily active after explantation . the trial is still recruiting ; a thorough assessment of the application safety of the novel therapy , including a comprehensive analysis of neurological , radiological , and laboratory parameters will be possible after completion of the trial including a total of 20 cases . according to the existing preclinical studies and the preliminary results of the ongoing clinical trial in ich patients , glp-1-secreting hmsc capsules might be an effective treatment for tbi patients as well . presumably , the neuroprotective and anti - inflammatory properties of the cell capsules are most effective in the acute stage after tbi preventing ongoing secondary brain injury . however , additional preclinical studies are required to ascertain that the transplantation of cell capsules does not increase the risk of edema or may cause increased icp . however , the preliminary radiological ( mri ) results in the ich patients suggest that the cell capsules may even decrease cerebral edema . currently the therapeutic value of intracerebral injection of cell capsules into a traumatic lesion , ie , cerebral contusion , or into the cerebral ventricles is not established . the intraventricular application has been shown to be effective in our rodent tbi model ; however , it is controversial as to whether this application route is also effective in humans . while the cerebroventricular administration of trophic factors has influenced the pathology of neurodegenerative disorders , the rapid clearance of csf into the venous circulation has been recognized as a substantial limitation to the pharmakokinetics of this drug delivery route . the only reported clinical study investigating intraventricular , hollow fiber encapsulated cell biodelivery revealed only minimally increased csf concentration of the delivered factor . however , microencapsulation , as used in our clinical study , allows for the transplantation of a significantly higher number of cells , ie , millions compared with only hundreds of thousands in the hollow fiber encapsulation . thus , the higher release rates that can be achieved with the microencapsulation technique might compensate for the rapid csf clearance , and thereby build up pharmacologically active csf factor concentrations . also , it is not clarified , whether an enclosure , similar to the mesh bag used in the ich trial , is necessary for intraventricular or intracerebral implantation . it might be safe and effective to inject the cell capsules without such containment . however , to validate this application , additional preclinical work addressing mainly acute and chronic safety issues is required . while encapsulated cell biodelivery has a reasonable perspective for a clinical application in traumatic brain injury , the translation of the existing findings requires extensive additional experimental studies .

traumatic brain injury remains a major cause of death and disability ; it is estimated that annually 10 million people are affected . preclinical studies have shown the potential therapeutic value of stem cell therapies . neuroprotective as well as regenerative properties of stem cells have been suggested to be the mechanism of action in preclinical studies . however , up to now stem cell therapy has not been studied extensively in clinical trials . this article summarizes the current experimental evidence and points out hurdles for clinical application . focusing on a cell therapy in the acute stage of head injury , the potential of encapsulated cell biodelivery as a novel cell - therapeutic approach will also be discussed .

Mechanisms of action of stem cell therapy in CNS injury Clinical translation of stem cell therapy in TBI Step 1: Deciding on an approach Step 2: Preclinical studies Step 3: Clinical translation of encapsulated mesenchymal cell biodelivery of GLP-1 Step 4: Encapsulated cell biodelivery in TBI Outlook

the neuroprotective effect of stem cells for the treatment of cns injury has been shown in several preclinical studies . lundberg et al administered human mesenchymal stem cells in the ipsilateral internal carotid artery of rats which had been subjected to experimental tbi . salazar et al transplanted human neural stem cells into immunodeficient nod - scid mice 30 days post spinal cord contusion injury . the transplanted neural stem cells exhibited long - term engraftment , migration , limited proliferation , and differentiation predominantly to oligodendrocytes and neurons . transplantation of the es cells activated both brainderived neurotrophic factor il-6 signaling pathways in the host tissue , leading to activation of camp / pka , phosporylation of cofilin and synapsin i , and promoting regenerative growth and neuronal survival . given the results of these preclinical studies , modulation of the proinflammatory environment could afford neuroprotection . models of tbi invariably show activation of microglial cells , although it is unclear whether such activation promotes neuronal survival , or exacerbates neuronal damage . for example cd4 + t cells are observed in the substantia nigra in tbi patients , and in a model of spinal cord injury , t cells isolated from diseased animals induced transient hind limb paralysis and spinal cord inflammation when injected into nave recipients . walker et al were able to show that the intravenous injection of multipotcnt adult progenitor cells after experimental tbi in rodents preserved splenic mass and increased the num - ber and proliferative rate of cd4 + t cells as well as the production of il-4 and il-10 in stimulated splenocyt.es . hence , the colocalization of transplanted mapc and resident cd4 + splenocyt.es seems to be associated with a global increase in il-4 and il-10 production and stabilization of the cerebral microvasculature tight junction proteins . nemeth et al administered bone marrow stromal cells to mice before or shortly after inducing sepsis by cecal ligation and puncture , and found monocytes and/or macrophages from septic lungs made more il-10 when prepared from mice treated with bone mesenchymal stem cells ( bmscs ) versus untreated mice , leading to reduced mortality and improved organ function . also , the transplantation of neuronal stem cells into the ipsilateral or contralateral corpus callosum of rats at 48 hours after severe experimental tbi failed to lead to proliferation of the implanted cells , regardless of the site of implantation . cao et al found pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord to be restricted to a glial lineage . the histological examination and immunohistochemistry revealed significant lymphocyte infiltration in the contusion , suggesting that immunosuppressive treatment is necessary following nsc transplantation . encapsulated cell biodelivery has been put forward as a novel clinical strategy for cell therapy in the cns . it appears that semipermeable hollow fibers , as well as spherical polymeric microcapsules , protect cells transplanted into the brain from the immunological graft - versus - host response . as the capsules permit the free passage of nutrients , oxygen , and , indeed , smaller molecules , the cells are maintained within the capsules , and can produce and deliver therapeutic peptides to the brain . encapsulated cells have already been used for the therapy of diabetes mellitus , amyotropic lateral sclerosis , chronic pain , huntington 's disease , and for the treatment of malignant brain tumors . our group conducted a preclinical study testing the effect of encapsulated native mscs and encapsulated glucagon -like pcptide-1 ( glp-1 ) transfected mscs in experimental traumatic brain injur } ' ( controlled cortical impact- cci ) . the blood - to - brain delivery of native glp-1 is , however , affected because glp-1 rapidly degrades , with a plasma half - life of between 1 and 2 min . hence , the cells were used as a bioreactor which constantly releases glp-1 , while simultaneously bypassing the blood - brain barrier . following transfection with a plasmid vector encoding a glp-1 fusion gene , the cells produced 8.7 kda of dimeric glp-1 . animals were randomized into five groups : controls ( no cci ) ; cci - only ; cci + native human bone - marrow derived mesenchymal stem cells ( hm'sc ) ; cci + glp1 producing hmsc ; and cci + empty capsules . in both of the stem cell - treated cci groups , hippocampal cell loss was reduced , along with an attenuation of cortical neuronal and glial abnormalities , as measured by map-2 and gfap expression . anti - neun staining demonstrated a major reduction of positively stained neurons in the hilus of the dentate gyrus in the cci - only and cci with empty capsule groups . similarly , both anti - gfap and anti - map-2 staining illustrated that the staining pattern in the animals with native and glp-1 producing stem cells were very similar to those of the healthy controls , whereas in the cci - only and cci with empty capsules groups , increased immunostaining was observed , indicating reactive neuronal and glial changes . however , the effects were more pronounced in animals treated with glp-1 secreting hmscs . in the cci animals with glp-1 producing hmscs , the csf concentration of glp-1 at day 14 was 17.3 + 3.4 pm.this concentration was significantly higher than that in the remaining groups : 3.1 1 .6 pm ( cci + capsules without cells ) , 3.32.9pm ( cci + native hmsc ) and 2.40.7pm ( cci - only ) . following a temporary cerebral implantation in healthyrats , the mean in vitro glp-1 production rate of the hmcs explanted at day 2 was 3.680.49 fmol / capsule / h . the in vitro production rate of the encapsulated glp-1 stem cells , retrieved after temporary implantation in healthy rats , was maintained at about half the rate of the nonimplantcd glp-1 secreting stem cells . independently of the duration of implantation , propidium iodide and sybr green fluorescence microscopy revealed that more than 95% of the stem cells were viable in the explanted capsules . mice earning mutations in the amyloid precursor protein and presenilin-1 and -2 genes develop ad - like deposits composed of a - beta at an early age . since a - beta deposits as well as inflammation of the cns arc visible at 3 months starting in the frontal cortex , stem cell implantation was performed at this age to test whether early treatment may prevent the onset of a - beta deposition and associated inflammation . cdllb immunostaining in the frontal lobes was significantly decreased in the glp-1 hmsc group compared with the untreated controls . also , the plaque - associated gfap immunoreactivity was only observed in one animal in the glp-1 msc group . according to these experimental findings , encapsulated native hmscs possess anti - inflammatory and neuroprotective properties , which seem to be enhanced by genetical engineering of the cells to secrete glp-1 . translating our experimental findings , intraccrcbal hemorrhage ( ich ) was chosen as disease model to investigate the safety of encapsulated mesenchymal cell biodelivery of glp-1 in a phase i / ii trial which is currently ongoing . the objective of this approach is to improve the outcome after surgery for ich ; the local , neuroprotective , and anti - inflammatory cell therapy is targeting the secondary neuronal injury in the perihematomal area occuring in the first weeks after the bleeding . in the clinical trial , each microcapsule contains about 3000 glp-1 hmsc capsules , and approximately 7.8 x 106 cells are implanted . since approval agencies are concerned about possible long - term side effects due to stem cell transplantation , the cells are not implanted into the brain directly , but filled into a 1.5 x 1.5 cm - sized bag that is manually sutured from a polypropylene mesh with pores of up to 300 urn . a 5-cm tether for fixation of the implant to the skull surface after surgical hematoma evacuation , this mesh bag is implanted into the hematoma cavity , and it is removed 2 weeks after implantation by a second surgery . also , up to 30% of the transplanted mscs survived the 2-weck implantation period and were still secretorily active after explantation . according to the existing preclinical studies and the preliminary results of the ongoing clinical trial in ich patients , glp-1-secreting hmsc capsules might be an effective treatment for tbi patients as well . presumably , the neuroprotective and anti - inflammatory properties of the cell capsules are most effective in the acute stage after tbi preventing ongoing secondary brain injury . however , additional preclinical studies are required to ascertain that the transplantation of cell capsules does not increase the risk of edema or may cause increased icp . however , the preliminary radiological ( mri ) results in the ich patients suggest that the cell capsules may even decrease cerebral edema . currently the therapeutic value of intracerebral injection of cell capsules into a traumatic lesion , ie , cerebral contusion , or into the cerebral ventricles is not established . the intraventricular application has been shown to be effective in our rodent tbi model ; however , it is controversial as to whether this application route is also effective in humans . while the cerebroventricular administration of trophic factors has influenced the pathology of neurodegenerative disorders , the rapid clearance of csf into the venous circulation has been recognized as a substantial limitation to the pharmakokinetics of this drug delivery route . the only reported clinical study investigating intraventricular , hollow fiber encapsulated cell biodelivery revealed only minimally increased csf concentration of the delivered factor . however , microencapsulation , as used in our clinical study , allows for the transplantation of a significantly higher number of cells , ie , millions compared with only hundreds of thousands in the hollow fiber encapsulation . also , it is not clarified , whether an enclosure , similar to the mesh bag used in the ich trial , is necessary for intraventricular or intracerebral implantation . however , to validate this application , additional preclinical work addressing mainly acute and chronic safety issues is required . also , the transplantation of neuronal stem cells into the ipsilateral or contralateral corpus callosum of rats at 48 hours after severe experimental tbi failed to lead to proliferation of the implanted cells , regardless of the site of implantation . cao et al found pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord to be restricted to a glial lineage . zheng et al implanted neural stem cells derived from wistar rats into traumatized sprague - dawley rats and studied the local lymphocyte infiltration . the histological examination and immunohistochemistry revealed significant lymphocyte infiltration in the contusion , suggesting that immunosuppressive treatment is necessary following nsc transplantation . encapsulated cell biodelivery has been put forward as a novel clinical strategy for cell therapy in the cns . it appears that semipermeable hollow fibers , as well as spherical polymeric microcapsules , protect cells transplanted into the brain from the immunological graft - versus - host response . as the capsules permit the free passage of nutrients , oxygen , and , indeed , smaller molecules , the cells are maintained within the capsules , and can produce and deliver therapeutic peptides to the brain . encapsulated cells have already been used for the therapy of diabetes mellitus , amyotropic lateral sclerosis , chronic pain , huntington 's disease , and for the treatment of malignant brain tumors . our group conducted a preclinical study testing the effect of encapsulated native mscs and encapsulated glucagon -like pcptide-1 ( glp-1 ) transfected mscs in experimental traumatic brain injur } ' ( controlled cortical impact- cci ) . the blood - to - brain delivery of native glp-1 is , however , affected because glp-1 rapidly degrades , with a plasma half - life of between 1 and 2 min . hence , the cells were used as a bioreactor which constantly releases glp-1 , while simultaneously bypassing the blood - brain barrier . following transfection with a plasmid vector encoding a glp-1 fusion gene , the cells produced 8.7 kda of dimeric glp-1 . animals were randomized into five groups : controls ( no cci ) ; cci - only ; cci + native human bone - marrow derived mesenchymal stem cells ( hm'sc ) ; cci + glp1 producing hmsc ; and cci + empty capsules . in both of the stem cell - treated cci groups , hippocampal cell loss was reduced , along with an attenuation of cortical neuronal and glial abnormalities , as measured by map-2 and gfap expression . anti - neun staining demonstrated a major reduction of positively stained neurons in the hilus of the dentate gyrus in the cci - only and cci with empty capsule groups . similarly , both anti - gfap and anti - map-2 staining illustrated that the staining pattern in the animals with native and glp-1 producing stem cells were very similar to those of the healthy controls , whereas in the cci - only and cci with empty capsules groups , increased immunostaining was observed , indicating reactive neuronal and glial changes . however , the effects were more pronounced in animals treated with glp-1 secreting hmscs . in the cci animals with glp-1 producing hmscs , the csf concentration of glp-1 at day 14 was 17.3 + 3.4 pm.this concentration was significantly higher than that in the remaining groups : 3.1 1 .6 pm ( cci + capsules without cells ) , 3.32.9pm ( cci + native hmsc ) and 2.40.7pm ( cci - only ) . no measurable glp-1 concentrations ( detection limit : 2 pm ) were found in the healthy control group . thus , the in vitro production rate of the encapsulated glp-1 stem cells , retrieved after temporary implantation in healthy rats , was maintained at about half the rate of the nonimplantcd glp-1 secreting stem cells . independently of the duration of implantation , propidium iodide and sybr green fluorescence microscopy revealed that more than 95% of the stem cells were viable in the explanted capsules . mice earning mutations in the amyloid precursor protein and presenilin-1 and -2 genes develop ad - like deposits composed of a - beta at an early age . since a - beta deposits as well as inflammation of the cns arc visible at 3 months starting in the frontal cortex , stem cell implantation was performed at this age to test whether early treatment may prevent the onset of a - beta deposition and associated inflammation . cdllb immunostaining in the frontal lobes was significantly decreased in the glp-1 hmsc group compared with the untreated controls . also , the plaque - associated gfap immunoreactivity was only observed in one animal in the glp-1 msc group . according to these experimental findings , encapsulated native hmscs possess anti - inflammatory and neuroprotective properties , which seem to be enhanced by genetical engineering of the cells to secrete glp-1 . translating our experimental findings , intraccrcbal hemorrhage ( ich ) was chosen as disease model to investigate the safety of encapsulated mesenchymal cell biodelivery of glp-1 in a phase i / ii trial which is currently ongoing . the objective of this approach is to improve the outcome after surgery for ich ; the local , neuroprotective , and anti - inflammatory cell therapy is targeting the secondary neuronal injury in the perihematomal area occuring in the first weeks after the bleeding . in the clinical trial , each microcapsule contains about 3000 glp-1 hmsc capsules , and approximately 7.8 x 106 cells are implanted . since approval agencies are concerned about possible long - term side effects due to stem cell transplantation , the cells are not implanted into the brain directly , but filled into a 1.5 x 1.5 cm - sized bag that is manually sutured from a polypropylene mesh with pores of up to 300 urn . a 5-cm tether for fixation of the implant to the skull surface after surgical hematoma evacuation , this mesh bag is implanted into the hematoma cavity , and it is removed 2 weeks after implantation by a second surgery . also , up to 30% of the transplanted mscs survived the 2-weck implantation period and were still secretorily active after explantation . according to the existing preclinical studies and the preliminary results of the ongoing clinical trial in ich patients , glp-1-secreting hmsc capsules might be an effective treatment for tbi patients as well . presumably , the neuroprotective and anti - inflammatory properties of the cell capsules are most effective in the acute stage after tbi preventing ongoing secondary brain injury . however , additional preclinical studies are required to ascertain that the transplantation of cell capsules does not increase the risk of edema or may cause increased icp . however , the preliminary radiological ( mri ) results in the ich patients suggest that the cell capsules may even decrease cerebral edema . currently the therapeutic value of intracerebral injection of cell capsules into a traumatic lesion , ie , cerebral contusion , or into the cerebral ventricles is not established . the intraventricular application has been shown to be effective in our rodent tbi model ; however , it is controversial as to whether this application route is also effective in humans . while the cerebroventricular administration of trophic factors has influenced the pathology of neurodegenerative disorders , the rapid clearance of csf into the venous circulation has been recognized as a substantial limitation to the pharmakokinetics of this drug delivery route . the only reported clinical study investigating intraventricular , hollow fiber encapsulated cell biodelivery revealed only minimally increased csf concentration of the delivered factor . however , microencapsulation , as used in our clinical study , allows for the transplantation of a significantly higher number of cells , ie , millions compared with only hundreds of thousands in the hollow fiber encapsulation . thus , the higher release rates that can be achieved with the microencapsulation technique might compensate for the rapid csf clearance , and thereby build up pharmacologically active csf factor concentrations . also , it is not clarified , whether an enclosure , similar to the mesh bag used in the ich trial , is necessary for intraventricular or intracerebral implantation . however , to validate this application , additional preclinical work addressing mainly acute and chronic safety issues is required . while encapsulated cell biodelivery has a reasonable perspective for a clinical application in traumatic brain injury , the translation of the existing findings requires extensive additional experimental studies .

the incidence of obesity defined as body mass index ( bmi ) 30 kg / m has increased drastically worldwide during recent decades . obesity is associated with a cluster of metabolic disorders , including increased risk of insulin resistance , type 2 diabetes ( t2 dm ) , hypertension , dyslipidemia , and cardiovascular disease ; these disorders and obesity per se constitute a serious threat , known as the metabolic syndrome ( ms ) . it is well known that chronic inflammation is a key feature of obesity ; this obesity - induced inflammation builds the common soil for the pathogenesis of ms . therefore , resetting the immunological balance in obesity is a crucial approach for the management of ms . white adipose tissue ( at ) has been regarded as the primary goal of pharmacological intervention since obesity - induced inflammation is mainly initiated and exacerbated in this organ [ 35 ] . although several prominent molecular mechanisms have been proposed to trigger inflammation in white at , including hypoxia , endoplasmic reticulum stress , lipotoxicity , and metabolic endotoxemia , these factors can not fully explain the origin of inflammation . on the other hand , current anti - inflammatory strategies are not sufficient enough in the treatment of ms . these facts suggest that there are lots of unknowns responsible for at inflammation in obesity . in recent years , a growing body of evidence has highlighted the modulating roles of mirnas in immune / inflammatory system [ 6 , 7 ] and their involvement in the obesity - related metabolic disorders including t2 dm and atherosclerosis [ 8 , 9 ] . as yet , whether these mirnas could be another mechanism for mediating at inflammation and whether these tiny molecules could serve as potent therapeutic anti - inflammatory targets for ms are still not fully settled . during the past decade , it became clear that inflammation is a key feature of obesity . the inflammatory response triggered by obesity involves many components of the classical inflammatory response to pathogens and includes the increases in circulating inflammatory cytokines and acute - phase proteins ( e.g. , c - reactive protein ) , recruitment of leukocytes to inflamed tissues , activation of tissue leukocytes , and generation of reparative tissue responses ( e.g. , fibrosis ) . however , the nature of obesity - induced inflammation , referred to as metainflammation ( metabolically triggered inflammation ) , is unique compared with other inflammatory paradigms ( e.g. , infection and autoimmune disease ) in several key aspects . first , the chronic nature of obesity produces a low - grade activation of the innate immune system that affects metabolic homeostasis over time . second , childhood obesity may place individuals at risk for lifelong inflammation , since inflammatory markers are elevated in obese children as young as 3 years old . third , this chronic inflammation is composed of recurrent acute episodes of nutrition - related immune activation induced by nutrient availability ( fasting or high - fat meals ) . this fluctuation may be associated with the induction of pro- or anti - inflammatory mediators . the metabolic syndrome ( ms ) refers to the clustering of cardiovascular risk factors that include central obesity , hyperglycemia , dyslipidemia , and hypertension . the ultimate importance of this cluster is to identify individuals at high risk of both t2 dm and cardiovascular disease . growing evidence implicates obesity - induced inflammation as an important mechanism linking obesity to the ms in metabolically active organs . assessment of gene expression networks in obese at has identified a robust pattern of overexpressed inflammatory genes associated with metabolic disease [ 12 , 13 ] . multiple inflammatory mediators abnormally secreted by at and the crosstalk between immune and metabolic cells can impair insulin signaling and induce oxidative stress and endothelial dysfunction , leading to systemic insulin resistance and cardiovascular disease . deregulated macrophage - myocyte and macrophage - hepatocyte signaling can impair insulin sensitivity as well [ 14 , 15 ] . hypothalamic inflammation , which is induced very rapidly by a high - fat diet may provoke hyperphagia and has been documented to impair insulin release from cells , peripheral insulin action , and potentiate hypertension [ 1719 ] . thus , chronic excess of nutrients , such as lipids and glucose may simultaneously trigger inflammatory responses , which further disrupt metabolic function , enhancing stress , and inflammation . therefore , breaking this vicious circle by resetting the immunological balance in obesity is a crucial approach for the management of ms . obesity is characterized by excessive expansion of white adipose tissue ( at ) , which has been thought to be the primary site for the initiation of obesity - associated inflammation . although at 's principal function deals with energy storage , it serves as an active secretory organ as well . a number of bioactive peptides or proteins , collectively named adipokines , are produced and secreted by fat and/or nonfat cells of white at . they act in an autocrine / paracrine manner to regulate local at function and also act in an endocrine manner to influence the functions of distant tissues such as liver , skeletal muscle , and cardiovascular and central nervous systems [ 5 , 20 ] . in obesity , white at is remodeled dynamically by adipocyte hypertrophy ( increased size ) , hyperplasia ( increased number ) , immune cell infiltration , endothelial cell overactivation , and extracellular matrix overproduction [ 2124 ] . this remodeling may trigger metabolic and hypoxic stress , resulting in activation of multiple inflammatory signaling pathways , ultimately leading to dysregulation of numerous adipokines including proinflammatory cytokines , chemokines , growth factors , acute - phase proteins , and complement - like factors . such a deregulation is the main feature of at low - grade inflammation and contributes to the pathogenesis of ms . the growing at is submitted to hypoxia induced by hypoperfusion at the earliest stages of expansion . observations have proved that at is poorly oxygenated in the obese state in humans and rodents due to a reduction in adipose tissue blood flow [ 25 , 26 ] . this hypoxia could be a determinant mediator of obesity - induced inflammation in at by activating multiple signaling pathways such as hypoxia inducible factor-1 ( hif-1 ) and nuclear factor kappa b ( nf-b ) in adipocytes and macrophages , thereby altering the expression of many proinflammatory adipokines [ 2729 ] . in addition , some studies reported that hypoxia also induces at inflammatory responses indirectly by causing adipocyte death ( e.g. , apoptosis and necrosis ) and lipolysis [ 3032 ] . the endoplasmic reticulum ( er ) is responsible for much of a cell 's protein synthesis and folding , but it has also a role in sensing cellular stress . obesity results in conditions that increase the demand on the er , such as hypoxia and excess of cytokines , lipids , or glucose . subsequently , the er stress induces a complex response known as the unfolded protein response ( upr ) , which alters a cell 's transcriptional and translational programs to cope with these stressful conditions and resolve the protein - folding defect . er stress and the upr lead to obesity - induced inflammation and metabolic abnormalities by several distinct mechanisms , including the activation of jnk - ap1 ( jun n - terminal kinase - activator protein 1 ) and ikk ( ib kinase)-nfb pathways , the induction of the acute - phase response , and the production of reactive oxygen species ( ros ) [ 34 , 35 ] . obesity is characterized by a positive energy balance , and the classical response to this nutrient oversupply is at hypertrophy . this limited storage capacity , coupled with the overstimulation of hormone - sensitive lipase , leads to massive increase in free fatty acid ( ffa ) release and accumulation ectopically . tlrs are a family of pattern - recognition receptors that play a critical role in the innate immune system by activating proinflammatory signaling pathways in response to microbial pathogens . at tlr expression in at is inducible by inflammatory stimulation and linked to downstream activation of nf-b or jnk and subsequent release of proinflammatory adipokines [ 37 , 38 , 40 ] . tlr4-deficient mice and c3h / hej mice ( which have a loss - of - function mutation in tlr4 ) are partially protected from fat - induced inflammation and insulin resistance in their visceral at [ 41 , 42 ] . ffas also increase the infiltration and activation of macrophages , especially the cd11c+ subset , thereby exacerbating their proinflammatory activity . high - fat diet given to mice chronically increased plasma lipopolysaccharide ( lps ) concentration two to three times ; a threshold that has been defined as metabolic endotoxemia . obese subjects with type 2 diabetes have 76% higher circulating lps than healthy controls and this high level of lps decreased significantly after surgical weight loss . elevated plasma lps levels result from increased absorption of lps across the intestinal barrier triggered by high - fat diet . recently , three underlying mechanisms have been proposed , including the changes in gut microbiota environment , the increased availability of chylomicrons , and the permeability of the gut epithelium [ 4749 ] . in murine adipocytes , lps initiates inflammation via tlr4 and induces the secretion of proinflammatory cytokines via downstream activation of nf-b or mitogen - activated protein kinases ( mapk ) signaling pathways . in human adipocytes , stimulation by lps increases release of tumor necrosis factor - alpha ( tnf- ) , interleukin-6 ( il-6 ) , and monocyte chemoattractant protein-1 ( mcp-1 ) by nf-b activation and upregulates tlr2 expression . micrornas ( mirnas ) are endogenous ~22 nt rnas that can bind to the 3-untranslated region ( 3-utr ) of target mrnas to repress mrna expression at the posttranscriptional level . as a group , mirnas may directly regulate expression of over 30% of human and mouse genes and more than 60% of human protein - coding genes have been under selective pressure to maintain pairing to mirnas . specific mirnas have been implicated in adipocyte differentiation and mature adipocyte function , including lipolysis , glucose - uptake , and insulin sensitivity [ 8 , 10 ] . on the other hand , mirnas have been defined as important immunomodulators by regulating the differentiation , induction , and function of immune cells and the expression of multiple cytokines in the immune system . these findings could shed light on the possible links between mirnas and adipose tissue immunity / inflammation . studies conducted by some groups have demonstrated that mirna regulation is indispensable for stable immune process . ablation of whole mirnas by deletion of argonaute 2 ( ago2 ) or dicer impairs pre - b - cell differentiation and the succeeding peripheral b - cell generation . several individual mirnas have also been reported to be involved in the b - cell biology . for example , overexpression of mir-17~92 clusters enhances b - cell proliferation and survival , while mir-150 profoundly impairs early b - cell differentiation and mature b - cell responses . mirnas have been shown to be key regulators of the t - cell lineage induction pathways . deletion of dicer at an early stage of t - cell development compromised the survival of t - cell lineage . the best evidence for mirnas playing a role in specific developmental stages of t - cell differentiation is from mir-181 , which reduces the number of t - cells in haematopoietic overexpression systems and also increases the sensitivity of t - cell receptor signaling . in addition , mirnas also play pivotal roles in the induction , function , and maintenance of the regulatory t - cell ( treg ) lineage . mirnas can enhance thymic and peripheral induction of treg cells [ 55 , 58 ] . several mirnas ( including mir-155 , mir-146a , mir-21 , and mir-9 ) have been consistently found to be rapidly induced by innate immune activation ( e.g. , toll - like receptor ) , indicating that they may regulate the innate immune response . target prediction analyses indicate that up to a half of innate immune genes could be under the direct regulation of mirnas . a study on 613 genes , which regulate immunity utilizing a computational approach , identified 285 genes as mirna targets . major targets include transcription factors , cofactors , and chromatin modifiers whereas upstream factors , such as ligands and receptors ( cytokines and chemokines ) were , in general , poor or nontargets . the mechanisms by which mirnas regulate cytokine expression include direct regulation by binding to seeding sites in mrna 3-utr and indirect regulation . for example , of the interleukin genes ( il1 - 29 ) studied , 9 had predicted mirna binding sites . out of 20 interleukin receptor genes examined , only 2 had high probability mirna target sites . these findings suggest that the regulation of cytokine genes by mirnas occurs often via indirect mechanism . recent studies indicate indeed that mirnas could indirectly regulate cytokine genes via au - rich elements ( are ) located in the mrna 3-utr by targeting are - binding proteins . are are the cis - acting structural rna motifs that are important determinants of cytokine message stability . are - mediated mrna degradation is regulated by a number of trans - acting factors , called the are - binding proteins ( which include tristetraprolin ( ttp ) , au rich binding factor 1 ( auf1 ) , and members of the hu protein r ( hur ) family ) . repression of several are components by mirnas may alter the levels of inflammatory cytokines as well as of other immune genes [ 60 , 62 ] . obesity - induced inflammation is associated with dysregulated expression of mirnas in white adipose tissue . a number of mirnas have been found dysregulated in white at during obesity and closely associated with obesity - related metabolic disorders . a recent study identified 21 mirnas , which were differentially expressed in epididymal at between lean mice fed a standard diet and mice rendered obese by a high - fat diet . ortega et al . performed mirna array on human subcutaneous at : 50 of the 799 mirnas tested ( 6.2% ) significantly differed between obese ( n = 9 ) and lean ( n = 6 ) subjects . among these 50 mirnas , 17 these data are concordant with those obtained in overweight or obese subjects by klting et al . they showed significant correlations between the expression of selected mirnas and both at morphology and key metabolic parameters , including visceral fat area , hba1c , fasting plasma glucose , and circulating leptin , adiponectin , and il-6 . another two mirnas ( mir-175p and mir-132 ) were significantly decreased in the omental fat and the circulation of obese subjects . a very recent study defined a set of eleven adipocyte - specific mirnas downregulated in obese subjects as all of them were concomitantly altered in both the white at and the isolated adipocytes . xie et al . showed that tnf- treatment of 3t3-l1 adipocytes mimicked the changes of mirna expression observed in at of obese mice . with obesity and inflammation being so intrinsically associated , the dysregulated expression of mirnas in inflammatory adipocytes highly suggests the particular importance of mirnas in obesity - induced inflammation . indeed , a few individual mirnas have been reported as playing a crucial role in the inflammatory state of at so far ( figure 1 ) . expression of mir-221 and mir-222 has been positively correlated to tnf- and negatively correlated to apn expression in white at of mice . yet , the direct effects of both mirnas on these pro- or anti - inflammatory adipokines are still unraveled . mir-132 , which was downregulated in human obese omental fat , has been reported to activate nf-b and the transcription of il-8 and mcp-1 in primary human preadipocytes and in in vitro differentiated adipocytes . zhuang et al . demonstrated that mir-223 played a crucial role in modulating macrophage polarization in a pattern that protects mice from diet - induced at inflammation and systemic insulin resistance . macrophages by targeting pknox1 in vitro , while mir-223 deficient mice fed a high - fat diet exhibited increased at inflammation characterized by enhanced proinflammatory activation of macrophages . the group of brichard has identified several mirnas , which were regulated by adiponectin ( a promising anti - inflammatory adipokine ) in white at in vivo . the mir883b-5p , which was upregulated by adiponectin and downregulated in human obesity repressed the lps - binding protein ( lbp ) and tlr4 signaling , acting therefore as a major mediator of the anti - inflammatory action of apn . moreover , mir883b - silencing in the de novo at formed from in vivo differentiation of preadipocytes also induced lbp production and tissue inflammation . very recently , arner et al . overexpressed individual mirnas ( which have been defined downregulated in at in human obesity ) in human adipocytes differentiated in vitro . they found that a set of nine mirnas significantly reduced the secretion of chemokine ( c - c motif ) ligand 2 ( ccl2 ) which is an initiator of at inflammation by attracting the migration of inflammatory cells into the tissue . among these mirnas , only mir-126 was predicted by in silico analysis and confirmed by luciferase reporter assay in 3t3-l1 cells to bind directly to the 3-utr of ccl2 , whereas mir-193b affected indirectly the ccl2 production through downregulating the transcription factors ( tfs ) of ccl2 ( relb , stat6 , and ets1 ) . thus , mirnas may mediate at inflammation by regulating either the activation of macrophages or the production of adipokines . the mechanism by which mirnas regulate adipokines in at seems via direct and indirect mechanisms . mirnas may act directly on the target inflammatory adipokines or indirectly by first regulating the intermediate machinery components like the tfs , which , in turn , control the expression of adipokines . in addition to the innate immunity , recent studies have disclosed the importance of the adaptive immunity in at inflammation . the subsets of the lymphocyte lineage , including cd8 + and cd4 + t - cells , tregs , natural killer t - cells ( nkt ) , and b - cells have been revealed to infiltrate into at which may orchestrate at inflammation . yet , the mechanisms underlying the infiltration of lymphocytes into at remain largely unknown ( figure 1 ) . whether mirnas could play roles in modulating the infiltration or activation of the lymphocytes in at may arouse research interest and requires to be investigated in the future . more recently , adipocytes have been reported to secrete mirnas in the form of adipocyte - derived microvesicles ( adms ) , which may regulate the function of distant or neighboring cells . one study found that microvesicles released from cultured 3t3-l1 adipocytes harbored 143 mirnas , most of them being adipocyte - specific and reflecting the abundance of their expression levels in the donor cells . interestingly , the mirnas - containing microvesicles were transported into cultured macrophages which were incubated with adms containing - medium for 24 h ; they were also present in rat serum in vivo . yet , whether the mirnas inside the vesicles were functional in their new location remains to be determined . furthermore , another group discovered that mirnas - microvesicle complexes derived from the larger primary rat adipocytes were transferred into and expressed in the smaller adipocytes and were involved in the transcription of multiple genes for lipid synthesis and cell growth . hypertrophied adipocytes are the main sources of at inflammation , the mirna - mediated interactions ( adipocytes - macrophages and larger adipocytes - smaller adipocytes ) may be novel mechanisms for mirnas contributing to at inflammation ( figure 1 ) . the facts that mirnas are largely dysregulated in obesity and that specific mirnas regulate obesity - associated inflammation suggests the potential of these molecules as targets for therapeutic intervention in obesity and obesity - related metabolic disorders . chen et al . screened serum mirnas of healthy chinese subjects and found over 100 and 91 serum mirnas in male and female subjects , respectively . yet , how these mirnas make their way into the circulation is still mysterious . new studies have revealed that circulating mirnas can reside in microvesicles ( exosomes , microparticles , and apoptotic bodies ) and in protein / lipoprotein complexes ( high density lipoprotein ( hdl ) and argonaute 2 ) ; these formations make the circulating mirnas protected and resistant to rnase activity and degradation . therefore , the levels of mirnas in serum are remarkable , stable , reproducible , and tissue - specific among individuals . multiple reports have noted the potential use of blood mirnas as biomarkers for the obesity , cardiovascular diseases , atherosclerosis , and t2 dm . heneghan et al . showed that circulating levels of mir-17 - 5p and mir-132 were significantly decreased in obese individuals compared to nonobese individuals and reflected mirna expression in omental fat . the mirna expression in blood and omental fat from obese patients correlated significantly with bmi , fasting blood glucose , and glycated hemoglobin . cardiac specific mir-1 , mir-208 , and mir-499 were induced consistently in blood of patients with myocardial infarction and strongly correlated to markers of cardiac damage including troponin t and creatine kinase - mb ( ck - mb ) activity [ 7881 ] . circulating mirnas have also been proposed as highly sensitive biomarkers for t2 dm . in the bruneck cohort of 822 subjects , decreased serum levels of mir-126 in t2 dm emerged as a significant predictor of t2 dm and correlated negatively with increasing glucose intolerance . silencing of key mirna and replacement of certain tissue - specific mirna whose expression is known to be decreased are potential therapeutic interventions . techniques . mirnas can be deactivated and silenced by anti - mirna oligonucleotides ( amos ) , mirna sponges , and mirna masking . amos are synthetic antisense oligonucleotides that competitively inhibit the interaction between mirnas and their mrna targets . the most widely employed types of amos are 2-o - methyl amo , 2-o - methoxyethyl amos , and locked nucleic acids ( lnas ) . given that mirnas have been observed to function often in clusters in pathological processes , the this technique requires a vector encoding large quantities of transcripts ( anti - mirnas ) which display numerous and tandem binding sites for a cluster of mirnas of interest . the mirna masking is an alternative mirna - knockdown strategy to the amo approach , with the advantage of targeting mirnas in a gene - specific manner . unlike amo , which binds to the endogenous mirna directly , a mirna mask binds to the mirna 's binding site located in the 3-utr of its target mrna , thereby avoiding off - target effects . conversely , with regard to those mirnas with decreased expression in disease , the fundamental principle strategy is to restore their expression . this can be achieved through mirna mimicry or plasmid / viral vector - encoded mirna replacement . mirna mimics are small chemically altered double - stranded rna molecules that imitate endogenous mirnas . plasmid / viral vectors encoding mirnas are encouraging strategies to replace mirna in vivo , with good transduction efficiency and minimal toxicity [ 85 , 86 ] . micrornas and therapeutics . since the obesity - induced inflammation is considered to build the common soil for the pathogenesis of ms , some potential anti - inflammatory therapies have been investigated . several drugs in current clinical practice show the anti - inflammatory properties : the peroxisome proliferator - activated receptor ( ppar ) family and nonacetylated salicylates . other strategies have been explored by targeting the cytokines and chemokines or their receptors ( e.g. , the anti - tnf or anti - ccr2 ( chemokine ( c c motif ) receptor 2 ) therapies ) . although there have been some encouraging results , it is likely that the benefits of these approaches are limited [ 87 , 88 ] . therefore , the development of mirna - based therapeutics has raised interest in this field . to date , the greatest efforts have been made in exploring the potential application of mirnas therapeutics in cancer and liver disorders . gain or loss of function of individual mirnas have been reported in almost every solid and hematological cancer , with therapeutic suppressing effect in tumor cell proliferation , progression of tumors , and the metastatic process . the liver specific mir-122 has been proved to regulate cholesterol biosynthesis and hepatitis c virus replication . silencing of mir-122 by intraperitoneal administration of high affinity lna anti - mir-122 has resulted in dose - dependent lowering of plasma cholesterol in mice and nonhuman primates ( monkeys ) . this was achieved without significant adverse reactions or hepatic toxicity [ 89 , 90 ] . likewise , induction or reduction of mirna expression that is known to be under / upper - expressed and involved in at inflammation using viral or liposomal delivery of tissue - specific mirnas to affected at yet , the application of mirnas therapeutics in at in vivo is poorly investigated because fat cells are insusceptible to selective transfer of exogenous nucleotide due to the containing lipid droplets of adipocytes . encouragingly , the discovery that mirnas could be secreted by adipocytes and accepted by neighboring adipocytes or macrophages in the form of adipocyte - derived microvesicles [ 75 , 76 ] raises the hope that it could be possible to import the targeting mirnas into at in vivo by the manner of microvesicles . this manner could construct the microvesicles containing specific mirnas and inject the constructed microvesicles into white at . although significant advances have been made in mirna - based therapy , various challenges remain to be overcome before clinical use . first , one individual mirna may have multiple potential targets , which may coordinate or antagonize each other 's functions . in addition , mirna - target interactions depend not only on the sequence of the target site but also on the cellular context , in which the interactions occur . this complexity explains the difficulties in predicting the spectrum of side effects and toxicity profiles , which may be associated with mirna - based therapeutics . high levels of exogenous mirna can compete with the endogenous mirna biogenesis processing , leading to cell toxicity [ 91 , 92 ] . another critical issue is the site - specific , safe , effective , and repeated delivery of mirna in vivo . the systemic intravenous delivery of viral or liposomal - mediated mirna oligonucleotides have been well achieved in cancer and liver disease . however , targeting certain mirnas in other specific tissues is poorly achieved due to the invasive nature of access for repeat delivery and lack of cell - specific up - take . restoration of tissue - specific mirna expression could be a more rational approach for managing local abnormalities with more efficiency and precision and with less systemic side effects [ 83 , 92 ] . evidence has provided that mirnas are largely dysregulated in white at in human obesity ; specific mirnas regulate the polarization of macrophages and the expression and secretion of adipokines in white at ; a subset of mirnas could be packaged into adipocyte - derived microvesicles and delivered into blood or neighboring cells , mediating intercellular communication . furthermore , with the recent discovery that mirnas are secreted into circulation in a stable manner and correlated closely with crucial metabolic parameters , the plasma mirna profiles could therefore be used as attractive biomarkers for the ms . finally , the establishment of mirnas - based techniques allows exploring further fascinating roles of mirnas and their applications in the management of ms . despite huge challenges lying in mirna - targeting therapeutics , especially in developing safe and effective delivery of targeting mirnas in specific tissues in vivo , the development of mirna - based therapeutics for ms is warranted because of the severe problem that metabolic disease poses to society .

obesity is associated closely with the metabolic syndrome ( ms ) . it is well known that obesity - induced chronic inflammation plays a fundamental role in the pathogenesis of ms . white adipose tissue ( at ) is the primary site for the initiation and exacerbation of obesity - associated inflammation . exploring the mechanisms of white at inflammation and resetting the immunological balance in white at could be crucial for the management of ms . several prominent molecular mechanisms have been proposed to mediate inflammation in white at , including hypoxia , endoplasmic reticulum stress , lipotoxicity , and metabolic endotoxemia . recently , a growing body of evidence supports the role of mirnas as a new important inflammatory mediator by regulating both the adaptive and innate immunity . this review will focus on the implication of mirnas in white at inflammation in obesity , and will also highlight the potential of mirnas as targets for therapeutic intervention in ms as well as the challenges lying in mirna - targeting therapeutics .

1. Introduction 2. Obesity, Inflammation, and Metabolic Syndrome 3. The Fundamental Role of White Adipose Tissue in Obesity-Induced Inflammation 4. Molecular Mechanisms Mediating White Adipose Tissue Inflammation 5. microRNAs as a New Mechanism Mediating White Adipose Tissue Inflammation 6. Therapeutic Potential of microRNAs in Metabolic Syndrome 7. Conclusion

the incidence of obesity defined as body mass index ( bmi ) 30 kg / m has increased drastically worldwide during recent decades . obesity is associated with a cluster of metabolic disorders , including increased risk of insulin resistance , type 2 diabetes ( t2 dm ) , hypertension , dyslipidemia , and cardiovascular disease ; these disorders and obesity per se constitute a serious threat , known as the metabolic syndrome ( ms ) . it is well known that chronic inflammation is a key feature of obesity ; this obesity - induced inflammation builds the common soil for the pathogenesis of ms . therefore , resetting the immunological balance in obesity is a crucial approach for the management of ms . white adipose tissue ( at ) has been regarded as the primary goal of pharmacological intervention since obesity - induced inflammation is mainly initiated and exacerbated in this organ [ 35 ] . although several prominent molecular mechanisms have been proposed to trigger inflammation in white at , including hypoxia , endoplasmic reticulum stress , lipotoxicity , and metabolic endotoxemia , these factors can not fully explain the origin of inflammation . on the other hand , current anti - inflammatory strategies are not sufficient enough in the treatment of ms . these facts suggest that there are lots of unknowns responsible for at inflammation in obesity . in recent years , a growing body of evidence has highlighted the modulating roles of mirnas in immune / inflammatory system [ 6 , 7 ] and their involvement in the obesity - related metabolic disorders including t2 dm and atherosclerosis [ 8 , 9 ] . as yet , whether these mirnas could be another mechanism for mediating at inflammation and whether these tiny molecules could serve as potent therapeutic anti - inflammatory targets for ms are still not fully settled . during the past decade , it became clear that inflammation is a key feature of obesity . however , the nature of obesity - induced inflammation , referred to as metainflammation ( metabolically triggered inflammation ) , is unique compared with other inflammatory paradigms ( e.g. first , the chronic nature of obesity produces a low - grade activation of the innate immune system that affects metabolic homeostasis over time . third , this chronic inflammation is composed of recurrent acute episodes of nutrition - related immune activation induced by nutrient availability ( fasting or high - fat meals ) . this fluctuation may be associated with the induction of pro- or anti - inflammatory mediators . the metabolic syndrome ( ms ) refers to the clustering of cardiovascular risk factors that include central obesity , hyperglycemia , dyslipidemia , and hypertension . growing evidence implicates obesity - induced inflammation as an important mechanism linking obesity to the ms in metabolically active organs . multiple inflammatory mediators abnormally secreted by at and the crosstalk between immune and metabolic cells can impair insulin signaling and induce oxidative stress and endothelial dysfunction , leading to systemic insulin resistance and cardiovascular disease . deregulated macrophage - myocyte and macrophage - hepatocyte signaling can impair insulin sensitivity as well [ 14 , 15 ] . hypothalamic inflammation , which is induced very rapidly by a high - fat diet may provoke hyperphagia and has been documented to impair insulin release from cells , peripheral insulin action , and potentiate hypertension [ 1719 ] . thus , chronic excess of nutrients , such as lipids and glucose may simultaneously trigger inflammatory responses , which further disrupt metabolic function , enhancing stress , and inflammation . therefore , breaking this vicious circle by resetting the immunological balance in obesity is a crucial approach for the management of ms . obesity is characterized by excessive expansion of white adipose tissue ( at ) , which has been thought to be the primary site for the initiation of obesity - associated inflammation . a number of bioactive peptides or proteins , collectively named adipokines , are produced and secreted by fat and/or nonfat cells of white at . they act in an autocrine / paracrine manner to regulate local at function and also act in an endocrine manner to influence the functions of distant tissues such as liver , skeletal muscle , and cardiovascular and central nervous systems [ 5 , 20 ] . in obesity , white at is remodeled dynamically by adipocyte hypertrophy ( increased size ) , hyperplasia ( increased number ) , immune cell infiltration , endothelial cell overactivation , and extracellular matrix overproduction [ 2124 ] . this remodeling may trigger metabolic and hypoxic stress , resulting in activation of multiple inflammatory signaling pathways , ultimately leading to dysregulation of numerous adipokines including proinflammatory cytokines , chemokines , growth factors , acute - phase proteins , and complement - like factors . such a deregulation is the main feature of at low - grade inflammation and contributes to the pathogenesis of ms . observations have proved that at is poorly oxygenated in the obese state in humans and rodents due to a reduction in adipose tissue blood flow [ 25 , 26 ] . this hypoxia could be a determinant mediator of obesity - induced inflammation in at by activating multiple signaling pathways such as hypoxia inducible factor-1 ( hif-1 ) and nuclear factor kappa b ( nf-b ) in adipocytes and macrophages , thereby altering the expression of many proinflammatory adipokines [ 2729 ] . the endoplasmic reticulum ( er ) is responsible for much of a cell 's protein synthesis and folding , but it has also a role in sensing cellular stress . obesity results in conditions that increase the demand on the er , such as hypoxia and excess of cytokines , lipids , or glucose . subsequently , the er stress induces a complex response known as the unfolded protein response ( upr ) , which alters a cell 's transcriptional and translational programs to cope with these stressful conditions and resolve the protein - folding defect . er stress and the upr lead to obesity - induced inflammation and metabolic abnormalities by several distinct mechanisms , including the activation of jnk - ap1 ( jun n - terminal kinase - activator protein 1 ) and ikk ( ib kinase)-nfb pathways , the induction of the acute - phase response , and the production of reactive oxygen species ( ros ) [ 34 , 35 ] . obesity is characterized by a positive energy balance , and the classical response to this nutrient oversupply is at hypertrophy . this limited storage capacity , coupled with the overstimulation of hormone - sensitive lipase , leads to massive increase in free fatty acid ( ffa ) release and accumulation ectopically . tlrs are a family of pattern - recognition receptors that play a critical role in the innate immune system by activating proinflammatory signaling pathways in response to microbial pathogens . tlr4-deficient mice and c3h / hej mice ( which have a loss - of - function mutation in tlr4 ) are partially protected from fat - induced inflammation and insulin resistance in their visceral at [ 41 , 42 ] . recently , three underlying mechanisms have been proposed , including the changes in gut microbiota environment , the increased availability of chylomicrons , and the permeability of the gut epithelium [ 4749 ] . as a group , mirnas may directly regulate expression of over 30% of human and mouse genes and more than 60% of human protein - coding genes have been under selective pressure to maintain pairing to mirnas . specific mirnas have been implicated in adipocyte differentiation and mature adipocyte function , including lipolysis , glucose - uptake , and insulin sensitivity [ 8 , 10 ] . on the other hand , mirnas have been defined as important immunomodulators by regulating the differentiation , induction , and function of immune cells and the expression of multiple cytokines in the immune system . these findings could shed light on the possible links between mirnas and adipose tissue immunity / inflammation . several individual mirnas have also been reported to be involved in the b - cell biology . mirnas have been shown to be key regulators of the t - cell lineage induction pathways . the best evidence for mirnas playing a role in specific developmental stages of t - cell differentiation is from mir-181 , which reduces the number of t - cells in haematopoietic overexpression systems and also increases the sensitivity of t - cell receptor signaling . in addition , mirnas also play pivotal roles in the induction , function , and maintenance of the regulatory t - cell ( treg ) lineage . several mirnas ( including mir-155 , mir-146a , mir-21 , and mir-9 ) have been consistently found to be rapidly induced by innate immune activation ( e.g. target prediction analyses indicate that up to a half of innate immune genes could be under the direct regulation of mirnas . major targets include transcription factors , cofactors , and chromatin modifiers whereas upstream factors , such as ligands and receptors ( cytokines and chemokines ) were , in general , poor or nontargets . recent studies indicate indeed that mirnas could indirectly regulate cytokine genes via au - rich elements ( are ) located in the mrna 3-utr by targeting are - binding proteins . are - mediated mrna degradation is regulated by a number of trans - acting factors , called the are - binding proteins ( which include tristetraprolin ( ttp ) , au rich binding factor 1 ( auf1 ) , and members of the hu protein r ( hur ) family ) . repression of several are components by mirnas may alter the levels of inflammatory cytokines as well as of other immune genes [ 60 , 62 ] . obesity - induced inflammation is associated with dysregulated expression of mirnas in white adipose tissue . a number of mirnas have been found dysregulated in white at during obesity and closely associated with obesity - related metabolic disorders . they showed significant correlations between the expression of selected mirnas and both at morphology and key metabolic parameters , including visceral fat area , hba1c , fasting plasma glucose , and circulating leptin , adiponectin , and il-6 . a very recent study defined a set of eleven adipocyte - specific mirnas downregulated in obese subjects as all of them were concomitantly altered in both the white at and the isolated adipocytes . with obesity and inflammation being so intrinsically associated , the dysregulated expression of mirnas in inflammatory adipocytes highly suggests the particular importance of mirnas in obesity - induced inflammation . indeed , a few individual mirnas have been reported as playing a crucial role in the inflammatory state of at so far ( figure 1 ) . expression of mir-221 and mir-222 has been positively correlated to tnf- and negatively correlated to apn expression in white at of mice . demonstrated that mir-223 played a crucial role in modulating macrophage polarization in a pattern that protects mice from diet - induced at inflammation and systemic insulin resistance . macrophages by targeting pknox1 in vitro , while mir-223 deficient mice fed a high - fat diet exhibited increased at inflammation characterized by enhanced proinflammatory activation of macrophages . the group of brichard has identified several mirnas , which were regulated by adiponectin ( a promising anti - inflammatory adipokine ) in white at in vivo . the mir883b-5p , which was upregulated by adiponectin and downregulated in human obesity repressed the lps - binding protein ( lbp ) and tlr4 signaling , acting therefore as a major mediator of the anti - inflammatory action of apn . very recently , arner et al . they found that a set of nine mirnas significantly reduced the secretion of chemokine ( c - c motif ) ligand 2 ( ccl2 ) which is an initiator of at inflammation by attracting the migration of inflammatory cells into the tissue . among these mirnas , only mir-126 was predicted by in silico analysis and confirmed by luciferase reporter assay in 3t3-l1 cells to bind directly to the 3-utr of ccl2 , whereas mir-193b affected indirectly the ccl2 production through downregulating the transcription factors ( tfs ) of ccl2 ( relb , stat6 , and ets1 ) . thus , mirnas may mediate at inflammation by regulating either the activation of macrophages or the production of adipokines . mirnas may act directly on the target inflammatory adipokines or indirectly by first regulating the intermediate machinery components like the tfs , which , in turn , control the expression of adipokines . in addition to the innate immunity , recent studies have disclosed the importance of the adaptive immunity in at inflammation . the subsets of the lymphocyte lineage , including cd8 + and cd4 + t - cells , tregs , natural killer t - cells ( nkt ) , and b - cells have been revealed to infiltrate into at which may orchestrate at inflammation . yet , the mechanisms underlying the infiltration of lymphocytes into at remain largely unknown ( figure 1 ) . whether mirnas could play roles in modulating the infiltration or activation of the lymphocytes in at may arouse research interest and requires to be investigated in the future . more recently , adipocytes have been reported to secrete mirnas in the form of adipocyte - derived microvesicles ( adms ) , which may regulate the function of distant or neighboring cells . one study found that microvesicles released from cultured 3t3-l1 adipocytes harbored 143 mirnas , most of them being adipocyte - specific and reflecting the abundance of their expression levels in the donor cells . furthermore , another group discovered that mirnas - microvesicle complexes derived from the larger primary rat adipocytes were transferred into and expressed in the smaller adipocytes and were involved in the transcription of multiple genes for lipid synthesis and cell growth . hypertrophied adipocytes are the main sources of at inflammation , the mirna - mediated interactions ( adipocytes - macrophages and larger adipocytes - smaller adipocytes ) may be novel mechanisms for mirnas contributing to at inflammation ( figure 1 ) . the facts that mirnas are largely dysregulated in obesity and that specific mirnas regulate obesity - associated inflammation suggests the potential of these molecules as targets for therapeutic intervention in obesity and obesity - related metabolic disorders . new studies have revealed that circulating mirnas can reside in microvesicles ( exosomes , microparticles , and apoptotic bodies ) and in protein / lipoprotein complexes ( high density lipoprotein ( hdl ) and argonaute 2 ) ; these formations make the circulating mirnas protected and resistant to rnase activity and degradation . therefore , the levels of mirnas in serum are remarkable , stable , reproducible , and tissue - specific among individuals . multiple reports have noted the potential use of blood mirnas as biomarkers for the obesity , cardiovascular diseases , atherosclerosis , and t2 dm . the mirna expression in blood and omental fat from obese patients correlated significantly with bmi , fasting blood glucose , and glycated hemoglobin . circulating mirnas have also been proposed as highly sensitive biomarkers for t2 dm . in the bruneck cohort of 822 subjects , decreased serum levels of mir-126 in t2 dm emerged as a significant predictor of t2 dm and correlated negatively with increasing glucose intolerance . mirnas can be deactivated and silenced by anti - mirna oligonucleotides ( amos ) , mirna sponges , and mirna masking . the most widely employed types of amos are 2-o - methyl amo , 2-o - methoxyethyl amos , and locked nucleic acids ( lnas ) . given that mirnas have been observed to function often in clusters in pathological processes , the this technique requires a vector encoding large quantities of transcripts ( anti - mirnas ) which display numerous and tandem binding sites for a cluster of mirnas of interest . the mirna masking is an alternative mirna - knockdown strategy to the amo approach , with the advantage of targeting mirnas in a gene - specific manner . unlike amo , which binds to the endogenous mirna directly , a mirna mask binds to the mirna 's binding site located in the 3-utr of its target mrna , thereby avoiding off - target effects . since the obesity - induced inflammation is considered to build the common soil for the pathogenesis of ms , some potential anti - inflammatory therapies have been investigated . other strategies have been explored by targeting the cytokines and chemokines or their receptors ( e.g. although there have been some encouraging results , it is likely that the benefits of these approaches are limited [ 87 , 88 ] . to date , the greatest efforts have been made in exploring the potential application of mirnas therapeutics in cancer and liver disorders . gain or loss of function of individual mirnas have been reported in almost every solid and hematological cancer , with therapeutic suppressing effect in tumor cell proliferation , progression of tumors , and the metastatic process . likewise , induction or reduction of mirna expression that is known to be under / upper - expressed and involved in at inflammation using viral or liposomal delivery of tissue - specific mirnas to affected at yet , the application of mirnas therapeutics in at in vivo is poorly investigated because fat cells are insusceptible to selective transfer of exogenous nucleotide due to the containing lipid droplets of adipocytes . encouragingly , the discovery that mirnas could be secreted by adipocytes and accepted by neighboring adipocytes or macrophages in the form of adipocyte - derived microvesicles [ 75 , 76 ] raises the hope that it could be possible to import the targeting mirnas into at in vivo by the manner of microvesicles . this manner could construct the microvesicles containing specific mirnas and inject the constructed microvesicles into white at . although significant advances have been made in mirna - based therapy , various challenges remain to be overcome before clinical use . in addition , mirna - target interactions depend not only on the sequence of the target site but also on the cellular context , in which the interactions occur . this complexity explains the difficulties in predicting the spectrum of side effects and toxicity profiles , which may be associated with mirna - based therapeutics . high levels of exogenous mirna can compete with the endogenous mirna biogenesis processing , leading to cell toxicity [ 91 , 92 ] . another critical issue is the site - specific , safe , effective , and repeated delivery of mirna in vivo . the systemic intravenous delivery of viral or liposomal - mediated mirna oligonucleotides have been well achieved in cancer and liver disease . however , targeting certain mirnas in other specific tissues is poorly achieved due to the invasive nature of access for repeat delivery and lack of cell - specific up - take . restoration of tissue - specific mirna expression could be a more rational approach for managing local abnormalities with more efficiency and precision and with less systemic side effects [ 83 , 92 ] . evidence has provided that mirnas are largely dysregulated in white at in human obesity ; specific mirnas regulate the polarization of macrophages and the expression and secretion of adipokines in white at ; a subset of mirnas could be packaged into adipocyte - derived microvesicles and delivered into blood or neighboring cells , mediating intercellular communication . furthermore , with the recent discovery that mirnas are secreted into circulation in a stable manner and correlated closely with crucial metabolic parameters , the plasma mirna profiles could therefore be used as attractive biomarkers for the ms . finally , the establishment of mirnas - based techniques allows exploring further fascinating roles of mirnas and their applications in the management of ms . despite huge challenges lying in mirna - targeting therapeutics , especially in developing safe and effective delivery of targeting mirnas in specific tissues in vivo , the development of mirna - based therapeutics for ms is warranted because of the severe problem that metabolic disease poses to society .

severe cardiovascular disease ( cvd ) is a condition that constitutes the highest mortality in the united states ( us ) . however the risk of perioperative death is real , and it is most acute for those with heart failure or advanced age . as such , surgeons routinely inform patients of the possibility of death due to surgery - related complications . the life and death stakes in these circumstance makes an existential challenge inevitable , and survival from such experiences can pave a way to spiritual growth . the present study explored experiencing divine love , as a form of personal growth or spiritual transformation , in a long - term followup of patients who survived cardiac surgery and participated in a two - wave preoperative study . for decades behavioral health research has focused primarily on pathology and negative affect traits ( e.g. , depression , pain ) as indices of recovery from life - threatening conditions . this symptom reduction refers to the concept of subjective well - being ( swb , the hedonic tradition ) , in contrast to that of psychological well - being ( pwb , the eudaimonic tradition ) in light of positive psychology that focus on the positive side of life even in adversity . the latter especially involves personal growth after trauma , such as worldview alteration ( e.g. , meaning in life , enhanced relations ) . thus , experiencing divine love after surviving cardiac surgery can be viewed as an affective form of pwb - related spiritual growth . as a potential protector for health and mental health , love is generally defined as positive affect plus absence of social isolation [ 4 , 5 ] . in contrast to the love shared through relationships between human beings , divine love refers to loving god or love received from god , the absolute or higher power [ 4 , 68 ] . post has written extensively on this topic in relation to historical concepts in mainstream religions ( e.g. , agape , universal love , altruistic love , and unlimited love ) . sociologist sorokin had in - depth elaboration on religious love in light of spiritual growth . evidence has linked positive affect with adaptive coping in response to crises [ 10 , 11 ] , which was predictive of cardiovascular recovery following anxiety - producing situations . in line with the positive - affect dimension , levin [ 13 , 14 ] associated religious love with better subjective health and lower distress in a sample of 205 outpatients . no study , however , has examined divine love as a long - term pwb outcome in cardiac surgery patients . exploring this form of growth is important in the face of such an existential challenge , given that , in general , positive psychology has endorsed a positive dimension in life . more specifically , ai and colleagues argue that the heart is a mystified meaning - laden organ in human history and is symbolically related to the sense of love in many cultural legacies , including western religions , in particular . 's review of near death experiences in patients revived from cardiac arrest suggested spiritual transformation of most survivors . despite a different mechanism , cardiac surgery often requires cardioplegia ( temporarily stopping of the heart ) , during which time the patient is kept alive using a heart - lung bypass machine . accordingly , the experience of cardiac surgery and its attendant cardioplegia may be an occasion for spiritual transform similar to that described by survivors of cardiac arrest . our first research question concerned which of religious factors measured preoperatively would stand out as stronger predictors of divine love . we assessed divine love with a religious love subscale by levin and kaplan which indicates experiencing god 's love and conviction of the infinite nature of such love . to be more inclusive , we instructed our respondents to replace the term god ' with the deity / deities ' or a higher power ' pertaining to your spiritual or religious beliefs . however , we did acknowledge that the concept of divine love may be more predominant in those religious traditions that conceive god personally ( e.g. , christianity , judaism , or islam ) when compared to traditions with impersonal ( or even secular ) concepts of deity or higher power . love , with hope and faith , is an abiding christian characteristic in the new testament ( i corinthians 13 : 13 ) , given the central role of love in christianity , experiencing divine love would likely be especially important for our mainly christian sample ( 86% ) . in particular , we hypothesized that preoperative religious coping would predict experiencing divine love , above and beyond existing predictors ( e.g. , demographics , health and mental health - related risk , and protective factors ) . out of multifaceted faith factors , one behavioral measures is particularly worth noting : frankl asserted that meaningful actions were essential to people confronted with severe threats . reaching out to god ( or higher powers ) for help is seen as a form of meaningful action in the face of a life - changing event . positive spiritual coping involves positive faith - based approaches ( e.g. , turning toward god and searching for significance ; p. 46 ) whereas negative spiritual coping involves faith - based struggles and doubt ( e.g. , questioning about or turning anger toward god ) . a meta - analytic review linked positive spiritual coping with positive outcomes and negative coping with negative outcomes . in an earlier study of our sample , we found similar associations between the two styles of religious coping and short - term postoperative adjustment to cardiac surgery ( e.g. , those with positive religious coping had less negative affect one month postoperatively , and those with negative religious coping had more negative affect ) . however , only positive spiritual coping mediated the indirect effect of general religious involvement on negative affect . accordingly , we hypothesized that religious / spiritual coping measured before the operation would predict the postoperative experience of divine love , and that positive and negative coping styles would influence the experience of divine love in opposite directions . furthermore , according to a 17th century rabbi , prayer is god 's gift of love [ 26 , 27 ] . based on data demonstrating that lower levels of negative affect were associated with using prayer for coping , we also hypothesized that preoperative use of prayer for coping would predict postoperative perception of divine love . our second research question addressed the possible mechanism for our hypothesized effect of religious / spiritual coping . according to baron and kenny , if the effect of predictors on outcomes vanishes to zero after controlling for the correlated mediator , then the latter factors explain the indirect effect of the former predictors . spiritual connectedness has been taken as a component in various concepts of spirituality [ 7 , 30 ] or as the key of its definition . according to pargament [ 31 , page 34 ] , deep connection with the sacred is at the core of understanding faith - based life [ 31 , page 34 ] . in this light , we expected that cardiac surgery patients who used religious / spiritual coping preoperatively would be more likely to perceive spiritual connectedness postoperatively , and this connectedness might , in turn , influence the affective spiritual growth at the followup . to assess the possible mediating influence of latent spiritual connectedness , ai et al . [ 19 , 32 ] developed a scale , termed perceived spiritual support , to measure the perception of multifaceted support from a deep connection with a higher power or a spiritual relationship in a faith [ 19 , 32 ] . in samples of nonmedical subjects , perceived spiritual support was shown to mediate the effect of faith and prayer coping on such positive outcomes as optimism and posttraumatic growth ( ptg ) [ 19 , 32 ] . accordingly , we hypothesized that the postoperatively perceived spiritual support would mediate or explain the hypothesized effect of preoperative religious / spiritual coping on divine love . the study was conducted at the heart center of the university of michigan health systems ( umhs , ann arbor ) . the section of cardiac surgery , umhs , is a specialty center for valve surgery in heart failure management with many severe cases referred from other hospital settings . the patient pool thus contains more advanced heart conditions , especially congestive heart failure ( chf ) , than would be found in typical surgery units . participants were a convenience sample of patients who completed a mailed survey 30 months after surviving nonurgent and nontransplant cardiac surgery at the university of michigan 's medical center ( ummc ) . cardiac data were obtained from the society of thoracic surgeons ' ( sts ) national database . for the previous short - term , three - wave survey , cardiac surgeons delivered the information package about this study [ 26 , 27 ] . the first wave of interviews was conducted by trained interviewers and assessed demographics , religious affiliation , religiousness , medical comorbidities , and mental health symptoms ( depression ) two weeks before surgery . the second wave via telephone assessed psychosocial protectors ( optimism , hope , and social support ) and religious coping styles approximately 48 hours before surgery . the current study reports results from the additional contact at 30 months postoperatively with the 335 patients who survived surgery and also responded to this request for long - term followup . attrition at this long - term followup was mostly due to mortality , outdated contact information , or patient refusal to participate . a computerized double - entry system was used for data entry to ensure high quality . eligibility criteria for the initial sample were as follows ( a ) aged 35 years or older ; ( b ) scheduled for admission for nonemergency , nontransplant cardiac surgery ( e.g. , coronary artery bypass grafting ( cabg ) , aneurysm repair , and valve repair / replacement ) , requiring cardiopulmonary bypass ; ( c ) able to speak and understand english ; ( d ) cognitively and physically capable of providing informed consent . the details of the initial sample , recruited for the previous these survey , including percentages of religious involvement , were reported elsewhere . for demographics , the majority were male ( 60% ) , white ( 92% ) , and married with spouse present ( 76% ) . average age was 62 years ( sd = 11.7 , range , 3585 ) . among patients who were asked a question concerning whether or not they had their affairs in order one - month postoperatively respondents who indicated yes to having their affairs in order were given the opportunity to specify the type of arrangements . among the 355 participants who provided open - ended answers , the majority ( 65% ) indicated that they had a legal document such as a will , a trust , and/or living will . but , only a small number noted that they had provided instructions to family members ( 5% ) or specified funeral arrangements ( 3% ) . a few respondents ( 7% ) specified spiritual arrangements , such as asked for certain bible messages to be read at funeral , peace with myself and god , or blessed prior to surgery . for this subsequent analysis , left ventricular ejection fraction ( lvef ) was used to measure preoperative cardiac function . lvefs were derived from preoperative catheterization and angiography , indicating the percentage of blood emptied from the ventricle at the end of cardiac contraction . in this sample , new york heart association ( nyha ) classification ranks the extent of heart failure from i ( no limitation of ordinary activity ) to iv ( severe symptoms even at rest , mostly bedbound ) . for this sample , the highest level for each patient was recorded with the following distribution : level i = 39.3% , ii = 34.7% , iii = 23.7% , iv = 1.5% . demographics included age ( years ) , gender ( male , female ) , race ( white , other ) , and marital status ( married with spouse present or living with significant other , and other ) . preoperative depression was assessed with the 20-item center for epidemiologic studies depression scale ( ces - d ) with individual items scored on a 4-level likert scale ( m = 11.84 , sd = 9.10 , = .88 ) . perceived social support was assessed with the 12-item multidimensional scale of perceived social support ( mspss ) , measuring perceived support from family , friends , and significant others , with individual items scored on a 4-level scale ( m = 61.54 , sd = 8.59 , = .89 ) . patients were asked about the extent to which they agree with each statement in the prior month on a 5-level scale ( preoperative : m = 22.19 , sd = 4.46 , = .73 ) . hope was assessed with the 12-item hope scale , which asks participants to report how they felt about each statement in the prior month on a 5-level scale ( preoperative : m = 31.06 , sd = 4.31 , = .81 ) . religiousness was assessed with the 11-item religiosity scale which measures : public religiosity ( m = 11.28 , sd = 4.60 , a = .87 ) , private religiosity ( m = 10.06 , sd = 3.86 , = .78 ) , and subjective religiosity ( m = 5.98 , sd = 1.71 , = .88 ) . patients were asked the extent to which they agreed with each of the following statements : ( a ) prayer is important in my life ; ( b ) prayer does not help me to cope with difficulties and stress in my life ( reverse - scored ) ; and ( c ) i will use private prayer to cope with difficulties and stress associated with my cardiac surgery . all questions were scored on a 4-level scale ( m = 9.97 , sd = 2.44 , = .89 ) . religious / spiritual coping styles was assessed with the 14-item brief religious / spiritual coping scale ( r - cope ) , with seven items each for positive and negative styles . patients responded to these items in terms of how they generally coped with stressors rather than with respect to the operation or heart disease . thus , religious coping was assessed as a dispositional or coping style variable rather than a situation - specific construct . examples of positive coping include forgiveness , seeking spiritual support , collaborative religious coping , spiritual connection , and benevolent religious reappraisal ( e.g. , asked forgiveness for my sins , m = 11.69 , sd = 6.45 , = .93 ) . examples of negative coping include spiritual discontent , punishing god reappraisals , interpersonal religious discontent , and demonic reappraisal ( e.g. , questioned the power of god , m = 1.12 , sd = 2.30 , = .83 ) . perceived spiritual support was assessed with the 12-item spiritual support scale ( sss ) , assessing spiritual relationships in diverse beliefs ( e.g. , i have been inspired by my religious or spiritual faith in the face of distress ) . to be more inclusive respondents stated on a 4-level scale concerning each statement following cardiac surgery ( m = 41.57 , sd = 14.25 , = .98 ) . divine love was assessed with the 4-item subscale of the sorokin multidimensional inventory of love , assessing religious love ( e.g. , respondents stated on a 5-level scale concerning each statement at the long - term followup ( m = 16.24 , sd = 4.18 , = .97 ) . to test our hypotheses , we conducted serial hierarchical regression analyses following preplanned steps . first , bivariate correlation analyses were conducted using spss-18 to determine univariate associations between variables of major interests ( including controls ) and the outcome ( divine love ) . we then estimated the direct effect of religious / spiritual coping on divine love , controlling for other relevant predictors ( e.g. , demographics , baseline cardiac health , negative affect , optimistic expectations , and social support ) . finally , perceived spiritual support as the mediator was entered at the final step . predictors were entered into the equation following the predetermined five steps : ( 1 ) demographics , ( 2 ) baseline cardiac health , ( 3 ) self - reported medical comorbidities , psychological symptoms ( depression or anxiety ) , and protectors ( optimism , hope , and social support ) , ( 4 ) major religious factors ( three - factor religiousness , use of prayer for coping , sense of reverence , and religious / spiritual coping ) , and ( 5 ) a mediator at followup ( perceived spiritual support ) . collinearity statistics were inspected , using a conservative value of variance inflation factors ( vif ) of 4 . the zero - order correlations of predictors with the outcome show that divine love at the long - term followup was significantly correlated positively with female gender ( p < .001 ) and negatively with lvef ( p < .05 ) . none of the other factors had significant correlations ( e.g. , demographics , health and mental health related risk , or protective factors ) . of the variables of major interest , not surprisingly , all faith factors except negative spiritual coping were correlated with divine love ( ps.<.001 ) . results from hierarchical regression analyses predicting perceived love at followup are presented in table 1 . the step 1 model involving demographics accounted for only 5.8% of the variance but was statistically significant . only gender was a significant predictor of divine love ( p < .01 ) , and female patients were more likely to perceive love than their male counterparts . step 2 included lvef and nyha classification . the effect of gender found in step 1 persisted , and lvef was inversely associated with divine love ( p < .05 ) . this indicates that the sickest patients with lower lvef perceived greater divine love than healthier patients with higher lvef . in step 3 , the influence of gender and lvef on divine love persisted after expanding the model to include preoperative risk and protective factors . no new variables ( i.e. , self - reported medical comorbidities , preoperative depression , optimism , hope , and social support ) were found to be related to divine love . although the model explained an additional 2.1% of the variance , this change was not statistically significant . the previously described influence of lvef remained significant , but the gender effect size was completely eliminated . among all the new variables , only the positive spiritual coping style was associated with divine love ( p < .05 ) . the three factors of religiousness ( private , subjective , and public ) , negative spiritual coping , reverence , and using prayer for coping were each unrelated to divine love . patients reporting positive spiritual coping styles perceived greater divine love at followup . in the fifth and final step , perceived spiritual support at followup was entered in the equation to determine if it mediated any of the previous predictors . perceived spiritual support was positively associated with divine love , indicating that those with higher levels of spiritual support also reported higher levels of divine love . perceived spiritual support completely eliminated the previously observed influences of both positive spiritual coping and lvef . thus , the effects of positive spiritual coping on divine love appear to be mediated by perceived spiritual support . the model was significant , accounting for 62.2% of the variance ( f(21 , n = 200 ) = 12.32 , p < the present study examines how preoperative faith factors predict the postoperative perception of divine love at long - term followup for patients surviving life - altering cardiac surgery . consistent with our first hypothesis , our data demonstrate that preoperative positive religious / spiritual coping does predict greater levels of experiencing divine love at 30 months after surgery . while an earlier report on this followup found that prayer coping predicted less negative affect ( depression , anxiety ) , this analysis did not associate this coping strategy with greater positive affect ( divine love ) . despite the absence of effects from other faith factors , our findings add to the existing data regarding the effect of positive religious coping on a new positive outcome , divine love [ 4 , 6 , 7 , 13 , 14 ] among cardiac - surgery patients who have faced existential issues . in fact , this study may be the first of its kind to use divine love as an indicator of affective spiritual growth or transformation for survival experiences in aging - related severe diseases . clinically , what remains to be tested is if experiencing divine love is associated with positive physical recovery over time , as demonstrated in other cross - sectional studies [ 4 , 13 , 15 ] . unlike the associated pattern between two coping styles and negative affect , as shown in short - term recovery [ 26 , 27 ] , the present study did not support the expected opposite influence of negative religious / spiritual coping on experiencing less divine love . this difference might suggest that , as a positive spiritual affect , divine love is not merely the inverse of negative affect . unlike the concept of people 's love described in positive psychology [ 3 , 15 ] , that of divine love , as explicitly noted in the measure , highlights its unconditional or unlimited nature [ 9 , 14 ] . divine love is based on an underpinning spiritual outlook in a faith of the divinity , be it god or other higher powers , which transcends time , space , and human conditions , including the threats of human crises and mortality . in other words , its presence is not conditioned on factors in the physical dimension of life , which often affect or test the quality or quantity of interpersonal love ( e.g. , presence or fatality of persons involved , health status or resources in the loving context ) . the conviction of divine love thus can be uniquely reassuring to and existentially meaningful for middle - life and older patients after their survival of cardiac surgery . supporting our second hypothesis , postoperatively perceived spiritual support appears to mediate the role of positive religious / spiritual coping on experiencing divine love at followup . cardiac surgery patients who report positive coping strategies seem to perceive greater levels of spiritual support and greater levels of divine love , and perceived spiritual support seems to mediate the effect of positive religious / spiritual coping on perceived divine love . the finding thus replicate the mediating role of spiritual support in cross - sectional and prospective data from nonmedical settings , such as natural disasters ( e.g. , hurricane katrina ; a.l.a . , unpublished data , 2010 ) or political conflicts ( e.g. , 9/11 terrorist attacks ) . a sense of deep connectedness refers to the profound relationship with the divine or transcendent that bestow meanings to life . as can be seen as one indicator of this latent spiritual connection , the perceived spiritual support scale essentially measures its resourceful nature , referring to the moment of assessment [ 19 , 32 ] . in general , all these studies have lent further support for the deep connection with the sacred as the key to understanding the core religious life [ 31 , page 34 ] . the present study , however , shows that perceived spiritual support may particularly reflect cardiac patients ' attribution of their survival as the divine 's supportive response to their spiritual coping practice , and , as such , it may mediate the effects of that meaningful action on health - related psychological outcomes . interestingly , the mediating role of perceived spiritual support here is similar to that of social support in both cardiac patients and nonmedial subjects in crisis . because perceived spiritual support is conceptually different from divine love , it also deserves more quantitative and qualitative investigation in order to better understand its role in the decision making for managing life - changing medical events such as open heart surgery ( a.l.a . , unpublished data , 2010 ) [ 19 , 42 ] . finally , our analysis found that the positive effect of female gender on perceived divine love vanished when faith factors were added into the equation . this is not entirely surprising because substantial research has repeatedly demonstrated that women score higher than men on most measures of religiousness . therefore , the initially observed gender effect on divine love is likely to be mediated by women 's higher levels of religious and spiritual involvement than men . the disappearance of the initial and unexpected impact of low lvef on experiencing divine love is perhaps more interesting ; this vanishing effect did not occur after entry of other faith factors but present at the presence of perceived spiritual support . indeed , park et al . found that many scales of faith - related measures , even well validated , were correlated ; still , they exerted differential influences on psychological outcomes of cardiac patients . our previous report has found no correlation between faith factors and this cardiac index ; an initial association of lvef with divine love is thus spurious . as noted , this study has its limitations , including a convenience sample , racial and religious homogeneity , and attrition of the sample at followup that adds selective bias to healthier and relatively younger survivors . we did not measure divine love preoperatively , nor perceived spiritual support , because both scales had not been created when the study was initiated . given the central role of love in monotheist religions , divine love , positive spiritual coping , and perceived spiritual support could share this component in assessing faith - related actions , perceptions , and affective experience , despite distinct conceptual frameworks . the same may be true for other measures of religious involvement ( e.g. , attending service was , in part , for experiencing the divine ) . clearly , these factors were correlated in this sample and so were other faith measures in park et al . still , the present study contributes to aging and cardiac health literature by examining complexly related concepts important to the spirituality or existential meaning in life of patients surviving cardiac surgery . although the primary outcome of cardiac surgery is properly focused on cardiac physiology , the recent emphasis on patient - centered or whole person indeed , the data discussed here suggest that spiritual growth may be an outcome of cardiac surgery relevant to many patients . as such , health care providers may wish to investigate ways to support patients ' spiritual growth in the postoperative period , and this will require better understanding of the pre- and postoperative traits and coping behaviors associated with spiritual growth . further empirical exploration of divine love in medical contexts may be of major interest for both patients of advanced illnesses and care providers . given the meaning - laden implications in these concepts , divine love and perceived spiritual support deserve further exploration in research on aging , perhaps especially in the setting of research on pwb after life - changing medical events .

we examined experiencing divine love as an indicator of affective spiritual growth in a prospective cohort of 200 patients surviving cardiac surgery . these patients previously completed two - wave preoperative interviews when standardized cardiac surgery data were also collected . the information included left ventricular ejection fraction , new york heart association classification , baseline health ( physical and mental ) , optimism , hope , religiousness , prayer coping , religious / spiritual coping , and demographics . we then measured divine love at 900 days postoperatively . hierarchical linear regression indicated the direct effect of positive religious coping on experiences of divine love , controlling for other key variables . postoperatively perceived spiritual support was entered at the final step as an explanatory factor , which appeared to mediate the coping effect . none of the other faith factors predicted divine love . further research regarding divine love and spiritual support may eventually guide clinical attempts to support patients ' spiritual growth as an independently relevant outcome of cardiac surgery .

1. Introduction 2. Method 3. Results 4. Discussion

however the risk of perioperative death is real , and it is most acute for those with heart failure or advanced age . as such , surgeons routinely inform patients of the possibility of death due to surgery - related complications . the life and death stakes in these circumstance makes an existential challenge inevitable , and survival from such experiences can pave a way to spiritual growth . the present study explored experiencing divine love , as a form of personal growth or spiritual transformation , in a long - term followup of patients who survived cardiac surgery and participated in a two - wave preoperative study . this symptom reduction refers to the concept of subjective well - being ( swb , the hedonic tradition ) , in contrast to that of psychological well - being ( pwb , the eudaimonic tradition ) in light of positive psychology that focus on the positive side of life even in adversity . thus , experiencing divine love after surviving cardiac surgery can be viewed as an affective form of pwb - related spiritual growth . in contrast to the love shared through relationships between human beings , divine love refers to loving god or love received from god , the absolute or higher power [ 4 , 68 ] . , agape , universal love , altruistic love , and unlimited love ) . sociologist sorokin had in - depth elaboration on religious love in light of spiritual growth . in line with the positive - affect dimension , levin [ 13 , 14 ] associated religious love with better subjective health and lower distress in a sample of 205 outpatients . no study , however , has examined divine love as a long - term pwb outcome in cardiac surgery patients . despite a different mechanism , cardiac surgery often requires cardioplegia ( temporarily stopping of the heart ) , during which time the patient is kept alive using a heart - lung bypass machine . accordingly , the experience of cardiac surgery and its attendant cardioplegia may be an occasion for spiritual transform similar to that described by survivors of cardiac arrest . our first research question concerned which of religious factors measured preoperatively would stand out as stronger predictors of divine love . we assessed divine love with a religious love subscale by levin and kaplan which indicates experiencing god 's love and conviction of the infinite nature of such love . however , we did acknowledge that the concept of divine love may be more predominant in those religious traditions that conceive god personally ( e.g. love , with hope and faith , is an abiding christian characteristic in the new testament ( i corinthians 13 : 13 ) , given the central role of love in christianity , experiencing divine love would likely be especially important for our mainly christian sample ( 86% ) . in particular , we hypothesized that preoperative religious coping would predict experiencing divine love , above and beyond existing predictors ( e.g. , demographics , health and mental health - related risk , and protective factors ) . out of multifaceted faith factors , one behavioral measures is particularly worth noting : frankl asserted that meaningful actions were essential to people confronted with severe threats . positive spiritual coping involves positive faith - based approaches ( e.g. , turning toward god and searching for significance ; p. 46 ) whereas negative spiritual coping involves faith - based struggles and doubt ( e.g. a meta - analytic review linked positive spiritual coping with positive outcomes and negative coping with negative outcomes . in an earlier study of our sample , we found similar associations between the two styles of religious coping and short - term postoperative adjustment to cardiac surgery ( e.g. , those with positive religious coping had less negative affect one month postoperatively , and those with negative religious coping had more negative affect ) . however , only positive spiritual coping mediated the indirect effect of general religious involvement on negative affect . accordingly , we hypothesized that religious / spiritual coping measured before the operation would predict the postoperative experience of divine love , and that positive and negative coping styles would influence the experience of divine love in opposite directions . based on data demonstrating that lower levels of negative affect were associated with using prayer for coping , we also hypothesized that preoperative use of prayer for coping would predict postoperative perception of divine love . our second research question addressed the possible mechanism for our hypothesized effect of religious / spiritual coping . according to baron and kenny , if the effect of predictors on outcomes vanishes to zero after controlling for the correlated mediator , then the latter factors explain the indirect effect of the former predictors . according to pargament [ 31 , page 34 ] , deep connection with the sacred is at the core of understanding faith - based life [ 31 , page 34 ] . in this light , we expected that cardiac surgery patients who used religious / spiritual coping preoperatively would be more likely to perceive spiritual connectedness postoperatively , and this connectedness might , in turn , influence the affective spiritual growth at the followup . [ 19 , 32 ] developed a scale , termed perceived spiritual support , to measure the perception of multifaceted support from a deep connection with a higher power or a spiritual relationship in a faith [ 19 , 32 ] . in samples of nonmedical subjects , perceived spiritual support was shown to mediate the effect of faith and prayer coping on such positive outcomes as optimism and posttraumatic growth ( ptg ) [ 19 , 32 ] . accordingly , we hypothesized that the postoperatively perceived spiritual support would mediate or explain the hypothesized effect of preoperative religious / spiritual coping on divine love . the study was conducted at the heart center of the university of michigan health systems ( umhs , ann arbor ) . the section of cardiac surgery , umhs , is a specialty center for valve surgery in heart failure management with many severe cases referred from other hospital settings . the patient pool thus contains more advanced heart conditions , especially congestive heart failure ( chf ) , than would be found in typical surgery units . participants were a convenience sample of patients who completed a mailed survey 30 months after surviving nonurgent and nontransplant cardiac surgery at the university of michigan 's medical center ( ummc ) . cardiac data were obtained from the society of thoracic surgeons ' ( sts ) national database . for the previous short - term , three - wave survey , cardiac surgeons delivered the information package about this study [ 26 , 27 ] . the first wave of interviews was conducted by trained interviewers and assessed demographics , religious affiliation , religiousness , medical comorbidities , and mental health symptoms ( depression ) two weeks before surgery . the second wave via telephone assessed psychosocial protectors ( optimism , hope , and social support ) and religious coping styles approximately 48 hours before surgery . eligibility criteria for the initial sample were as follows ( a ) aged 35 years or older ; ( b ) scheduled for admission for nonemergency , nontransplant cardiac surgery ( e.g. , coronary artery bypass grafting ( cabg ) , aneurysm repair , and valve repair / replacement ) , requiring cardiopulmonary bypass ; ( c ) able to speak and understand english ; ( d ) cognitively and physically capable of providing informed consent . the details of the initial sample , recruited for the previous these survey , including percentages of religious involvement , were reported elsewhere . for demographics , the majority were male ( 60% ) , white ( 92% ) , and married with spouse present ( 76% ) . for this subsequent analysis , left ventricular ejection fraction ( lvef ) was used to measure preoperative cardiac function . lvefs were derived from preoperative catheterization and angiography , indicating the percentage of blood emptied from the ventricle at the end of cardiac contraction . in this sample , new york heart association ( nyha ) classification ranks the extent of heart failure from i ( no limitation of ordinary activity ) to iv ( severe symptoms even at rest , mostly bedbound ) . demographics included age ( years ) , gender ( male , female ) , race ( white , other ) , and marital status ( married with spouse present or living with significant other , and other ) . perceived social support was assessed with the 12-item multidimensional scale of perceived social support ( mspss ) , measuring perceived support from family , friends , and significant others , with individual items scored on a 4-level scale ( m = 61.54 , sd = 8.59 , = .89 ) . hope was assessed with the 12-item hope scale , which asks participants to report how they felt about each statement in the prior month on a 5-level scale ( preoperative : m = 31.06 , sd = 4.31 , = .81 ) . religiousness was assessed with the 11-item religiosity scale which measures : public religiosity ( m = 11.28 , sd = 4.60 , a = .87 ) , private religiosity ( m = 10.06 , sd = 3.86 , = .78 ) , and subjective religiosity ( m = 5.98 , sd = 1.71 , = .88 ) . patients were asked the extent to which they agreed with each of the following statements : ( a ) prayer is important in my life ; ( b ) prayer does not help me to cope with difficulties and stress in my life ( reverse - scored ) ; and ( c ) i will use private prayer to cope with difficulties and stress associated with my cardiac surgery . religious / spiritual coping styles was assessed with the 14-item brief religious / spiritual coping scale ( r - cope ) , with seven items each for positive and negative styles . thus , religious coping was assessed as a dispositional or coping style variable rather than a situation - specific construct . examples of positive coping include forgiveness , seeking spiritual support , collaborative religious coping , spiritual connection , and benevolent religious reappraisal ( e.g. examples of negative coping include spiritual discontent , punishing god reappraisals , interpersonal religious discontent , and demonic reappraisal ( e.g. perceived spiritual support was assessed with the 12-item spiritual support scale ( sss ) , assessing spiritual relationships in diverse beliefs ( e.g. to be more inclusive respondents stated on a 4-level scale concerning each statement following cardiac surgery ( m = 41.57 , sd = 14.25 , = .98 ) . divine love was assessed with the 4-item subscale of the sorokin multidimensional inventory of love , assessing religious love ( e.g. , respondents stated on a 5-level scale concerning each statement at the long - term followup ( m = 16.24 , sd = 4.18 , = .97 ) . first , bivariate correlation analyses were conducted using spss-18 to determine univariate associations between variables of major interests ( including controls ) and the outcome ( divine love ) . we then estimated the direct effect of religious / spiritual coping on divine love , controlling for other relevant predictors ( e.g. , demographics , baseline cardiac health , negative affect , optimistic expectations , and social support ) . finally , perceived spiritual support as the mediator was entered at the final step . predictors were entered into the equation following the predetermined five steps : ( 1 ) demographics , ( 2 ) baseline cardiac health , ( 3 ) self - reported medical comorbidities , psychological symptoms ( depression or anxiety ) , and protectors ( optimism , hope , and social support ) , ( 4 ) major religious factors ( three - factor religiousness , use of prayer for coping , sense of reverence , and religious / spiritual coping ) , and ( 5 ) a mediator at followup ( perceived spiritual support ) . the zero - order correlations of predictors with the outcome show that divine love at the long - term followup was significantly correlated positively with female gender ( p < .001 ) and negatively with lvef ( p < .05 ) . none of the other factors had significant correlations ( e.g. , demographics , health and mental health related risk , or protective factors ) . of the variables of major interest , not surprisingly , all faith factors except negative spiritual coping were correlated with divine love ( ps.<.001 ) . results from hierarchical regression analyses predicting perceived love at followup are presented in table 1 . the step 1 model involving demographics accounted for only 5.8% of the variance but was statistically significant . only gender was a significant predictor of divine love ( p < .01 ) , and female patients were more likely to perceive love than their male counterparts . the effect of gender found in step 1 persisted , and lvef was inversely associated with divine love ( p < .05 ) . this indicates that the sickest patients with lower lvef perceived greater divine love than healthier patients with higher lvef . in step 3 , the influence of gender and lvef on divine love persisted after expanding the model to include preoperative risk and protective factors . , self - reported medical comorbidities , preoperative depression , optimism , hope , and social support ) were found to be related to divine love . although the model explained an additional 2.1% of the variance , this change was not statistically significant . among all the new variables , only the positive spiritual coping style was associated with divine love ( p < .05 ) . the three factors of religiousness ( private , subjective , and public ) , negative spiritual coping , reverence , and using prayer for coping were each unrelated to divine love . patients reporting positive spiritual coping styles perceived greater divine love at followup . in the fifth and final step , perceived spiritual support at followup was entered in the equation to determine if it mediated any of the previous predictors . perceived spiritual support was positively associated with divine love , indicating that those with higher levels of spiritual support also reported higher levels of divine love . perceived spiritual support completely eliminated the previously observed influences of both positive spiritual coping and lvef . thus , the effects of positive spiritual coping on divine love appear to be mediated by perceived spiritual support . the model was significant , accounting for 62.2% of the variance ( f(21 , n = 200 ) = 12.32 , p < the present study examines how preoperative faith factors predict the postoperative perception of divine love at long - term followup for patients surviving life - altering cardiac surgery . consistent with our first hypothesis , our data demonstrate that preoperative positive religious / spiritual coping does predict greater levels of experiencing divine love at 30 months after surgery . while an earlier report on this followup found that prayer coping predicted less negative affect ( depression , anxiety ) , this analysis did not associate this coping strategy with greater positive affect ( divine love ) . despite the absence of effects from other faith factors , our findings add to the existing data regarding the effect of positive religious coping on a new positive outcome , divine love [ 4 , 6 , 7 , 13 , 14 ] among cardiac - surgery patients who have faced existential issues . in fact , this study may be the first of its kind to use divine love as an indicator of affective spiritual growth or transformation for survival experiences in aging - related severe diseases . clinically , what remains to be tested is if experiencing divine love is associated with positive physical recovery over time , as demonstrated in other cross - sectional studies [ 4 , 13 , 15 ] . unlike the associated pattern between two coping styles and negative affect , as shown in short - term recovery [ 26 , 27 ] , the present study did not support the expected opposite influence of negative religious / spiritual coping on experiencing less divine love . this difference might suggest that , as a positive spiritual affect , divine love is not merely the inverse of negative affect . unlike the concept of people 's love described in positive psychology [ 3 , 15 ] , that of divine love , as explicitly noted in the measure , highlights its unconditional or unlimited nature [ 9 , 14 ] . divine love is based on an underpinning spiritual outlook in a faith of the divinity , be it god or other higher powers , which transcends time , space , and human conditions , including the threats of human crises and mortality . in other words , its presence is not conditioned on factors in the physical dimension of life , which often affect or test the quality or quantity of interpersonal love ( e.g. the conviction of divine love thus can be uniquely reassuring to and existentially meaningful for middle - life and older patients after their survival of cardiac surgery . supporting our second hypothesis , postoperatively perceived spiritual support appears to mediate the role of positive religious / spiritual coping on experiencing divine love at followup . cardiac surgery patients who report positive coping strategies seem to perceive greater levels of spiritual support and greater levels of divine love , and perceived spiritual support seems to mediate the effect of positive religious / spiritual coping on perceived divine love . the finding thus replicate the mediating role of spiritual support in cross - sectional and prospective data from nonmedical settings , such as natural disasters ( e.g. as can be seen as one indicator of this latent spiritual connection , the perceived spiritual support scale essentially measures its resourceful nature , referring to the moment of assessment [ 19 , 32 ] . the present study , however , shows that perceived spiritual support may particularly reflect cardiac patients ' attribution of their survival as the divine 's supportive response to their spiritual coping practice , and , as such , it may mediate the effects of that meaningful action on health - related psychological outcomes . interestingly , the mediating role of perceived spiritual support here is similar to that of social support in both cardiac patients and nonmedial subjects in crisis . because perceived spiritual support is conceptually different from divine love , it also deserves more quantitative and qualitative investigation in order to better understand its role in the decision making for managing life - changing medical events such as open heart surgery ( a.l.a . finally , our analysis found that the positive effect of female gender on perceived divine love vanished when faith factors were added into the equation . therefore , the initially observed gender effect on divine love is likely to be mediated by women 's higher levels of religious and spiritual involvement than men . the disappearance of the initial and unexpected impact of low lvef on experiencing divine love is perhaps more interesting ; this vanishing effect did not occur after entry of other faith factors but present at the presence of perceived spiritual support . our previous report has found no correlation between faith factors and this cardiac index ; an initial association of lvef with divine love is thus spurious . as noted , this study has its limitations , including a convenience sample , racial and religious homogeneity , and attrition of the sample at followup that adds selective bias to healthier and relatively younger survivors . we did not measure divine love preoperatively , nor perceived spiritual support , because both scales had not been created when the study was initiated . given the central role of love in monotheist religions , divine love , positive spiritual coping , and perceived spiritual support could share this component in assessing faith - related actions , perceptions , and affective experience , despite distinct conceptual frameworks . clearly , these factors were correlated in this sample and so were other faith measures in park et al . still , the present study contributes to aging and cardiac health literature by examining complexly related concepts important to the spirituality or existential meaning in life of patients surviving cardiac surgery . although the primary outcome of cardiac surgery is properly focused on cardiac physiology , the recent emphasis on patient - centered or whole person indeed , the data discussed here suggest that spiritual growth may be an outcome of cardiac surgery relevant to many patients . as such , health care providers may wish to investigate ways to support patients ' spiritual growth in the postoperative period , and this will require better understanding of the pre- and postoperative traits and coping behaviors associated with spiritual growth . further empirical exploration of divine love in medical contexts may be of major interest for both patients of advanced illnesses and care providers . given the meaning - laden implications in these concepts , divine love and perceived spiritual support deserve further exploration in research on aging , perhaps especially in the setting of research on pwb after life - changing medical events .

mesa is a large , multicenter , prospective cohort study that has been described previously.7 participants in mesa completed as many as 5 clinic visits and 14 follow - up phone calls . timing of clinic visits was as follows : exam 1 in 2000 to 2002 , exam 2 in 2002 to 2004 , exam 3 in 2004 to 2006 , exam 4 in 2005 to 2007 , and exam 5 in 2010 to 2012 . this study was approved by the institutional review boards at each center and at the university of washington , and all subjects gave written informed consent . all participants were scanned by noncontrast cardiac computed tomography ( ct ) during exam 1 using methods that have been described previously.6,8 by design , about half of the participants were scanned again during exam 2 ( n = 2914 ) , and the other half were scanned during exam 3 ( n = 2925 ) . the exam 4 selection strategy prioritized participants without exam 3 scans and included ct scans for 1349 participants . during exam 5 , a total of 3304 participants received ct scans , preferentially including participants with scans from exam 3 and/or exam 4 . scans that were obtained subsequent to coronary revascularization procedures that were performed after exam 1 were excluded from this analysis . participants were advised of their cac scores following each examination , and 76% of participants requested that these results be released to their physicians , along with an interpretation of the score as average or as below or above average for age and sex , without recommendations for management.9 scanner models varied across centers and time . during exams 1 to 3 , 3 centers used electron beam ct technology and 3 used multidetector ct technology.6 the latter technology was in use at all centers by exam 5 . phantom that was described previously.8 cac scores were calculated according to the methodology of agatston et al.1016 calcified lesions of at least 4 adjacent voxels above a threshold of 130 hounsfield units ( hu ) were identified ; below this , scans were assigned a zero value . volume of each calcified lesion was then multiplied by a coefficient based on the brightest voxel it contained ( coefficient of 1 for a maximum of 130 to 199 hu , 2 for 200 to 299 hu , 3 for 300 to 399 hu , 4 for a maximum > 400 hu ) . the agatston score was calculated as the sum of the within - plane scores across all calcified lesions . the risk factors of interest included age , sex , race / ethnicity , education , household income , neighborhood socioeconomic status index,17 systolic and diastolic blood pressure , body mass index ( bmi ) , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , triglycerides , total cholesterol , statin use , antihypertensive use , smoking status , pack - years of smoking , exposure to second - hand smoke ( yes / no ) , diabetes status , family history of cvd , fibrinogen , ( high sensitivity ) c - reactive protein , and creatinine . the questionnaires and physical examinations used in mesa were described previously.7 family history of cvd was ascertained by questionnaire during the second in - clinic follow - up examination , and family history of premature cvd was defined as myocardial infarction / heart attack , stroke / brain attack , or cardiovascular procedure ( coronary bypass or balloon angioplasty ) in a female primary relative ( parent , sibling , or child ) aged < 65 years or a male primary relative aged < 55 years . neighborhood socioeconomic status index was calculated for the census tract in which participants lived during exam 1.17 most health metrics and demographics were defined at exam 1 and considered to be static . potential risk factors that were assessed at later exams but that were also considered to be static included family history of premature cvd ( assessed at exam 2 ) and household income ( defined as average midpoint of self - reported income category across all available examinations , with a value of $ 125 000 used for participants indicating an income greater than $ 100 000 ) . as a sensitivity analysis , we examined the relationship between cac progression and smoking status as classified by the mesa lung substudy . the mesa lung substudy confirmed smoking status by urinary cotinine levels ( immulite 2000 nicotine metabolite assay ; diagnostic products corp ) . participants with cotinine levels > 500 ng / ml were classified as current smokers , regardless of self - report . never - smokers at exam 1 that reported being former smokers at later examinations were also reclassified as former smokers.18 we analyzed the association between cvd risk factors and cac progression using a mixed - effects model . risk factors for cac progression were analyzed in both minimally and more fully adjusted models ( described in detail in minimally adjusted models ) ; in both cases , cac was modeled continuously on the natural scale . the use of statins was considered as both static , defined as in use at exam 1 , and time varying , defined as in use at the time of the clinical examination concurrent with ct scan . to further investigate the association between statin use and cac progression , we created a categorical variable delineating participants who were using statins at exam 1 , those who had never used statins , and those who had started using statins during follow - up . we analyzed the association between each risk factor and cac progression in models that , in all cases , included age at exam 1 , sex , race / ethnicity , metropolitan area or study site , and scanner type . in addition , in evaluating the association between cac progression and cholesterol ( total , hdl - c , ldl - c , and triglycerides ) , we further adjusted for statin use at exam 1 . in evaluating the association between cac progression and systolic or diastolic blood pressure , we adjusted for antihypertensive medication use at exam 1 . in evaluating the association between cac progression and antihypertensive medication use , we adjusted for hypertensive diagnosis the associations between cac progression and both pack - years of cigarette smoking and second - hand smoke exposure were adjusted for smoking status ( never , former , current ) . our secondary approach considered a more fully adjusted model , chosen using backward selection . because backward selection algorithms are optimized for ordinary least squares regression , we used a simplified outcome individually calculated annual cac progression to select variables for a fully adjusted model . variables considered included age at exam 1 , sex , race / ethnicity , metropolitan area or study site , bmi , smoking status , total cholesterol , hdl - c , ldl - c , diabetes status , statin use at exam 1 , systolic and diastolic blood pressure , hypertension , education , income , neighborhood socioeconomic status index , and family history of cvd . we assessed the sensitivity of the results to the outcome distribution by excluding the participants with the highest cac progression , using cutoffs of 250 and 150 agatston units / year , and by restricting the analysis to participants with > 0 agatston units of cac at exam 1 . we evaluated the sensitivity of the association between cac progression and statin use by comparing statin use at exam 1 with time - varying statin use . the mixed - effects model that we used in this analysis jointly modeled the cross - sectional and longitudinal relationships between cvd risk factors and cac . the cross - sectional relationships between cvd risk factors and cac can produce biased results in a progression analysis that controls for measured baseline.19,20 any measurement error exacerbates this bias.19,20 adjusting for an estimated baseline allowed us to control for cross - sectional confounding without inducing bias . the mixed - effects model provides 2 additional benefits : ( 1 ) participants with a variable number of observations , or even a single observation , can be included in the analysis ; and ( 2 ) the assumption that data are missing completely at random is not required . consequently , selection bias is of less concern using this method compared with methods that require full follow - up for all participants or the missing completely at random assumption , such as generalized estimating equations or repeated - measures analysis of variance . the specific form of the model is as follows , with participants indexed by i and exams indexed by v : this model uses the following definitions : yiv indicates cac measurement for subject i at vth follow - up exam ; xi0 , time - invariant cross - sectional confounders and risk factors at exam 1 for participant i ; wiv , possibly time - varying longitudinal confounders and risk factors at exam v for participant i ; uiv , time - varying variables to adjust measurements at exam v for participant i ; tiv , time in years from exam 1 to the vth follow - up exam for participant i ; 0 , cac progression ( annual rate of change ) in average participants in the reference group ; 1 , coefficients for associations between confounders and risk factors and rate of cac progression ( this is the term of interest ) ; 0 , average cac measurement at exam 1 for participants in the reference group ; 1 , coefficients for associations between exam 1 cac measurements and risk factors or confounders ; 1 , coefficients for cross - sectional associations between time - varying variables and cac measurements at all exams ; ai , participant - specific random intercept ; bi , participant - specific random slope ; iv , error associated with yiv . the model is composed of 3 parts , separated above by square brackets : ( 1 ) the cross - sectional relationship between the amount of cac at exam 1 and values of covariates at exam 1 , ( 2 ) the longitudinal relationship to model rate of change , and ( 3 ) time - varying transient terms that adjust for variables relevant to specific measurements . the cross - sectional terms in the model are equivalent in interpretation to terms from a cross - sectional model of the outcome at exam 1 . these fixed effects ( 1 ) , together with the random intercepts ( ai ) , model participant - specific intercepts . the longitudinal terms model an overall progression rate ( 0 ) , interpreted as the rate of change of cac for a participant with no additional risk factors ( ie , all terms wi = 0 ) , and incorporate terms that adjust that rate ( 1 ) according to the association between progression rate and risk factors . values of covariates included in the transient part ( uiv ) are time varying , but the transient adjustment does not modify the slope . the transient terms serve to adjust follow - up measurements that were measured under different conditions from the original . removing systematic differences due to different conditions allows the slope to be estimated based on the measurements as if they had been taken under the same conditions . this mixed - effects model leverages information from all participants , including those without follow - up measurements , to jointly model the amount of cac at exam 1 and cac progression over time , thereby adjusting progression for modeled baseline extent . each cvd risk factor was included as both a cross - sectional term and with an interaction with time . continuous covariates other than follow - up time were mean centered and scaled to a reasonable increment prior to analysis . p values were calculated using satterthwaite s approximation of denominator degrees of freedom for f statistics . all analyses were conducted using r 3.0.1 ( r foundation).21 as a sensitivity analysis and for completeness , a simplified model using annual change between exams 1 and 5 as the outcome was fit to the subset of participants with measurements available at exam 5 . these results were compared with those derived from fitting the above mixed - effects model to the same subset of participants . mesa is a large , multicenter , prospective cohort study that has been described previously.7 participants in mesa completed as many as 5 clinic visits and 14 follow - up phone calls . timing of clinic visits was as follows : exam 1 in 2000 to 2002 , exam 2 in 2002 to 2004 , exam 3 in 2004 to 2006 , exam 4 in 2005 to 2007 , and exam 5 in 2010 to 2012 . this study was approved by the institutional review boards at each center and at the university of washington , and all subjects gave written informed consent . all participants were scanned by noncontrast cardiac computed tomography ( ct ) during exam 1 using methods that have been described previously.6,8 by design , about half of the participants were scanned again during exam 2 ( n = 2914 ) , and the other half were scanned during exam 3 ( n = 2925 ) . the exam 4 selection strategy prioritized participants without exam 3 scans and included ct scans for 1349 participants . during exam 5 , a total of 3304 participants received ct scans , preferentially including participants with scans from exam 3 and/or exam 4 . scans that were obtained subsequent to coronary revascularization procedures that were performed after exam 1 were excluded from this analysis . participants were advised of their cac scores following each examination , and 76% of participants requested that these results be released to their physicians , along with an interpretation of the score as average or as below or above average for age and sex , without recommendations for management.9 scanner models varied across centers and time . during exams 1 to 3 , 3 centers used electron beam ct technology and 3 used multidetector ct technology.6 the latter technology was in use at all centers by exam 5 . phantom that was described previously.8 cac scores were calculated according to the methodology of agatston et al.1016 calcified lesions of at least 4 adjacent voxels above a threshold of 130 hounsfield units ( hu ) were identified ; below this , scans were assigned a zero value . volume of each calcified lesion was then multiplied by a coefficient based on the brightest voxel it contained ( coefficient of 1 for a maximum of 130 to 199 hu , 2 for 200 to 299 hu , 3 for 300 to 399 hu , 4 for a maximum > 400 hu ) . the agatston score was calculated as the sum of the within - plane scores across all calcified lesions . the risk factors of interest included age , sex , race / ethnicity , education , household income , neighborhood socioeconomic status index,17 systolic and diastolic blood pressure , body mass index ( bmi ) , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , triglycerides , total cholesterol , statin use , antihypertensive use , smoking status , pack - years of smoking , exposure to second - hand smoke ( yes / no ) , diabetes status , family history of cvd , fibrinogen , ( high sensitivity ) c - reactive protein , and creatinine . the questionnaires and physical examinations used in mesa were described previously.7 family history of cvd was ascertained by questionnaire during the second in - clinic follow - up examination , and family history of premature cvd was defined as myocardial infarction / heart attack , stroke / brain attack , or cardiovascular procedure ( coronary bypass or balloon angioplasty ) in a female primary relative ( parent , sibling , or child ) aged < 65 years or a male primary relative aged < 55 years . neighborhood socioeconomic status index was calculated for the census tract in which participants lived during exam 1.17 most health metrics and demographics were defined at exam 1 and considered to be static . potential risk factors that were assessed at later exams but that were also considered to be static included family history of premature cvd ( assessed at exam 2 ) and household income ( defined as average midpoint of self - reported income category across all available examinations , with a value of $ 125 000 used for participants indicating an income greater than $ 100 000 ) . as a sensitivity analysis , we examined the relationship between cac progression and smoking status as classified by the mesa lung substudy . the mesa lung substudy confirmed smoking status by urinary cotinine levels ( immulite 2000 nicotine metabolite assay ; diagnostic products corp ) . participants with cotinine levels > 500 ng / ml were classified as current smokers , regardless of self - report . never - smokers at exam 1 that reported being former smokers at later examinations were also reclassified as former smokers.18 we analyzed the association between cvd risk factors and cac progression using a mixed - effects model . risk factors for cac progression were analyzed in both minimally and more fully adjusted models ( described in detail in minimally adjusted models ) ; in both cases , cac was modeled continuously on the natural scale . the use of statins was considered as both static , defined as in use at exam 1 , and time varying , defined as in use at the time of the clinical examination concurrent with ct scan . to further investigate the association between statin use and cac progression , we created a categorical variable delineating participants who were using statins at exam 1 , those who had never used statins , and those who had started using statins during follow - up . we analyzed the association between each risk factor and cac progression in models that , in all cases , included age at exam 1 , sex , race / ethnicity , metropolitan area or study site , and scanner type . in addition , in evaluating the association between cac progression and cholesterol ( total , hdl - c , ldl - c , and triglycerides ) , we further adjusted for statin use at exam 1 . in evaluating the association between cac progression and systolic or diastolic blood pressure , we adjusted for antihypertensive medication use at exam 1 . in evaluating the association between cac progression and antihypertensive medication use , we adjusted for hypertensive diagnosis . the associations between cac progression and both pack - years of cigarette smoking and second - hand smoke exposure were adjusted for smoking status ( never , former , current ) . our secondary approach considered a more fully adjusted model , chosen using backward selection . because backward selection algorithms are optimized for ordinary least squares regression , we used a simplified outcome individually calculated annual cac progression to select variables for a fully adjusted model . variables considered included age at exam 1 , sex , race / ethnicity , metropolitan area or study site , bmi , smoking status , total cholesterol , hdl - c , ldl - c , diabetes status , statin use at exam 1 , systolic and diastolic blood pressure , hypertension , education , income , neighborhood socioeconomic status index , and family history of cvd . we assessed the sensitivity of the results to the outcome distribution by excluding the participants with the highest cac progression , using cutoffs of 250 and 150 agatston units / year , and by restricting the analysis to participants with > 0 agatston units of cac at exam 1 . we evaluated the sensitivity of the association between cac progression and statin use by comparing statin use at exam 1 with time - varying statin use . the mixed - effects model that we used in this analysis jointly modeled the cross - sectional and longitudinal relationships between cvd risk factors and cac . the cross - sectional relationships between cvd risk factors and cac can produce biased results in a progression analysis that controls for measured baseline.19,20 any measurement error exacerbates this bias.19,20 adjusting for an estimated baseline allowed us to control for cross - sectional confounding without inducing bias . the mixed - effects model provides 2 additional benefits : ( 1 ) participants with a variable number of observations , or even a single observation , can be included in the analysis ; and ( 2 ) the assumption that data are missing completely at random is not required . consequently , selection bias is of less concern using this method compared with methods that require full follow - up for all participants or the missing completely at random assumption , such as generalized estimating equations or repeated - measures analysis of variance . the specific form of the model is as follows , with participants indexed by i and exams indexed by v : this model uses the following definitions : yiv indicates cac measurement for subject i at vth follow - up exam ; xi0 , time - invariant cross - sectional confounders and risk factors at exam 1 for participant i ; wiv , possibly time - varying longitudinal confounders and risk factors at exam v for participant i ; uiv , time - varying variables to adjust measurements at exam v for participant i ; tiv , time in years from exam 1 to the vth follow - up exam for participant i ; 0 , cac progression ( annual rate of change ) in average participants in the reference group ; 1 , coefficients for associations between confounders and risk factors and rate of cac progression ( this is the term of interest ) ; 0 , average cac measurement at exam 1 for participants in the reference group ; 1 , coefficients for associations between exam 1 cac measurements and risk factors or confounders ; 1 , coefficients for cross - sectional associations between time - varying variables and cac measurements at all exams ; ai , participant - specific random intercept ; bi , participant - specific random slope ; iv , error associated with yiv . the model is composed of 3 parts , separated above by square brackets : ( 1 ) the cross - sectional relationship between the amount of cac at exam 1 and values of covariates at exam 1 , ( 2 ) the longitudinal relationship to model rate of change , and ( 3 ) time - varying transient terms that adjust for variables relevant to specific measurements . the cross - sectional terms in the model are equivalent in interpretation to terms from a cross - sectional model of the outcome at exam 1 . these fixed effects ( 1 ) , together with the random intercepts ( ai ) , model participant - specific intercepts . the longitudinal terms model an overall progression rate ( 0 ) , interpreted as the rate of change of cac for a participant with no additional risk factors ( ie , all terms wi = 0 ) , and incorporate terms that adjust that rate ( 1 ) according to the association between progression rate and risk factors . values of covariates included in the transient part ( uiv ) are time varying , but the transient adjustment does not modify the slope . the transient terms serve to adjust follow - up measurements that were measured under different conditions from the original . removing systematic differences due to different conditions allows the slope to be estimated based on the measurements as if they had been taken under the same conditions . this mixed - effects model leverages information from all participants , including those without follow - up measurements , to jointly model the amount of cac at exam 1 and cac progression over time , thereby adjusting progression for modeled baseline extent . each cvd risk factor was included as both a cross - sectional term and with an interaction with time . continuous covariates other than follow - up time were mean centered and scaled to a reasonable increment prior to analysis . p values were calculated using satterthwaite s approximation of denominator degrees of freedom for f statistics . all analyses were conducted using r 3.0.1 ( r foundation).21 as a sensitivity analysis and for completeness , a simplified model using annual change between exams 1 and 5 as the outcome was fit to the subset of participants with measurements available at exam 5 . these results were compared with those derived from fitting the above mixed - effects model to the same subset of participants . of the 6814 participants recruited to mesa , 744 had cac scores only at exam 1 , and 6066 had a score at both exam 1 and at least 1 follow - up exam . there were 166 participants that were scanned at least once after a coronary revascularization procedure and whose postprocedure measurements were excluded . the overall average cac progression was 23.9 agatston units / year among participants with at least 2 measurements ( range 365 to 834 , iqr 0.3 to 21.7 , with a median progression rate of 3.0 agatston units / year ) . at baseline , 50% of the cohort had no cac ; by exam 5 , this proportion had dropped to 31% . total number of participants included and excluded , distinguished by number of participant - specific observations used in the analysis . no participants were scanned during both exam 2 ( e2 ) and exam 3 ( e3 ) . scores after coronary revascularization procedures were excluded . characteristics of the study population are provided in table1 . participants with at least some follow - up were similar to the initial cohort in terms of race / ethnicity , sex ratio , bmi , cholesterol , baseline statin use , and smoking behavior . participants whose last follow - up was at exam 5 tended to be slightly younger and more educated and to have higher income , less prevalence of diabetes , and lower blood pressure compared with participants who were lost to follow - up or died . participants with longer follow - up also had noticeably less cac at exam 1 . selected participant characteristics at exam 1 data are shown mean ( sd ) or count ( percentage ) . bmi indicates body mass index ; cac , coronary artery calcium ; cvd , cardiovascular disease ; hdl - c , high - density lipoprotein cholesterol ; ldl - c , low - density lipoprotein cholesterol . cvd in a male parent , child , or sibling before age 55 years or in a female relative before age 65 years . reported during exam 2 . the primary results for this analysis are based on minimally adjusted models to show the most direct associations between the risk factors and cac progression . age at exam 1 and sex were both strongly associated with increased cac progression . among the racial / ethnic groups , black race was associated with decreased cac progression compared with white race , whereas cac progression among participants with hispanic ethnicity or chinese ancestry were not significantly different from cac progression among white participants in the minimally adjusted model . age , site , and scanner - adjusted progression rates for men and women in each racial ethnic group are provided in table3 . associations between risk factors and cac progression ( agatston units / year ) all risk factors were measured at exam 1 except as indicated . cac indicates coronary artery calcium ; cvd , cardiovascular disease ; ct , computed tomography ; hdl - c , high - density lipoprotein cholesterol ; ldl - c , low - density lipoprotein cholesterol ; ses , socioeconomic status . minimally adjusted models include age , sex , race / ethnicity , site , and ct scanner type along with the reported term . adjustment model determined by backward selection on participants slopes as outcome ; all terms reported in the table , plus site and ct scanner type were in the model . cvd in a male parent , child , or sibling before age 55 years or in a female relative before age 65 years . reported during exam 2 . annual progression rates ( agatston units / year ) for men and women in each racial / ethnic group rates are based on a mixed - effects model including interactions between race / ethnicity and sex and are adjusted for age , site , and scanner . time - varying statin use was less strongly associated with increased cac . to fully explore the relationship between cac progression and statin use , we also evaluated statin use categorically ( described in cac progression among participants who were prescribed statins over the course of follow - up was 5.2 agatston units / year faster than among participants who never took statins ( 95% ci 3.3 to 7.1 ) . among those who were taking statins at exam 1 , cac progression was even higher . both smoking status and pack - years of smoking were associated with increased cac progression ( table2 ) . overall , 401 participants reported never smoking at exam 1 but later reported being former smokers . using the mesa lung smoking status classification , this strengthened the association between smoking status and cac progression for former smokers compared with never smokers ( 5.3 agatston units / year [ 95% ci 2.5 to 8.1 ] ) and had a minimal impact for current smokers compared with never smokers ( 7.4 agatston units / year [ 95% ci 3.5 to 11.3 ] ) . second - hand smoke exposure was not associated with cac progression ( data not shown ) . as shown in table2 , we observed associations between cac progression and many other cvd risk factors and participant characteristics . these included family history of premature cvd , bmi , diabetes , hypertension , blood pressure , triglycerides , and hdl - c . income , education , or neighborhood socioeconomic status index were associated with cac progression . elevated c - reactive protein and fibrinogen were associated with increased cac progression , but creatinine was not . associations between cac progression and age at exam 1 , sex , bmi , hypertension , statin use , diabetes , and family history of premature cvd were still strong but slightly attenuated . the association between smoking status and cac progression was similar to the minimally adjusted result . we performed a large number of sensitivity analyses , which are shown in tables4 and 5 . our results were robust for changes such as participant exclusions and variations in the set of adjustment variables . we controlled for clinical center in all primary analyses because the study design stratified participant recruitment by race / ethnicity and study site ; however , site adjustment might not be necessary if the differences were captured by control for other risk factors . we found that site adjustment was not influential for most risk factors but affected associations between race / ethnicity and cac progression ( table4 ) . associations between race / ethnicity and cac progression : sensitivity to adjustment model values are differences in annual cac progression in agatston units / year . other risk factors investigated were not substantially ( < 20% change ) affected by the adjustment models specified . associations between risk factors and cac progression : sensitivity to participant exclusions values are differences in annual cac progression in agatston units / year . models included age , sex , race / ethnicity , site , and scanner type . cac indicates coronary artery calcium ; cvd , cardiovascular disease ; hdl - c , high - density lipoprotein cholesterol ; ldl - c , low - density lipoprotein cholesterol ; ses , socioeconomic status . cvd in a male parent , child , or sibling before age 55 years or in a female relative before age 65 years . other sensitivity analyses excluded participants with greater cac progression to better understand their influence on the results . , most relationships observed in the full data set persisted , but estimates were reduced by 25% among participants with cac progression < 250 agatston units / year ( table5 ) or by about 40% among participants with cac progression < 150 agatston units / year . excluding participants with cac below the scanner s threshold ( 0 agatston units ) at baseline , estimates were generally 20% to 50% more extreme . different relationships among the racial / ethnic groups were observed in this subset than in the cohort overall , and that may be related to a preferential exclusion of nonwhite participants . specifically , cac progression was not significantly different by race among participants with measureable cac at baseline . results from the analysis of annual change between exams 1 and 5 are presented in table6 . these results are highly consistent with results obtained from a mixed - effects model analysis of the same subset of participants that attended exams 1 and 5 , although many of the estimates are somewhat lower than those in the analysis of the whole cohort . comparison to analysis change between exams 1 and 5 results from the primary analysis are repeated in this table for easy comparison ( left ) alongside the results from multiple linear regression of risk factors on the annual change between exams 1 and 5 ( center ) . results from mixed - effects models restricted to the subset of participants with measurements at both exams 1 and 5 are also presented ( right ) . cvd indicates cardiovascular disease ; ct , computed tomography ; hdl - c , high - density lipoprotein cholesterol ; ldl - c , low - density lipoprotein cholesterol ; ses , socioeconomic status . annual change = ( exam 5exam 1)/(time in years ) . results are adjusted for the ct scanner model used at exams 1 and 5 and for the interaction between the 2 scanner types . cvd in a male parent , child , or sibling before age 55 years or in a female relative before age 65 years . reported during exam 2 . we found strong relationships between known risk factors for cvd and progression of cac over a 10-year period in a large multiethnic population . we adopted an innovative approach to statistical modeling to fully incorporate all data available from > 6000 subjects , regardless of their baseline cac levels and numbers of available scans . our findings were robust for a range of different data exclusions , refinements to the outcome , and modeling details . these results do not inform us about the prognoses for specific participants but rather provide a framework that can be used to describe and evaluate the importance of population - level characteristics , interventions , and potential risk factors for atherosclerosis . these advanced methods have expanded our understanding of the progression of subclinical atherosclerosis , as measured by coronary artery calcification . the associations that we observed between cac progression and risk factors were consistent with those of kronmal et al for bmi , family history of cvd , triglycerides , systolic blood pressure , and pack - years of smoking.6 also consistent with that study was an observed lack of association between cac progression and ldl - c , education , or creatinine . evidence from sensitivity analyses indicates that this discrepancy is attributable primarily to adjustment for site in our study . in both studies , race / ethnicity was more strongly related to cac progression in more fully adjusted models , suggesting confounding between race / ethnicity and risk factors such as smoking and bmi . this is further suggested by the lack of robustness in the associations observed between race / ethnicity and cac progression in our sensitivity analyses ( tables4 and 5 ) . the fact that race / ethnicity is more strongly associated with cac progression in some subsets of participants warrants further research . we observed higher magnitudes of association in cac progression for age , sex , cigarette smoking status , hdl - c , diabetes , antihypertensive medications , and statin use at exam 1 compared with kronmal et al.6 because atherosclerosis is a gradual process , a longer follow - up time presumably enables more accurate estimation of the progression rate . other prior studies with shorter follow - up observed associations between cac progression and white race,22 hypertension,22 diabetes,22 fibrinogen,23 hdl - c,24 and smoking.6,25 it is difficult to make meaningful comparisons among these studies due to differences in the study populations or transformations of the outcome ; however , we found that our results were consistent with these studies in direction of association . our findings regarding statin use in this cohort are mixed , likely reflecting the current practice of prescribing statins for those with multiple existing cvd risk factors and for those with evidence of atherosclerosis . others have also observed uneven impact of lipid - lowering medications on cac progression.3,24,2630 some prior studies have suggested that statin use does not affect cac progression,26,27,31,32 although these studies were limited by relatively small sample sizes and/or short follow - up times . in mesa , evidence from early examinations indicated that participants on statins had increased cac progression compared with those not on statins , presumably because they were already at higher risk.4,6 in our study , statin use at exam 1 was strongly associated with subsequent cac progression , supporting the explanation that the high - risk group was those who had already been prescribed statins in the years 20002002 . the association with time - varying statin use over the subsequent 10 years , when statin use became much more prevalent , was less strong . at least 1 study suggested that statin therapy could increase the density of calcification by removing the lipids from calcified plaques.33 this defatting process would thereby increase pixel brightness and could inflate agatston cac scores disproportionately relative to the amount of plaque present . this implies that statins could prevent the deposition of new cac while still increasing the agatston score . if this were true , we might expect to observe that those prescribed statins during the course of follow - up would have the highest cac progression because , presumably , much of this change in plaque architecture would have occurred prior to scanning among the participants that were already on statins at exam 1 . instead , we observed that cac progression was fastest among participants using statins at exam 1 . this would suggest that statins promote coronary atheroma calcification , even though they are known to regress plaque extent ; statins might help stabilize plaques by increasing the density of calcium . a recent evaluation of 3495 participants imaged with intravascular ultrasound in clinical trials indicated that high - intensity statin therapy was associated with increases in coronary atheroma calcification independent of the plaque - regressive effects.34 the attenuation of the association between statin use and cac progression in the fully adjusted model suggests some residual confounding . whatever mechanism is responsible for the observed association between cac progression and statin use would also induce a lack of association with ldl - c . because participants were advised of their cac scores , it raises the prospect that this information led to changes in risk factors and treatments , which in turn affects subsequent progression rates . in fact , prior investigators9 found that higher cac scores at baseline in mesa were associated with increased initiation of lipid - lowering agents . the use of these agents and other preventive therapies during the period of follow - up ( as at baseline ) is subject to confounding by indication35 because cac at baseline is associated with subsequent progression . this may make it difficult to interpret the use of these agents in the progression model and to interpret the measures that are strongly affected by these treatments such as total cholesterol and ldl - c . because our model adjusts fully , in a time - varying manner , for the use of lipid - lowering agents , the interpretation of factors not directly related to these therapies which are the focus of this paper should not be confounded . although participants with no follow - up contribute to our analysis in the cross - sectional exam 1 characterization , they do not directly contribute to the estimation of the progression rate . participants who had no follow - up , particularly those who died or became too sick to continue the study between exams 1 and 2 , may differ from those with follow - up measurements ; however , the overall retention rate was high , and the mixed - effects model approach allows the analysis to be adjusted for variables related to health status . consequently , we expected the impact of selection bias on these results to be minimal . cac data are well known to be highly skewed , which makes individual - level modeling difficult . the parameter that we investigated in this study , a mean summarizing a skewed distribution of progression , describes population - level atherosclerotic burden . it is necessarily true that the estimate of the coefficient relevant to this parameter is highly influenced by the relative number of participants with high progression rates and by the magnitude of those rates . studies that use this method of assessing subclinical atherosclerotic progression will need to examine the relative magnitudes of progression of traditional risk factors to create the appropriate context for results for novel risk factors . the fact that these results are consistent with those of kronmal et al despite the inclusion of a large number of participants with no cac at baseline lends credence to the idea that cac progression modeled as a single process is informative . this result is important because no concise summary could be generated from separately modeled time to progression and progression rate , as would be necessary for data that are unbalanced in time . we noted that results from the mixed - effects model were also consistent with the results obtained from a simple change analysis . because both methods should lead to valid conclusions there are 2 main drawbacks to the change analysis : ( 1 ) no time - varying information can be included , and ( 2 ) the analysis must be restricted to a more selected subset of participants than the mixed - effects model analysis . these 2 facts mean that simple adjustment is less accurate ( due to less control of scanner and other time - varying effects ) , and the results are less generalizable than those derived from the full mixed - effects model analysis . the major strengths of this analysis include the substantial follow - up time , the well - characterized prospective cohort , and our application of mixed - effects models to this question . baseline cac and cac progression rate are associated , but control for measured baseline can lead to bias.19,20 the linear mixed - effects model effectively controls for the baseline cac and permits an unbiased understanding of both cross - sectional and longitudinal relationships between risk factors and cac . it also allows us to appropriately account for repeated measures and variable follow - up times . including 3 or 4 measurements for more than half the cohort permits more accurate estimation of uncertainty compared with study designs that include only 2 measurements per participant . finally , by not using a logarithmically transformed cac score in our analysis , as others have used,28,30 we did not give extra weight to the lower cac scores , which convey less information about the quantity of interest and may be subject to more impact of transitions in scanner technology . we examined the associations among the lower calcium scores in a sensitivity analysis by calculating spatially weighted calcium scores36 for participants with an agatston score of 0 , but the results were essentially identical to those obtained using the agatston score alone . coronary artery calcification by ct scan is a useful and noninvasive measure of the extent of atherosclerosis . this study examined a comprehensive set of risk factors for cac progression over a long period of follow - up in a well - studied population that was selected to be free of clinical cvd at baseline . it relied on a statistical approach for progression analysis that is both less prone to bias and more flexible than other methods used previously to study atherosclerosis progression . the mixed - effects model method can also be applied to evaluating novel risk factors . we found that cac progression over 10 years was strongly associated with most cvd risk factors , confirming that risk factors for clinical coronary disease and the progression of cac are overlapping . this research was supported by contracts n01-hc-95159 , n01-hc-95160 , n01-hc-95161 , n01-hc-95162 , n01-hc-95163 , n01-hc-95164 , n01-hc-95165 , n01-hc-95166 , n01-hc-95167 , n01-hc-95168 and n01-hc-95169 , and grants r01 hl-077612 and hl-075476 from the national heart , lung , and blood institute , by grants ul1-rr-024156 and ul1-rr-025005 from the national center for research resources ( ncrr ) , and by grant k24es013195 from the national institute of environmental health sciences . the authors thank the other investigators , the staff , and the participants of the mesa study for their valuable contributions . the views expressed in this document are solely those of the authors and the epa does not endorse any products or commercial services mentioned in this publication .

backgroundcoronary artery calcium ( cac ) detected by noncontrast cardiac computed tomography scanning is a measure of coronary atherosclerosis burden . increasing cac levels have been strongly associated with increased coronary events . prior studies of cardiovascular disease risk factors and cac progression have been limited by short follow - up or restricted to patients with advanced disease.methods and resultswe examined cardiovascular disease risk factors and cac progression in a prospective multiethnic cohort study . cac was measured 1 to 4 times ( mean 2.5 scans ) over 10 years in 6810 adults without preexisting cardiovascular disease . mean cac progression was 23.9 agatston units / year . an innovative application of mixed - effects models investigated associations between cardiovascular disease risk factors and cac progression . this approach adjusted for time - varying factors , was flexible with respect to follow - up time and number of observations per participant , and allowed simultaneous control of factors associated with both baseline cac and cac progression . models included age , sex , study site , scanner type , and race / ethnicity . associations were observed between cac progression and age ( 14.2 agatston units / year per 10 years [ 95% ci 13.0 to 15.5 ] ) , male sex ( 17.8 agatston units / year [ 95% ci 15.3 to 20.3 ] ) , hypertension ( 13.8 agatston units / year [ 95% ci 11.2 to 16.5 ] ) , diabetes ( 31.3 agatston units / year [ 95% ci 27.4 to 35.3 ] ) , and other factors.conclusionscac progression analyzed over 10 years of follow - up , with a novel analytical approach , demonstrated strong relationships with risk factors for incident cardiovascular events . longitudinal cac progression analyzed in this framework can be used to evaluate novel cardiovascular risk factors .

Methods Study Population Measurement of CAC Measurement of Cardiovascular Risk Factors Analysis Minimally Adjusted Models Fully Adjusted Models Additional Sensitivity Analyses Mixed-effects Model Results Discussion Conclusions Sources of Funding Disclosures

mesa is a large , multicenter , prospective cohort study that has been described previously.7 participants in mesa completed as many as 5 clinic visits and 14 follow - up phone calls . all participants were scanned by noncontrast cardiac computed tomography ( ct ) during exam 1 using methods that have been described previously.6,8 by design , about half of the participants were scanned again during exam 2 ( n = 2914 ) , and the other half were scanned during exam 3 ( n = 2925 ) . the risk factors of interest included age , sex , race / ethnicity , education , household income , neighborhood socioeconomic status index,17 systolic and diastolic blood pressure , body mass index ( bmi ) , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , triglycerides , total cholesterol , statin use , antihypertensive use , smoking status , pack - years of smoking , exposure to second - hand smoke ( yes / no ) , diabetes status , family history of cvd , fibrinogen , ( high sensitivity ) c - reactive protein , and creatinine . the questionnaires and physical examinations used in mesa were described previously.7 family history of cvd was ascertained by questionnaire during the second in - clinic follow - up examination , and family history of premature cvd was defined as myocardial infarction / heart attack , stroke / brain attack , or cardiovascular procedure ( coronary bypass or balloon angioplasty ) in a female primary relative ( parent , sibling , or child ) aged < 65 years or a male primary relative aged < 55 years . never - smokers at exam 1 that reported being former smokers at later examinations were also reclassified as former smokers.18 we analyzed the association between cvd risk factors and cac progression using a mixed - effects model . risk factors for cac progression were analyzed in both minimally and more fully adjusted models ( described in detail in minimally adjusted models ) ; in both cases , cac was modeled continuously on the natural scale . to further investigate the association between statin use and cac progression , we created a categorical variable delineating participants who were using statins at exam 1 , those who had never used statins , and those who had started using statins during follow - up . we analyzed the association between each risk factor and cac progression in models that , in all cases , included age at exam 1 , sex , race / ethnicity , metropolitan area or study site , and scanner type . in addition , in evaluating the association between cac progression and cholesterol ( total , hdl - c , ldl - c , and triglycerides ) , we further adjusted for statin use at exam 1 . in evaluating the association between cac progression and antihypertensive medication use , we adjusted for hypertensive diagnosis the associations between cac progression and both pack - years of cigarette smoking and second - hand smoke exposure were adjusted for smoking status ( never , former , current ) . variables considered included age at exam 1 , sex , race / ethnicity , metropolitan area or study site , bmi , smoking status , total cholesterol , hdl - c , ldl - c , diabetes status , statin use at exam 1 , systolic and diastolic blood pressure , hypertension , education , income , neighborhood socioeconomic status index , and family history of cvd . we assessed the sensitivity of the results to the outcome distribution by excluding the participants with the highest cac progression , using cutoffs of 250 and 150 agatston units / year , and by restricting the analysis to participants with > 0 agatston units of cac at exam 1 . we evaluated the sensitivity of the association between cac progression and statin use by comparing statin use at exam 1 with time - varying statin use . the mixed - effects model that we used in this analysis jointly modeled the cross - sectional and longitudinal relationships between cvd risk factors and cac . the cross - sectional relationships between cvd risk factors and cac can produce biased results in a progression analysis that controls for measured baseline.19,20 any measurement error exacerbates this bias.19,20 adjusting for an estimated baseline allowed us to control for cross - sectional confounding without inducing bias . the mixed - effects model provides 2 additional benefits : ( 1 ) participants with a variable number of observations , or even a single observation , can be included in the analysis ; and ( 2 ) the assumption that data are missing completely at random is not required . the specific form of the model is as follows , with participants indexed by i and exams indexed by v : this model uses the following definitions : yiv indicates cac measurement for subject i at vth follow - up exam ; xi0 , time - invariant cross - sectional confounders and risk factors at exam 1 for participant i ; wiv , possibly time - varying longitudinal confounders and risk factors at exam v for participant i ; uiv , time - varying variables to adjust measurements at exam v for participant i ; tiv , time in years from exam 1 to the vth follow - up exam for participant i ; 0 , cac progression ( annual rate of change ) in average participants in the reference group ; 1 , coefficients for associations between confounders and risk factors and rate of cac progression ( this is the term of interest ) ; 0 , average cac measurement at exam 1 for participants in the reference group ; 1 , coefficients for associations between exam 1 cac measurements and risk factors or confounders ; 1 , coefficients for cross - sectional associations between time - varying variables and cac measurements at all exams ; ai , participant - specific random intercept ; bi , participant - specific random slope ; iv , error associated with yiv . this mixed - effects model leverages information from all participants , including those without follow - up measurements , to jointly model the amount of cac at exam 1 and cac progression over time , thereby adjusting progression for modeled baseline extent . mesa is a large , multicenter , prospective cohort study that has been described previously.7 participants in mesa completed as many as 5 clinic visits and 14 follow - up phone calls . all participants were scanned by noncontrast cardiac computed tomography ( ct ) during exam 1 using methods that have been described previously.6,8 by design , about half of the participants were scanned again during exam 2 ( n = 2914 ) , and the other half were scanned during exam 3 ( n = 2925 ) . the risk factors of interest included age , sex , race / ethnicity , education , household income , neighborhood socioeconomic status index,17 systolic and diastolic blood pressure , body mass index ( bmi ) , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , triglycerides , total cholesterol , statin use , antihypertensive use , smoking status , pack - years of smoking , exposure to second - hand smoke ( yes / no ) , diabetes status , family history of cvd , fibrinogen , ( high sensitivity ) c - reactive protein , and creatinine . the questionnaires and physical examinations used in mesa were described previously.7 family history of cvd was ascertained by questionnaire during the second in - clinic follow - up examination , and family history of premature cvd was defined as myocardial infarction / heart attack , stroke / brain attack , or cardiovascular procedure ( coronary bypass or balloon angioplasty ) in a female primary relative ( parent , sibling , or child ) aged < 65 years or a male primary relative aged < 55 years . never - smokers at exam 1 that reported being former smokers at later examinations were also reclassified as former smokers.18 we analyzed the association between cvd risk factors and cac progression using a mixed - effects model . risk factors for cac progression were analyzed in both minimally and more fully adjusted models ( described in detail in minimally adjusted models ) ; in both cases , cac was modeled continuously on the natural scale . to further investigate the association between statin use and cac progression , we created a categorical variable delineating participants who were using statins at exam 1 , those who had never used statins , and those who had started using statins during follow - up . we analyzed the association between each risk factor and cac progression in models that , in all cases , included age at exam 1 , sex , race / ethnicity , metropolitan area or study site , and scanner type . in addition , in evaluating the association between cac progression and cholesterol ( total , hdl - c , ldl - c , and triglycerides ) , we further adjusted for statin use at exam 1 . in evaluating the association between cac progression and systolic or diastolic blood pressure , we adjusted for antihypertensive medication use at exam 1 . in evaluating the association between cac progression and antihypertensive medication use , we adjusted for hypertensive diagnosis . the associations between cac progression and both pack - years of cigarette smoking and second - hand smoke exposure were adjusted for smoking status ( never , former , current ) . variables considered included age at exam 1 , sex , race / ethnicity , metropolitan area or study site , bmi , smoking status , total cholesterol , hdl - c , ldl - c , diabetes status , statin use at exam 1 , systolic and diastolic blood pressure , hypertension , education , income , neighborhood socioeconomic status index , and family history of cvd . we assessed the sensitivity of the results to the outcome distribution by excluding the participants with the highest cac progression , using cutoffs of 250 and 150 agatston units / year , and by restricting the analysis to participants with > 0 agatston units of cac at exam 1 . we evaluated the sensitivity of the association between cac progression and statin use by comparing statin use at exam 1 with time - varying statin use . the mixed - effects model that we used in this analysis jointly modeled the cross - sectional and longitudinal relationships between cvd risk factors and cac . the mixed - effects model provides 2 additional benefits : ( 1 ) participants with a variable number of observations , or even a single observation , can be included in the analysis ; and ( 2 ) the assumption that data are missing completely at random is not required . the specific form of the model is as follows , with participants indexed by i and exams indexed by v : this model uses the following definitions : yiv indicates cac measurement for subject i at vth follow - up exam ; xi0 , time - invariant cross - sectional confounders and risk factors at exam 1 for participant i ; wiv , possibly time - varying longitudinal confounders and risk factors at exam v for participant i ; uiv , time - varying variables to adjust measurements at exam v for participant i ; tiv , time in years from exam 1 to the vth follow - up exam for participant i ; 0 , cac progression ( annual rate of change ) in average participants in the reference group ; 1 , coefficients for associations between confounders and risk factors and rate of cac progression ( this is the term of interest ) ; 0 , average cac measurement at exam 1 for participants in the reference group ; 1 , coefficients for associations between exam 1 cac measurements and risk factors or confounders ; 1 , coefficients for cross - sectional associations between time - varying variables and cac measurements at all exams ; ai , participant - specific random intercept ; bi , participant - specific random slope ; iv , error associated with yiv . this mixed - effects model leverages information from all participants , including those without follow - up measurements , to jointly model the amount of cac at exam 1 and cac progression over time , thereby adjusting progression for modeled baseline extent . the overall average cac progression was 23.9 agatston units / year among participants with at least 2 measurements ( range 365 to 834 , iqr 0.3 to 21.7 , with a median progression rate of 3.0 agatston units / year ) . participants with at least some follow - up were similar to the initial cohort in terms of race / ethnicity , sex ratio , bmi , cholesterol , baseline statin use , and smoking behavior . participants whose last follow - up was at exam 5 tended to be slightly younger and more educated and to have higher income , less prevalence of diabetes , and lower blood pressure compared with participants who were lost to follow - up or died . the primary results for this analysis are based on minimally adjusted models to show the most direct associations between the risk factors and cac progression . age at exam 1 and sex were both strongly associated with increased cac progression . associations between risk factors and cac progression ( agatston units / year ) all risk factors were measured at exam 1 except as indicated . minimally adjusted models include age , sex , race / ethnicity , site , and ct scanner type along with the reported term . annual progression rates ( agatston units / year ) for men and women in each racial / ethnic group rates are based on a mixed - effects model including interactions between race / ethnicity and sex and are adjusted for age , site , and scanner . time - varying statin use was less strongly associated with increased cac . to fully explore the relationship between cac progression and statin use , we also evaluated statin use categorically ( described in cac progression among participants who were prescribed statins over the course of follow - up was 5.2 agatston units / year faster than among participants who never took statins ( 95% ci 3.3 to 7.1 ) . both smoking status and pack - years of smoking were associated with increased cac progression ( table2 ) . using the mesa lung smoking status classification , this strengthened the association between smoking status and cac progression for former smokers compared with never smokers ( 5.3 agatston units / year [ 95% ci 2.5 to 8.1 ] ) and had a minimal impact for current smokers compared with never smokers ( 7.4 agatston units / year [ 95% ci 3.5 to 11.3 ] ) . as shown in table2 , we observed associations between cac progression and many other cvd risk factors and participant characteristics . associations between cac progression and age at exam 1 , sex , bmi , hypertension , statin use , diabetes , and family history of premature cvd were still strong but slightly attenuated . we controlled for clinical center in all primary analyses because the study design stratified participant recruitment by race / ethnicity and study site ; however , site adjustment might not be necessary if the differences were captured by control for other risk factors . we found that site adjustment was not influential for most risk factors but affected associations between race / ethnicity and cac progression ( table4 ) . associations between race / ethnicity and cac progression : sensitivity to adjustment model values are differences in annual cac progression in agatston units / year . associations between risk factors and cac progression : sensitivity to participant exclusions values are differences in annual cac progression in agatston units / year . models included age , sex , race / ethnicity , site , and scanner type . , most relationships observed in the full data set persisted , but estimates were reduced by 25% among participants with cac progression < 250 agatston units / year ( table5 ) or by about 40% among participants with cac progression < 150 agatston units / year . we found strong relationships between known risk factors for cvd and progression of cac over a 10-year period in a large multiethnic population . these results do not inform us about the prognoses for specific participants but rather provide a framework that can be used to describe and evaluate the importance of population - level characteristics , interventions , and potential risk factors for atherosclerosis . the associations that we observed between cac progression and risk factors were consistent with those of kronmal et al for bmi , family history of cvd , triglycerides , systolic blood pressure , and pack - years of smoking.6 also consistent with that study was an observed lack of association between cac progression and ldl - c , education , or creatinine . in both studies , race / ethnicity was more strongly related to cac progression in more fully adjusted models , suggesting confounding between race / ethnicity and risk factors such as smoking and bmi . this is further suggested by the lack of robustness in the associations observed between race / ethnicity and cac progression in our sensitivity analyses ( tables4 and 5 ) . the fact that race / ethnicity is more strongly associated with cac progression in some subsets of participants warrants further research . we observed higher magnitudes of association in cac progression for age , sex , cigarette smoking status , hdl - c , diabetes , antihypertensive medications , and statin use at exam 1 compared with kronmal et al.6 because atherosclerosis is a gradual process , a longer follow - up time presumably enables more accurate estimation of the progression rate . other prior studies with shorter follow - up observed associations between cac progression and white race,22 hypertension,22 diabetes,22 fibrinogen,23 hdl - c,24 and smoking.6,25 it is difficult to make meaningful comparisons among these studies due to differences in the study populations or transformations of the outcome ; however , we found that our results were consistent with these studies in direction of association . others have also observed uneven impact of lipid - lowering medications on cac progression.3,24,2630 some prior studies have suggested that statin use does not affect cac progression,26,27,31,32 although these studies were limited by relatively small sample sizes and/or short follow - up times . the association with time - varying statin use over the subsequent 10 years , when statin use became much more prevalent , was less strong . if this were true , we might expect to observe that those prescribed statins during the course of follow - up would have the highest cac progression because , presumably , much of this change in plaque architecture would have occurred prior to scanning among the participants that were already on statins at exam 1 . the use of these agents and other preventive therapies during the period of follow - up ( as at baseline ) is subject to confounding by indication35 because cac at baseline is associated with subsequent progression . because our model adjusts fully , in a time - varying manner , for the use of lipid - lowering agents , the interpretation of factors not directly related to these therapies which are the focus of this paper should not be confounded . participants who had no follow - up , particularly those who died or became too sick to continue the study between exams 1 and 2 , may differ from those with follow - up measurements ; however , the overall retention rate was high , and the mixed - effects model approach allows the analysis to be adjusted for variables related to health status . because both methods should lead to valid conclusions there are 2 main drawbacks to the change analysis : ( 1 ) no time - varying information can be included , and ( 2 ) the analysis must be restricted to a more selected subset of participants than the mixed - effects model analysis . these 2 facts mean that simple adjustment is less accurate ( due to less control of scanner and other time - varying effects ) , and the results are less generalizable than those derived from the full mixed - effects model analysis . the major strengths of this analysis include the substantial follow - up time , the well - characterized prospective cohort , and our application of mixed - effects models to this question . baseline cac and cac progression rate are associated , but control for measured baseline can lead to bias.19,20 the linear mixed - effects model effectively controls for the baseline cac and permits an unbiased understanding of both cross - sectional and longitudinal relationships between risk factors and cac . this study examined a comprehensive set of risk factors for cac progression over a long period of follow - up in a well - studied population that was selected to be free of clinical cvd at baseline . we found that cac progression over 10 years was strongly associated with most cvd risk factors , confirming that risk factors for clinical coronary disease and the progression of cac are overlapping .

the current study was performed as part of the maastricht aging study ( maas ) , a longitudinal study of determinants of cognitive aging ( 12 ) . a total of 10,396 people were sampled from the registration network family practices ( rnh ) , a patient register of collaborating general practitioners in the province of limburg , the netherlands ( 13 ) . patients in the rnh register are considered representative of the limburg and dutch population with respect to demographic characteristics ( age , sex , and educational level ) ( 12 ) . of these 10,396 people , 4,490 ( 43.2% ) were willing to participate , 3,531 ( 34% ) refused participation , and 2,375 ( 22.8% ) did not respond to the written request . medical exclusion criteria for baseline assessment were defined as active or inactive medical conditions in the rnh problem list that could interfere with normal cognitive function , including coma ( only active ) , cerebrovascular pathology , a tumor of the nervous system , congenital malformation of the nervous system , multiple sclerosis , parkinsonism , epilepsy ( all types ) , dementia , organic psychosis , schizophrenia , affective psychosis , and mental retardation . before participation in maas , all 4,490 participants were screened by telephone for the following medical conditions not documented in the rnh database : history of transient ischemic attacks , brain surgery , hemodialysis for renal failure , electroconvulsive therapy , and regular use of psychotropic drugs . based on this interview , were randomly selected , stratified for age ( 12 discrete age groups from 2481 years ) , sex , and level of general ability ( low / high ) and equally distributed over four demographically identical test panels . the local ethics committee approved the study , and all participants gave informed consent . at baseline , these 1,823 participants underwent a comprehensive assessment of medical status , lifestyle , and anthropomorphic and cognitive functioning measures . the baseline assessment took place from 19931996 and was repeated 6 and 12 years after baseline . only participants aged 40 years presence of type 2 diabetes was based on a diagnosis made by a physician ( as reported by the study participant ) and current medication use for diabetes reported in a questionnaire . diagnosis of type 2 diabetes based on self - report has previously been used in large cohort studies ( 10,14 ) and has been shown to be a reliable estimate of the actual prevalence of diabetes ( 15 ) . to make a distinction between type 1 and type 2 diabetes , participants were considered to have type 2 diabetes when they used oral antidiabetic drugs , started using insulin at or after the age of 40 years , or were diagnosed with diabetes at or after the age of 40 years . diabetes status was assessed at baseline and at the 6- and 12-year follow - ups . several potentially health - related conditions that might influence the association between type 2 diabetes and cognitive change over time were taken into account . these included self - report of present or past history of smoking ( yes / no ) , alcohol intake ( according to world health organization guidelines , less / more than 21 standard consumption per week for females , less / more than 35 consumptions per week for males ) ( 16 ) , a history of cardiovascular disease ( yes / no ) as reported in a questionnaire , and bmi ( weight in kg / length in m ) . blood pressure was measured three times at 5-min intervals on the left arm using an automatic recording device ( critikon dinamap 8100 ; critikon , tampa , fl ) . hypertension ( yes / no ) was defined as a mean systolic blood pressure of 140 mmhg , a mean diastolic blood pressure of 90 mmhg ( 17 ) , or current use of antihypertensive medication as reported in a questionnaire . depressive symptoms were assessed with the 16-item depression subscale of the revised symptom checklist-90 ( score range 1680 ) ( 18 ) . apolipoprotein ( apoe ) genotyping was determined on genomic dna extracted from edta - anticoagulated blood using a pcr technique ( 19 ) . the presence ( yes / no ) of the apoe 4 ( apoe - e4 ) risk allele , which is associated with an increased risk of dementia ( 20 ) , was used in the current study . furthermore , age , sex , and educational level [ ranging from primary education , 1 , to university degree , 8 ] ( 21 ) were included as demographic covariates . three groups were made for educational level : low ( levels 1 to 2 ) , middle ( levels 35 ) , and high ( levels 68 ) . hypertension , a history of cardiovascular disease , bmi , and depression were measured at baseline and each follow - up assessment . cognitive function was measured at baseline and the 6- and 12-year follow - ups with a battery of neuropsychological tests administered by psychologists and trained test assistants . an a priori selection of these neuropsychological tests was used to assess cognitive function in the current study . the visual verbal learning test ( 22 ) was used to assess verbal memory . in this test , 15 nonrelated monosyllabic words are presented in five subsequent trials on a computer screen , followed by a recall phase immediately after each trial and a delayed recall phase 20 min after the test . the total number of correctly reproduced words in five trials was measured ( immediate word recall ) , together with the number of correctly reproduced words 20 min after the last trial ( delayed word recall ) . the concept shifting test ( cst ) ( 23 ) was used to measure executive function . in three trials , the participants have to cross out as fast as possible 16 digits in ascending order ( cst part a ) , 16 letters in alphabetical order ( cst part b ) , and finally 8 digits and 8 letters in alternating order ( cst part c ) . the shifting score is calculated by subtracting the average time needed to finish part a and b from the time needed to finish part c. the letter digit substitution test ( 24 ) was used to assess information - processing speed . participants were instructed to match digits to letters , according to a key of letter / digit combinations at the top of the sheet , as quickly as possible within 90 s. the mini mental state examination ( mmse ) ( 25 ) broadly assesses several domains of cognitive functioning and was used to measure global cognitive functioning . differences between group characteristics at baseline were tested using independent sample t tests for quantitative variables ( age , bmi , depression score , mmse score , and baseline scores on cognitive tests ) , tests for qualitative variables ( sex , history of cardiovascular disease , hypertension , history of smoking , alcohol intake , and apoe - e4 allele ) , and the mann - whitney u test for the ordinal variable educational level . linear regression was used to estimate the effect of baseline type 2 diabetes on cognitive performance at baseline . next , separate linear mixed models ( random - effects models ) were run to measure the crude and adjusted effect of baseline diabetes on change in cognition from baseline to 6-year ( f1 ) and to 12-year follow - up ( f2 ) . an advantage of mixed models over anova with repeated measures is that it models time trajectories without restricting analyses to study completers . terms for the fixed effects included the intercept , baseline diabetes , age , age ( in order to control for nonlinear trends ) , sex , educational level , baseline history of smoking , baseline alcohol intake , hypertension , history of cardiovascular disease , bmi , depression score , type 2 diabetes , time ( f1 , f2 ) , as well as the interaction terms between f1 and diabetes and f2 and diabetes . apoe - e4 allele status ( risk allele present / absent ) was included as a covariate in an additional analysis , as this was only measured in a subsample . a second set of linear mixed models tested the crude and adjusted effect of incident type 2 diabetes on change in cognitive performance over time , excluding participants with baseline diabetes . this yielded three groups for comparison ( 1 , no diabetes [ reference ] ; 2 , incident diabetes at f1 ; and 3 , incident diabetes at f2 ) . hypertension , past or present cardiovascular disease , bmi , and depression score were now treated as time - dependent covariates , thereby allowing them to covary with diabetes status over time . to test whether loss to follow - up could be predicted by the observed variables , a logistic regression with lost to follow - up ( yes / no ) as a dependent variable was performed . all analyses were done in ibm spss statistics 19 ( spss , chicago , il ) . the current study was performed as part of the maastricht aging study ( maas ) , a longitudinal study of determinants of cognitive aging ( 12 ) . a total of 10,396 people were sampled from the registration network family practices ( rnh ) , a patient register of collaborating general practitioners in the province of limburg , the netherlands ( 13 ) . patients in the rnh register are considered representative of the limburg and dutch population with respect to demographic characteristics ( age , sex , and educational level ) ( 12 ) . of these 10,396 people , 4,490 ( 43.2% ) were willing to participate , 3,531 ( 34% ) refused participation , and 2,375 ( 22.8% ) did not respond to the written request . medical exclusion criteria for baseline assessment were defined as active or inactive medical conditions in the rnh problem list that could interfere with normal cognitive function , including coma ( only active ) , cerebrovascular pathology , a tumor of the nervous system , congenital malformation of the nervous system , multiple sclerosis , parkinsonism , epilepsy ( all types ) , dementia , organic psychosis , schizophrenia , affective psychosis , and mental retardation . before participation in maas , all 4,490 participants were screened by telephone for the following medical conditions not documented in the rnh database : history of transient ischemic attacks , brain surgery , hemodialysis for renal failure , electroconvulsive therapy , and regular use of psychotropic drugs . based on this interview , were randomly selected , stratified for age ( 12 discrete age groups from 2481 years ) , sex , and level of general ability ( low / high ) and equally distributed over four demographically identical test panels . the local ethics committee approved the study , and all participants gave informed consent . at baseline , these 1,823 participants underwent a comprehensive assessment of medical status , lifestyle , and anthropomorphic and cognitive functioning measures . the baseline assessment took place from 19931996 and was repeated 6 and 12 years after baseline . only participants aged 40 years presence of type 2 diabetes was based on a diagnosis made by a physician ( as reported by the study participant ) and current medication use for diabetes reported in a questionnaire . diagnosis of type 2 diabetes based on self - report has previously been used in large cohort studies ( 10,14 ) and has been shown to be a reliable estimate of the actual prevalence of diabetes ( 15 ) . to make a distinction between type 1 and type 2 diabetes , participants were considered to have type 2 diabetes when they used oral antidiabetic drugs , started using insulin at or after the age of 40 years , or were diagnosed with diabetes at or after the age of 40 years . diabetes status was assessed at baseline and at the 6- and 12-year follow - ups . several potentially health - related conditions that might influence the association between type 2 diabetes and cognitive change over time were taken into account . these included self - report of present or past history of smoking ( yes / no ) , alcohol intake ( according to world health organization guidelines , less / more than 21 standard consumption per week for females , less / more than 35 consumptions per week for males ) ( 16 ) , a history of cardiovascular disease ( yes / no ) as reported in a questionnaire , and bmi ( weight in kg / length in m ) . blood pressure was measured three times at 5-min intervals on the left arm using an automatic recording device ( critikon dinamap 8100 ; critikon , tampa , fl ) . hypertension ( yes / no ) was defined as a mean systolic blood pressure of 140 mmhg , a mean diastolic blood pressure of 90 mmhg ( 17 ) , or current use of antihypertensive medication as reported in a questionnaire . depressive symptoms were assessed with the 16-item depression subscale of the revised symptom checklist-90 ( score range 1680 ) ( 18 ) . apolipoprotein ( apoe ) genotyping was determined on genomic dna extracted from edta - anticoagulated blood using a pcr technique ( 19 ) . the presence ( yes / no ) of the apoe 4 ( apoe - e4 ) risk allele , which is associated with an increased risk of dementia ( 20 ) , was used in the current study . furthermore , age , sex , and educational level [ ranging from primary education , 1 , to university degree , 8 ] ( 21 ) were included as demographic covariates . three groups were made for educational level : low ( levels 1 to 2 ) , middle ( levels 35 ) , and high ( levels 68 ) . hypertension , a history of cardiovascular disease , bmi , and depression were measured at baseline and each follow - up assessment . cognitive function was measured at baseline and the 6- and 12-year follow - ups with a battery of neuropsychological tests administered by psychologists and trained test assistants . an a priori selection of these neuropsychological tests was used to assess cognitive function in the current study . the visual verbal learning test ( 22 ) was used to assess verbal memory . in this test , 15 nonrelated monosyllabic words are presented in five subsequent trials on a computer screen , followed by a recall phase immediately after each trial and a delayed recall phase 20 min after the test . the total number of correctly reproduced words in five trials was measured ( immediate word recall ) , together with the number of correctly reproduced words 20 min after the last trial ( delayed word recall ) . the concept shifting test ( cst ) ( 23 ) was used to measure executive function . in three trials , the participants have to cross out as fast as possible 16 digits in ascending order ( cst part a ) , 16 letters in alphabetical order ( cst part b ) , and finally 8 digits and 8 letters in alternating order ( cst part c ) . the shifting score is calculated by subtracting the average time needed to finish part a and b from the time needed to finish part c. the letter digit substitution test ( 24 ) was used to assess information - processing speed . participants were instructed to match digits to letters , according to a key of letter / digit combinations at the top of the sheet , as quickly as possible within 90 s. the mini mental state examination ( mmse ) ( 25 ) broadly assesses several domains of cognitive functioning and was used to measure global cognitive functioning . differences between group characteristics at baseline were tested using independent sample t tests for quantitative variables ( age , bmi , depression score , mmse score , and baseline scores on cognitive tests ) , tests for qualitative variables ( sex , history of cardiovascular disease , hypertension , history of smoking , alcohol intake , and apoe - e4 allele ) , and the mann - whitney u test for the ordinal variable educational level . linear regression was used to estimate the effect of baseline type 2 diabetes on cognitive performance at baseline . next , separate linear mixed models ( random - effects models ) were run to measure the crude and adjusted effect of baseline diabetes on change in cognition from baseline to 6-year ( f1 ) and to 12-year follow - up ( f2 ) . an advantage of mixed models over anova with repeated measures is that it models time trajectories without restricting analyses to study completers . terms for the fixed effects included the intercept , baseline diabetes , age , age ( in order to control for nonlinear trends ) , sex , educational level , baseline history of smoking , baseline alcohol intake , hypertension , history of cardiovascular disease , bmi , depression score , type 2 diabetes , time ( f1 , f2 ) , as well as the interaction terms between f1 and diabetes and f2 and diabetes . apoe - e4 allele status ( risk allele present / absent ) was included as a covariate in an additional analysis , as this was only measured in a subsample . a second set of linear mixed models tested the crude and adjusted effect of incident type 2 diabetes on change in cognitive performance over time , excluding participants with baseline diabetes . this yielded three groups for comparison ( 1 , no diabetes [ reference ] ; 2 , incident diabetes at f1 ; and 3 , incident diabetes at f2 ) . hypertension , past or present cardiovascular disease , bmi , and depression score were now treated as time - dependent covariates , thereby allowing them to covary with diabetes status over time . to test whether loss to follow - up could be predicted by the observed variables , a logistic regression with lost to follow - up ( yes / no ) as a dependent variable was performed . all analyses were done in ibm spss statistics 19 ( spss , chicago , il ) . at baseline , 1,290 participants ( aged 40.082.7 years ) were tested , of whom 68 ( 5.3% ) had diabetes . at f1 , 925 ( 75.7% ) participants without type 2 diabetes at baseline and 41 ( 60.3% ) participants with diabetes at baseline were still in the study . at f2 , the sample sizes were 761 ( 62.3% ) and 21 ( 30.9% ) for the two groups , respectively . of the 1,222 participants without type 2 diabetes at baseline , 54 ( 4.4% ) participants developed type 2 diabetes between baseline and f1 , and 57 ( 4.7% ) participants developed type 2 diabetes between f1 and f2 . having type 2 diabetes at baseline , being older , having a history of smoking , cardiovascular disease , higher bmi , or lower baseline mmse score increased the risk of study dropout . baseline characteristics stratified by diabetes status are shown in table 1 . compared with participants without diabetes , participants with diabetes were older , had a lower educational level , had a higher bmi , were more likely to have hypertension and a history of cardiovascular disease , and performed worse on all cognitive measures . these between group differences in cognitive performance were still significant after adjustment for demographic variables , history of smoking , alcohol use , and comorbid conditions . there were no statistically significant differences in sex , alcohol intake , history of smoking , presence of apoe - e4 allele , and depression score between groups . baseline characteristics of the study group , stratified by diabetes status at baseline and follow - up participants with incident diabetes at f1 were more likely to be male , were significantly older , had a higher bmi , were more likely to have a history of cardiovascular disease and hypertension , and had a lower depression score than participants who were free of diabetes during the whole follow - up period . participants with incident diabetes at f2 were more likely to have hypertension and had a significantly higher bmi than participants without diabetes during the whole follow - up period . at baseline ( i.e. , before a diagnosis of diabetes was made ) , there were no significant differences in measures for information - processing speed ( p = 0.24 ) , concept shifting ( p = 0.28 ) , immediate word recall ( p = 0.08 ) , and delayed word recall ( p = 0.06 ) between participants with incident diabetes at f1 and control subjects . likewise , there were no significant differences in information - processing speed ( p = 0.57 ) , concept shifting ( p = 0.35 ) , immediate word recall ( p = 0.26 ) , and delayed word recall ( p = 0.06 ) between participants with incident diabetes at f2 and control subjects . however , participants with incident diabetes at f2 had a significantly higher mmse score than participants without diabetes . results of the association between baseline diabetes and cognitive change over time are presented in table 2 . in crude analyses ( model 1 ) , participants with diabetes showed a significantly larger decline in information - processing speed and concept shifting from baseline to f1 and to f2 compared with those without diabetes . this was virtually unchanged after adjusting for demographic variables , history of smoking , and alcohol intake ( model 2 ) and after further adjustments for comorbid conditions ( model 3 ) . the association between diabetes and change in delayed word recall from baseline to f1 and to f2 was not significant in models 1 and 2 , but became modestly significant in the fully adjusted model 3 . in contrast , there were no associations with decline in immediate word recall . to examine the effect of carrying the apoe - e4 allele on the association between diabetes and cognitive change over time , apoe - e4 was included as a covariate in a restricted subgroup of 699 individuals without and 36 with baseline diabetes . results were similar to the analyses in the full sample ( data not shown ) , except that the effect of diabetes on decline in delayed word recall from baseline to f1 was not statistically significant anymore ( p = 0.14 ) . however , the magnitude of this effect was similar to the analysis without apoe - e4 . the results per cognitive outcome domain of the fully adjusted models ( without apoe - e4 ) are presented in fig . 1 . decline in information - processing speed from baseline to f2 was three times larger for participants with diabetes compared with those without and decline in set shifting was four times larger . although participants without diabetes did not decline in delayed word recall , participants with diabetes declined by 14% . change in cognitive performance ( 95% ci ) from baseline to 6-year follow - up ( f1 ) and to 12-year follow - up ( f2 ) in participants with baseline diabetes relative to participants without diabetes before and after adjustment for various covariates cognitive performance ( mean domain score adjusted for demographics , history of smoking , alcohol intake , and comorbid conditions ) for participants with baseline type 2 diabetes ( white circles ) and participants without baseline diabetes ( black circles ) at baseline ( 0 ) , 6-year follow - up ( 6 ) , and 12-year follow - up ( 12 ) . for information - processing speed and immediate and delayed word recall , participants with incident diabetes at f1 showed a larger decline in information - processing speed from baseline to f1 and to f2 ( table 3 , model 1 ) . no differences were observed between participants who developed diabetes at follow - up and control subjects . change in cognitive performance ( 95% ci ) from baseline to 6-year follow - up ( f1 ) and to 12-year follow - up ( f2 ) in participants with incident diabetes relative to participants without diabetes before and after adjustment for various covariates to better understand the effects of incident diabetes on cognitive decline , we performed two post hoc analyses . in the first analysis , the two groups with participants with incident diabetes were pooled ( n = 111 ) to increase power . in the fully adjusted model , the incident diabetes group showed a significantly larger decline in information processing speed from baseline to f2 compared with the control group ( estimate 1.62 ; p = 0.04 ) . next , we explored whether disease - exposure time plays a role in the development of cognitive decline . we performed a post hoc analysis including only diabetic patients and measured the effect of disease duration on cognitive decline . data on disease duration ( based on self - report ) were available at baseline for 67 participants with baseline diabetes , at follow - up for 44 participants with incident diabetes at f1 , and for 53 participants with incident diabetes at f2 . missing values were replaced with the strata - specific mean of the observed values of disease duration within each of the three diabetes strata ( baseline , at f1 , and at f2 ) . diabetes had been diagnosed 1.01 9.15 years ( mean sd ) after baseline assessment . in line with our expectation , we found that , after adjustment for all covariates described previously , increasing disease duration had a significant effect on decline in information - processing speed from baseline to 12-year follow - up ( estimate 0.19 ; p < 0.05 ) . we did not find a significant effect of disease duration on immediate recall ( estimate 0.04 ; p = 0.72 ) , delayed recall ( estimate 0.03 ; p = 0.41 ) , or concept shifting ( estimate 0.59 ; p = 0.05 ) . baseline characteristics stratified by diabetes status are shown in table 1 . compared with participants without diabetes , participants with diabetes were older , had a lower educational level , had a higher bmi , were more likely to have hypertension and a history of cardiovascular disease , and performed worse on all cognitive measures . these between group differences in cognitive performance were still significant after adjustment for demographic variables , history of smoking , alcohol use , and comorbid conditions . there were no statistically significant differences in sex , alcohol intake , history of smoking , presence of apoe - e4 allele , and depression score between groups . baseline characteristics of the study group , stratified by diabetes status at baseline and follow - up participants with incident diabetes at f1 were more likely to be male , were significantly older , had a higher bmi , were more likely to have a history of cardiovascular disease and hypertension , and had a lower depression score than participants who were free of diabetes during the whole follow - up period . participants with incident diabetes at f2 were more likely to have hypertension and had a significantly higher bmi than participants without diabetes during the whole follow - up period . at baseline ( i.e. , before a diagnosis of diabetes was made ) , there were no significant differences in measures for information - processing speed ( p = 0.24 ) , concept shifting ( p = 0.28 ) , immediate word recall ( p = 0.08 ) , and delayed word recall ( p = 0.06 ) between participants with incident diabetes at f1 and control subjects . likewise , there were no significant differences in information - processing speed ( p = 0.57 ) , concept shifting ( p = 0.35 ) , immediate word recall ( p = 0.26 ) , and delayed word recall ( p = 0.06 ) between participants with incident diabetes at f2 and control subjects . however , participants with incident diabetes at f2 had a significantly higher mmse score than participants without diabetes . results of the association between baseline diabetes and cognitive change over time are presented in table 2 . in crude analyses ( model 1 ) , participants with diabetes showed a significantly larger decline in information - processing speed and concept shifting from baseline to f1 and to f2 compared with those without diabetes . this was virtually unchanged after adjusting for demographic variables , history of smoking , and alcohol intake ( model 2 ) and after further adjustments for comorbid conditions ( model 3 ) . the association between diabetes and change in delayed word recall from baseline to f1 and to f2 was not significant in models 1 and 2 , but became modestly significant in the fully adjusted model 3 . in contrast , there were no associations with decline in immediate word recall . to examine the effect of carrying the apoe - e4 allele on the association between diabetes and cognitive change over time , apoe - e4 was included as a covariate in a restricted subgroup of 699 individuals without and 36 with baseline diabetes . results were similar to the analyses in the full sample ( data not shown ) , except that the effect of diabetes on decline in delayed word recall from baseline to f1 was not statistically significant anymore ( p = 0.14 ) . however , the magnitude of this effect was similar to the analysis without apoe - e4 . the results per cognitive outcome domain of the fully adjusted models ( without apoe - e4 ) are presented in fig . 1 . decline in information - processing speed from baseline to f2 was three times larger for participants with diabetes compared with those without and decline in set shifting was four times larger . although participants without diabetes did not decline in delayed word recall , participants with diabetes declined by 14% . change in cognitive performance ( 95% ci ) from baseline to 6-year follow - up ( f1 ) and to 12-year follow - up ( f2 ) in participants with baseline diabetes relative to participants without diabetes before and after adjustment for various covariates cognitive performance ( mean domain score adjusted for demographics , history of smoking , alcohol intake , and comorbid conditions ) for participants with baseline type 2 diabetes ( white circles ) and participants without baseline diabetes ( black circles ) at baseline ( 0 ) , 6-year follow - up ( 6 ) , and 12-year follow - up ( 12 ) . for information - processing speed and immediate and delayed word recall , participants with incident diabetes at f1 showed a larger decline in information - processing speed from baseline to f1 and to f2 ( table 3 , model 1 ) . no differences were observed between participants who developed diabetes at follow - up and control subjects . change in cognitive performance ( 95% ci ) from baseline to 6-year follow - up ( f1 ) and to 12-year follow - up ( f2 ) in participants with incident diabetes relative to participants without diabetes before and after adjustment for various covariates to better understand the effects of incident diabetes on cognitive decline , we performed two post hoc analyses . in the first analysis , the two groups with participants with incident diabetes were pooled ( n = 111 ) to increase power . in the fully adjusted model , the incident diabetes group showed a significantly larger decline in information processing speed from baseline to f2 compared with the control group ( estimate 1.62 ; p = 0.04 ) . next , we explored whether disease - exposure time plays a role in the development of cognitive decline . we performed a post hoc analysis including only diabetic patients and measured the effect of disease duration on cognitive decline . data on disease duration ( based on self - report ) were available at baseline for 67 participants with baseline diabetes , at follow - up for 44 participants with incident diabetes at f1 , and for 53 participants with incident diabetes at f2 . missing values were replaced with the strata - specific mean of the observed values of disease duration within each of the three diabetes strata ( baseline , at f1 , and at f2 ) . diabetes had been diagnosed 1.01 9.15 years ( mean sd ) after baseline assessment . in line with our expectation , we found that , after adjustment for all covariates described previously , increasing disease duration had a significant effect on decline in information - processing speed from baseline to 12-year follow - up ( estimate 0.19 ; p < 0.05 ) . we did not find a significant effect of disease duration on immediate recall ( estimate 0.04 ; p = 0.72 ) , delayed recall ( estimate 0.03 ; p = 0.41 ) , or concept shifting ( estimate 0.59 ; p = 0.05 ) . this study examined the effect of baseline and incident diabetes on change in multiple cognitive functions over a long follow - up period of 12 years . participants with baseline diabetes performed worse on all cognitive measures at baseline and also showed a three times larger decline in information - processing speed and a four times larger decline in executive function than participants without diabetes . the effects on speed and executive function were largely independent of apoe - e4 genotype status . interestingly , no significant effect of incident diabetes at f1 and f2 on cognitive decline was observed , although the coefficients suggested a small effect on decline in information - processing speed from baseline to 12-year follow - up . few previous studies examined the effect of incident diabetes on cognitive decline ( 26,27 ) . ( 26 ) was performed in a restricted sample of middle - aged individuals followed for 5 years . ( 27 ) used two cognitive measures and was performed in older adults followed for 9 years . though both studies did not find an independent effect of incident diabetes on cognitive decline , both point out that the decline in the incident diabetes group was intermediate between the control group and the prevalent diabetes group . in a post hoc analysis in which we pooled the two groups with incident diabetes at f1 and f2 , incident diabetes indeed had a significant effect on decline in information - processing speed . this might indicate that our initial model had missed the small differences in cognitive decline between incident diabetic patients and control subjects . so , diabetes might have a subtle effect on cognition even in the early stages of the disease . the finding that baseline diabetes had an effect on cognitive decline is in line with numerous previous studies with shorter follow - up duration ( 8,9 ) , although the rate of decline in our study was larger than reported before , and some studies did not find an effect of diabetes on cognitive decline ( 57 ) . given the differences in the effect of baseline diabetes and incident diabetes , it appears that effects of diabetes on cognition take time to evolve and that profound differences in cognition between people with diabetes and people without diabetes become apparent only in the long run . this is consistent with the finding that incident diabetes only had an effect on a measure of information - processing speed , which is known to be most sensitive to diabetes - related cognitive decline ( 28 ) and with the finding that disease duration had a significant effect on decline in information - processing speed . these results seem to indicate that duration of disease exposure plays an important role in the development of cognitive decline , which is in line with previous research showing that longer duration of diabetes is associated with increased odds of mild cognitive impairment ( 29 ) , a possible prodromal stage of dementia . few studies examined the effect of baseline diabetes on cognitive decline over a comparable follow - up period ( 10,3032 ) . in the baltimore longitudinal study of aging , no effect of diabetes on cognitive decline over a 12-year follow - up period however , this study only included men and assessed other cognitive domains , namely visual memory and vocabulary performance ( 31 ) . the study of osteoporotic fractures has shown that lack of diabetes was predictive of maintaining optimal cognitive function over a 15-year follow - up as opposed to minor cognitive decline in older women ( 32 ) . the indianapolis - ibadan dementia project and the atherosclerosis risk in communities studies showed a modest effect of diabetes on change in cognition over a 15-year and 14-year follow - up period , respectively ( 10,30 ) . stronger associations might have been missed because measures of general cognitive functioning were used ( 10 ) , which are less sensitive to change , and because the study sample was restricted to middle - aged individuals ( 30 ) , in whom the association between diabetes and cognitive decline might be less obvious ( 26 ) . in contrast , the current study had no upper age restriction and used a comprehensive and validated multicognitive domain test battery . in line with previous studies ( 3335 ) , we found that diabetes was most strongly related to decline in information - processing speed and executive function , although in these studies , the follow - up durations were shorter and the age ranges were smaller than in our study . in the absence of major cerebrovascular disease or neurodegenerative processes , decline in these domains has been linked to cerebral small vessel disease ( 36,37 ) . indeed , patients with diabetes have been shown to have more white matter lesions , which have been related to cognitive functioning in these patients ( 38 ) . furthermore , microvascular disease seems to play a role in the development of dementia in diabetic patients ( 1,39 ) . other putative factors include hyperglycemia , which can have toxic effects on the brain by glycation of important proteins ( 1,2 ) , and insulin , which inhibits degradation of amyloid- peptide ( the main product of the alzheimer 's disease process ) by competition for insulin - degrading enzyme in the brain ( 1,2 ) . first , the size of the diabetes groups was relatively small due to the population - based nature of maas , especially the incident diabetes groups . we are thus likely to have missed more subtle associations between incident diabetes and cognitive decline , especially for the group with incident diabetes at f1 and decline from baseline to f2 . next , participants with diabetes and poorer global cognition were more likely to drop out of the study , which might have led to selection bias . hence , our results most likely are an underestimation of the true effect of diabetes on cognitive decline in the population . however , we partially accounted for this attrition by using mixed models , which does not restrict analysis to study completers . furthermore , the diagnosis of diabetes was based on self - report or on antidiabetic medication use . as a diagnosis of diabetes is often missed ( 40 ) , some participants with diabetes might have been wrongly assigned to the nondiabetic group . it is unknown how this would have affected our estimates , but it seems unlikely that this accounts for the robust differences in the rate of cognitive decline between groups . finally , because we did not have data on hba1c levels , we were unable to examine the effect of glucose control on cognitive decline . these include multiple measures of cognitive functions , assessment of separate cognitive domains by a comprehensive neuropsychological test battery , inclusion of important potential confounders , and a long follow - up time . furthermore , we had the opportunity to measure the effect of incident diabetes and comorbid conditions at each follow - up moment . to conclude , this study indicates that patients with baseline type 2 diabetes show accelerated cognitive decline , particularly in information - processing speed and executive function , compared with individuals without diabetes and that patients with incident diabetes show signs of early decline in information - processing speed . it seems that disease - exposure time plays an important role in the development of cognitive decline . this might provide a window of opportunity for prevention and early treatment of diabetes - related cognitive deficits . for this , it is important to assess cognitive status at an early stage of the disease and on a regular basis . studies that focus on the underlying mechanisms between diabetes and cognitive decline are highly desirable in order to develop adequate treatment for cognitive decline .

objectiveto examine the effects of baseline and incident diabetes on change in cognitive function over 12 years.research design and methodsa sample of 1,290 individuals aged 40 years at baseline , participating in the maastricht aging study , were cognitively tested at baseline , after 6 years , and after 12 years . of these , 68 participants had type 2 diabetes at baseline , and 54 and 57 had incident diabetes at the 6- and 12-year follow - up , respectively . changes in performance on tests of information - processing speed , executive function , and verbal memory from baseline to 6- and 12-year follow - up were compared between groups using linear mixed models . effects of diabetes on cognitive decline were adjusted for demographic variables , history of smoking , alcohol intake , and comorbid conditions , including hypertension , cardiovascular disease , bmi , and depression.resultsparticipants with baseline diabetes showed larger decline in information - processing speed ( estimate 7.64 ; p < 0.01 ) , executive function ( 21.82 ; p < 0.01 ) , and delayed word recall ( 1.35 ; p < 0.05 ) over the 12-year follow - up compared with control subjects . no significant difference in decline was observed for immediate word recall . compared with control subjects , participants with incident diabetes showed subtle early decline in information - processing speed only . interestingly , they did not show larger decline in any other cognitive domain.conclusionsindividuals with baseline type 2 diabetes show accelerated cognitive decline , particularly in information - processing speed and executive function , compared with individuals without diabetes . in incident diabetes , decline in speed becomes detectable first , and cognitive decline seems to increase with increasing exposure time .

RESEARCH DESIGN AND METHODS Sample Diabetes status Covariates Assessment of cognitive function Statistical analysis RESULTS Baseline differences Baseline diabetes and 12-year cognitive decline Incident diabetes and 12-year cognitive decline Post hoc analyses CONCLUSIONS

medical exclusion criteria for baseline assessment were defined as active or inactive medical conditions in the rnh problem list that could interfere with normal cognitive function , including coma ( only active ) , cerebrovascular pathology , a tumor of the nervous system , congenital malformation of the nervous system , multiple sclerosis , parkinsonism , epilepsy ( all types ) , dementia , organic psychosis , schizophrenia , affective psychosis , and mental retardation . to make a distinction between type 1 and type 2 diabetes , participants were considered to have type 2 diabetes when they used oral antidiabetic drugs , started using insulin at or after the age of 40 years , or were diagnosed with diabetes at or after the age of 40 years . diabetes status was assessed at baseline and at the 6- and 12-year follow - ups . these included self - report of present or past history of smoking ( yes / no ) , alcohol intake ( according to world health organization guidelines , less / more than 21 standard consumption per week for females , less / more than 35 consumptions per week for males ) ( 16 ) , a history of cardiovascular disease ( yes / no ) as reported in a questionnaire , and bmi ( weight in kg / length in m ) . hypertension , a history of cardiovascular disease , bmi , and depression were measured at baseline and each follow - up assessment . cognitive function was measured at baseline and the 6- and 12-year follow - ups with a battery of neuropsychological tests administered by psychologists and trained test assistants . differences between group characteristics at baseline were tested using independent sample t tests for quantitative variables ( age , bmi , depression score , mmse score , and baseline scores on cognitive tests ) , tests for qualitative variables ( sex , history of cardiovascular disease , hypertension , history of smoking , alcohol intake , and apoe - e4 allele ) , and the mann - whitney u test for the ordinal variable educational level . linear regression was used to estimate the effect of baseline type 2 diabetes on cognitive performance at baseline . next , separate linear mixed models ( random - effects models ) were run to measure the crude and adjusted effect of baseline diabetes on change in cognition from baseline to 6-year ( f1 ) and to 12-year follow - up ( f2 ) . terms for the fixed effects included the intercept , baseline diabetes , age , age ( in order to control for nonlinear trends ) , sex , educational level , baseline history of smoking , baseline alcohol intake , hypertension , history of cardiovascular disease , bmi , depression score , type 2 diabetes , time ( f1 , f2 ) , as well as the interaction terms between f1 and diabetes and f2 and diabetes . a second set of linear mixed models tested the crude and adjusted effect of incident type 2 diabetes on change in cognitive performance over time , excluding participants with baseline diabetes . hypertension , past or present cardiovascular disease , bmi , and depression score were now treated as time - dependent covariates , thereby allowing them to covary with diabetes status over time . medical exclusion criteria for baseline assessment were defined as active or inactive medical conditions in the rnh problem list that could interfere with normal cognitive function , including coma ( only active ) , cerebrovascular pathology , a tumor of the nervous system , congenital malformation of the nervous system , multiple sclerosis , parkinsonism , epilepsy ( all types ) , dementia , organic psychosis , schizophrenia , affective psychosis , and mental retardation . to make a distinction between type 1 and type 2 diabetes , participants were considered to have type 2 diabetes when they used oral antidiabetic drugs , started using insulin at or after the age of 40 years , or were diagnosed with diabetes at or after the age of 40 years . diabetes status was assessed at baseline and at the 6- and 12-year follow - ups . these included self - report of present or past history of smoking ( yes / no ) , alcohol intake ( according to world health organization guidelines , less / more than 21 standard consumption per week for females , less / more than 35 consumptions per week for males ) ( 16 ) , a history of cardiovascular disease ( yes / no ) as reported in a questionnaire , and bmi ( weight in kg / length in m ) . hypertension , a history of cardiovascular disease , bmi , and depression were measured at baseline and each follow - up assessment . cognitive function was measured at baseline and the 6- and 12-year follow - ups with a battery of neuropsychological tests administered by psychologists and trained test assistants . differences between group characteristics at baseline were tested using independent sample t tests for quantitative variables ( age , bmi , depression score , mmse score , and baseline scores on cognitive tests ) , tests for qualitative variables ( sex , history of cardiovascular disease , hypertension , history of smoking , alcohol intake , and apoe - e4 allele ) , and the mann - whitney u test for the ordinal variable educational level . linear regression was used to estimate the effect of baseline type 2 diabetes on cognitive performance at baseline . next , separate linear mixed models ( random - effects models ) were run to measure the crude and adjusted effect of baseline diabetes on change in cognition from baseline to 6-year ( f1 ) and to 12-year follow - up ( f2 ) . terms for the fixed effects included the intercept , baseline diabetes , age , age ( in order to control for nonlinear trends ) , sex , educational level , baseline history of smoking , baseline alcohol intake , hypertension , history of cardiovascular disease , bmi , depression score , type 2 diabetes , time ( f1 , f2 ) , as well as the interaction terms between f1 and diabetes and f2 and diabetes . a second set of linear mixed models tested the crude and adjusted effect of incident type 2 diabetes on change in cognitive performance over time , excluding participants with baseline diabetes . hypertension , past or present cardiovascular disease , bmi , and depression score were now treated as time - dependent covariates , thereby allowing them to covary with diabetes status over time . at f1 , 925 ( 75.7% ) participants without type 2 diabetes at baseline and 41 ( 60.3% ) participants with diabetes at baseline were still in the study . of the 1,222 participants without type 2 diabetes at baseline , 54 ( 4.4% ) participants developed type 2 diabetes between baseline and f1 , and 57 ( 4.7% ) participants developed type 2 diabetes between f1 and f2 . having type 2 diabetes at baseline , being older , having a history of smoking , cardiovascular disease , higher bmi , or lower baseline mmse score increased the risk of study dropout . compared with participants without diabetes , participants with diabetes were older , had a lower educational level , had a higher bmi , were more likely to have hypertension and a history of cardiovascular disease , and performed worse on all cognitive measures . these between group differences in cognitive performance were still significant after adjustment for demographic variables , history of smoking , alcohol use , and comorbid conditions . there were no statistically significant differences in sex , alcohol intake , history of smoking , presence of apoe - e4 allele , and depression score between groups . baseline characteristics of the study group , stratified by diabetes status at baseline and follow - up participants with incident diabetes at f1 were more likely to be male , were significantly older , had a higher bmi , were more likely to have a history of cardiovascular disease and hypertension , and had a lower depression score than participants who were free of diabetes during the whole follow - up period . participants with incident diabetes at f2 were more likely to have hypertension and had a significantly higher bmi than participants without diabetes during the whole follow - up period . , before a diagnosis of diabetes was made ) , there were no significant differences in measures for information - processing speed ( p = 0.24 ) , concept shifting ( p = 0.28 ) , immediate word recall ( p = 0.08 ) , and delayed word recall ( p = 0.06 ) between participants with incident diabetes at f1 and control subjects . likewise , there were no significant differences in information - processing speed ( p = 0.57 ) , concept shifting ( p = 0.35 ) , immediate word recall ( p = 0.26 ) , and delayed word recall ( p = 0.06 ) between participants with incident diabetes at f2 and control subjects . however , participants with incident diabetes at f2 had a significantly higher mmse score than participants without diabetes . in crude analyses ( model 1 ) , participants with diabetes showed a significantly larger decline in information - processing speed and concept shifting from baseline to f1 and to f2 compared with those without diabetes . this was virtually unchanged after adjusting for demographic variables , history of smoking , and alcohol intake ( model 2 ) and after further adjustments for comorbid conditions ( model 3 ) . the association between diabetes and change in delayed word recall from baseline to f1 and to f2 was not significant in models 1 and 2 , but became modestly significant in the fully adjusted model 3 . results were similar to the analyses in the full sample ( data not shown ) , except that the effect of diabetes on decline in delayed word recall from baseline to f1 was not statistically significant anymore ( p = 0.14 ) . decline in information - processing speed from baseline to f2 was three times larger for participants with diabetes compared with those without and decline in set shifting was four times larger . although participants without diabetes did not decline in delayed word recall , participants with diabetes declined by 14% . change in cognitive performance ( 95% ci ) from baseline to 6-year follow - up ( f1 ) and to 12-year follow - up ( f2 ) in participants with baseline diabetes relative to participants without diabetes before and after adjustment for various covariates cognitive performance ( mean domain score adjusted for demographics , history of smoking , alcohol intake , and comorbid conditions ) for participants with baseline type 2 diabetes ( white circles ) and participants without baseline diabetes ( black circles ) at baseline ( 0 ) , 6-year follow - up ( 6 ) , and 12-year follow - up ( 12 ) . for information - processing speed and immediate and delayed word recall , participants with incident diabetes at f1 showed a larger decline in information - processing speed from baseline to f1 and to f2 ( table 3 , model 1 ) . change in cognitive performance ( 95% ci ) from baseline to 6-year follow - up ( f1 ) and to 12-year follow - up ( f2 ) in participants with incident diabetes relative to participants without diabetes before and after adjustment for various covariates to better understand the effects of incident diabetes on cognitive decline , we performed two post hoc analyses . in the fully adjusted model , the incident diabetes group showed a significantly larger decline in information processing speed from baseline to f2 compared with the control group ( estimate 1.62 ; p = 0.04 ) . data on disease duration ( based on self - report ) were available at baseline for 67 participants with baseline diabetes , at follow - up for 44 participants with incident diabetes at f1 , and for 53 participants with incident diabetes at f2 . in line with our expectation , we found that , after adjustment for all covariates described previously , increasing disease duration had a significant effect on decline in information - processing speed from baseline to 12-year follow - up ( estimate 0.19 ; p < 0.05 ) . we did not find a significant effect of disease duration on immediate recall ( estimate 0.04 ; p = 0.72 ) , delayed recall ( estimate 0.03 ; p = 0.41 ) , or concept shifting ( estimate 0.59 ; p = 0.05 ) . compared with participants without diabetes , participants with diabetes were older , had a lower educational level , had a higher bmi , were more likely to have hypertension and a history of cardiovascular disease , and performed worse on all cognitive measures . these between group differences in cognitive performance were still significant after adjustment for demographic variables , history of smoking , alcohol use , and comorbid conditions . there were no statistically significant differences in sex , alcohol intake , history of smoking , presence of apoe - e4 allele , and depression score between groups . baseline characteristics of the study group , stratified by diabetes status at baseline and follow - up participants with incident diabetes at f1 were more likely to be male , were significantly older , had a higher bmi , were more likely to have a history of cardiovascular disease and hypertension , and had a lower depression score than participants who were free of diabetes during the whole follow - up period . participants with incident diabetes at f2 were more likely to have hypertension and had a significantly higher bmi than participants without diabetes during the whole follow - up period . , before a diagnosis of diabetes was made ) , there were no significant differences in measures for information - processing speed ( p = 0.24 ) , concept shifting ( p = 0.28 ) , immediate word recall ( p = 0.08 ) , and delayed word recall ( p = 0.06 ) between participants with incident diabetes at f1 and control subjects . likewise , there were no significant differences in information - processing speed ( p = 0.57 ) , concept shifting ( p = 0.35 ) , immediate word recall ( p = 0.26 ) , and delayed word recall ( p = 0.06 ) between participants with incident diabetes at f2 and control subjects . however , participants with incident diabetes at f2 had a significantly higher mmse score than participants without diabetes . in crude analyses ( model 1 ) , participants with diabetes showed a significantly larger decline in information - processing speed and concept shifting from baseline to f1 and to f2 compared with those without diabetes . this was virtually unchanged after adjusting for demographic variables , history of smoking , and alcohol intake ( model 2 ) and after further adjustments for comorbid conditions ( model 3 ) . the association between diabetes and change in delayed word recall from baseline to f1 and to f2 was not significant in models 1 and 2 , but became modestly significant in the fully adjusted model 3 . results were similar to the analyses in the full sample ( data not shown ) , except that the effect of diabetes on decline in delayed word recall from baseline to f1 was not statistically significant anymore ( p = 0.14 ) . decline in information - processing speed from baseline to f2 was three times larger for participants with diabetes compared with those without and decline in set shifting was four times larger . although participants without diabetes did not decline in delayed word recall , participants with diabetes declined by 14% . change in cognitive performance ( 95% ci ) from baseline to 6-year follow - up ( f1 ) and to 12-year follow - up ( f2 ) in participants with baseline diabetes relative to participants without diabetes before and after adjustment for various covariates cognitive performance ( mean domain score adjusted for demographics , history of smoking , alcohol intake , and comorbid conditions ) for participants with baseline type 2 diabetes ( white circles ) and participants without baseline diabetes ( black circles ) at baseline ( 0 ) , 6-year follow - up ( 6 ) , and 12-year follow - up ( 12 ) . for information - processing speed and immediate and delayed word recall , participants with incident diabetes at f1 showed a larger decline in information - processing speed from baseline to f1 and to f2 ( table 3 , model 1 ) . change in cognitive performance ( 95% ci ) from baseline to 6-year follow - up ( f1 ) and to 12-year follow - up ( f2 ) in participants with incident diabetes relative to participants without diabetes before and after adjustment for various covariates to better understand the effects of incident diabetes on cognitive decline , we performed two post hoc analyses . in the fully adjusted model , the incident diabetes group showed a significantly larger decline in information processing speed from baseline to f2 compared with the control group ( estimate 1.62 ; p = 0.04 ) . data on disease duration ( based on self - report ) were available at baseline for 67 participants with baseline diabetes , at follow - up for 44 participants with incident diabetes at f1 , and for 53 participants with incident diabetes at f2 . in line with our expectation , we found that , after adjustment for all covariates described previously , increasing disease duration had a significant effect on decline in information - processing speed from baseline to 12-year follow - up ( estimate 0.19 ; p < 0.05 ) . we did not find a significant effect of disease duration on immediate recall ( estimate 0.04 ; p = 0.72 ) , delayed recall ( estimate 0.03 ; p = 0.41 ) , or concept shifting ( estimate 0.59 ; p = 0.05 ) . this study examined the effect of baseline and incident diabetes on change in multiple cognitive functions over a long follow - up period of 12 years . participants with baseline diabetes performed worse on all cognitive measures at baseline and also showed a three times larger decline in information - processing speed and a four times larger decline in executive function than participants without diabetes . interestingly , no significant effect of incident diabetes at f1 and f2 on cognitive decline was observed , although the coefficients suggested a small effect on decline in information - processing speed from baseline to 12-year follow - up . though both studies did not find an independent effect of incident diabetes on cognitive decline , both point out that the decline in the incident diabetes group was intermediate between the control group and the prevalent diabetes group . in a post hoc analysis in which we pooled the two groups with incident diabetes at f1 and f2 , incident diabetes indeed had a significant effect on decline in information - processing speed . the finding that baseline diabetes had an effect on cognitive decline is in line with numerous previous studies with shorter follow - up duration ( 8,9 ) , although the rate of decline in our study was larger than reported before , and some studies did not find an effect of diabetes on cognitive decline ( 57 ) . given the differences in the effect of baseline diabetes and incident diabetes , it appears that effects of diabetes on cognition take time to evolve and that profound differences in cognition between people with diabetes and people without diabetes become apparent only in the long run . this is consistent with the finding that incident diabetes only had an effect on a measure of information - processing speed , which is known to be most sensitive to diabetes - related cognitive decline ( 28 ) and with the finding that disease duration had a significant effect on decline in information - processing speed . few studies examined the effect of baseline diabetes on cognitive decline over a comparable follow - up period ( 10,3032 ) . in the baltimore longitudinal study of aging , no effect of diabetes on cognitive decline over a 12-year follow - up period however , this study only included men and assessed other cognitive domains , namely visual memory and vocabulary performance ( 31 ) . the study of osteoporotic fractures has shown that lack of diabetes was predictive of maintaining optimal cognitive function over a 15-year follow - up as opposed to minor cognitive decline in older women ( 32 ) . the indianapolis - ibadan dementia project and the atherosclerosis risk in communities studies showed a modest effect of diabetes on change in cognition over a 15-year and 14-year follow - up period , respectively ( 10,30 ) . in line with previous studies ( 3335 ) , we found that diabetes was most strongly related to decline in information - processing speed and executive function , although in these studies , the follow - up durations were shorter and the age ranges were smaller than in our study . we are thus likely to have missed more subtle associations between incident diabetes and cognitive decline , especially for the group with incident diabetes at f1 and decline from baseline to f2 . to conclude , this study indicates that patients with baseline type 2 diabetes show accelerated cognitive decline , particularly in information - processing speed and executive function , compared with individuals without diabetes and that patients with incident diabetes show signs of early decline in information - processing speed .

endotracheal intubation ( et ) is a critical airway management technique used during anesthetic practice and emergency and critical care . conventional et requires laryngoscopy to clearly visualize the glottis . however , laryngoscopy is associated with many potential complications , including significant hemodynamic changes , tooth loss , oropharyngeal injury , cricoarytenoid joint dislocation , infection , and brain damage . difficult ( incidence , 118% ) or failed intubation ( incidence , 0.050.35% ) may also occur because of poor laryngoscopic exposure of the posterior glottis [ 36 ] . although the use of visualization technology in recent years has significantly reduced complications and difficulties associated with laryngoscopy and et [ 710 ] , the use of such technology has been greatly limited by high costs , low availability of proficient operators , and interference by oropharyngeal secretions . moreover , laryngoscopy may not always be available in a timely manner in urgent situations . blind tracheal intubation is an alternative to laryngoscopy and is recommended to minimize invasive intubation during management of difficult airways [ 1114 ] . in 1975 , magill developed this technique while observing an overlap of the pharyngeal and laryngeal axes . it is also easily affected by anesthesia and the patient s state of consciousness . during deep anesthesia , inhibition of respiration influences the accuracy of catheter localization ; however , when conscious , patients are usually unable to tolerate blind nasotracheal intubation . because of the angle between the thyromental line and laryngeal axis , this method is usually contraindicated in clinical practice . due to the aforementioned difficulties encountered during et , a novel blind orotracheal intubation method ( zhong s blind orotracheal intubation ) was created with the assistance of pyriform sinus localization . anatomically , the epiglottis is located between the upper edge of the laryngopharynx and lower edge of the cricoid cartilage . on either side of the laryngeal orifice the pyriform sinus is a subsite of the hypopharynx and has a fixed anatomical position in relation to the throat and rima glottidis . this distinction is important for localization of the throat and rima glottidis ( figure 1 ) . the aim of the present study was to compare the efficacy and safety of pyriform sinus localization - assisted blind orotracheal intubation with those of conventional laryngoscopic orotracheal intubation . the study revealed that blind orotracheal intubation with the assistance of pyriform sinus localization is equally or slightly more effective than conventional laryngoscopic orotracheal intubation . after approval by our institution s research ethics committee , informed consent was obtained from all patients . the inclusion criteria were : 1 ) american society of anesthesiologists ( asa ) physical status of i or ii in accordance with the definitions of the asa physical status ( asa ps ) class levels , 2 ) age 18 years , 3 ) no serious cardiovascular or pulmonary disease , 4 ) requirement for general anesthesia and et , and 5 ) no coagulation dysfunction . the exclusion criteria were : 1 ) history of oral , ear , nose , or throat operations ; 2 ) upper respiratory disease ( e.g. , tumors , polyps , severe inflammation or trauma , abscesses , or foreign objects ) ; 3 ) symptoms of sore throat or hoarseness ; 4 ) a mouth opening of < 1.5 cm ; 5 ) inability to tolerate mask ventilation ; and 6 ) anticipated requirement for > 5 h of anesthesia . the modified mallampati scoring system utilizes the following criteria as previously described : class i : visible soft palate , uvula , fauces , and pillars ; class ii : visible soft palate , uvula , and fauces ; class iii : visible soft palate and base of uvula ; and class iv : visible hard palate only . a total of 300 patients who underwent various operations from march 2012 to september 2012 met the inclusion criteria . according to the operation sequence , the patients were randomly divided into the laryngoscope group and blind group in a 1-vs.-2 style in which 1 patient was assigned to the conventional direct laryngoscope et group ( n=100 ; model h33060 shanghai medical devices co. , ltd . , shanghai , china ) and 2 patients were assigned to the pyriform sinus - assisted blind et group ( n=200 ) . all patients were required to fast for at least 8 h before the operation , and no premedication was given . upon arrival in the operating room , preoperative clinical data were recorded ( table 1 ) . standard monitoring was instituted , including noninvasive blood pressure measurement , pulse oximetry , electrocardiography , capnography , and body temperature measurement . after adequate preoxygenation , the patients were induced with 0.1 mg / kg of midazolam , 5.0 g / kg of fentanyl , 1.0 mg / kg of propofol , and 0.1 mg / kg of vecuronium . plastic endotracheal tubes with inside diameter ( i d ) of 7.5 mm ( male patients ) and 7.0 mm ( female patients ) were used . patients in the laryngoscopy group were intubated with conventional direct laryngoscopic orotracheal intubation , as previously described . patients in the blind group were intubated with pyriform sinus localization - assisted blind orotracheal intubation . the key points of this method are : 1 ) shape of endotracheal tube : the proximal end of an endotracheal tube with an ordinary core was bent to an angle of 90135 in a j-shape . the length of the tube at the proximal end was equal to the patient s thyromental distance . the arc of the tube was nearly equal to the angle between the thyromental line and the laryngeal axis ( figure 2 ) . the patient was placed in the supine position without a pillow and with the head slightly extended , or the mandibular angle of the patient was pushed upward to keep the mouth open . 3 ) intubation : the j-shaped tracheal tube was inserted into the mouth from the midline , close to the posterior region of the tongue . when the proximal end of the tube reached the posterior pharyngeal wall , it was swung to one side for placement at a 1015 angle and continuously advanced until it encountered resistance , which indicated that the tube had reached the homolateral pyriform sinus ( figure 3 ) . the tube was then withdrawn 58 mm , and the proximal end of the tube was swung back to midline and continuously advanced as the core was pulled out of the tube . a sense of emptiness indicated that the tube had successfully entered the trachea ( figure 4 ) . 4 ) fixation of the tube : after the endotracheal tube was connected to the anesthesia machine and correct position had been ensured by auscultation and confirmation of the end - tidal carbon dioxide partial pressure , it was fixed using the conventional method as previously described . the following 2 key points should be kept in mind when implementing this technique : 1 ) shape of the endotracheal tube : the angle between the oral and laryngeal axes changes with head movement . we usually determine the radius of the endotracheal tube according to the angle between the thyromental line and laryngeal axis . this angle is usually 90135 and j-shaped , and it increases with extension of the head . 2 ) location of pyriform sinus : when the proximal end of the endotracheal tube reaches the posterior pharyngeal wall , the tube should be swung to one side for 1015 angle placement and continuously advanced . in this way if resistance is felt , the tube has most likely reached the homolateral pyriform sinus . at this point , the tube should be slightly withdrawn and swung to the midline , after which the tube can be easily inserted into the trachea . 3 ) a localized outpouching of the neck can be seen when the proximal end of the endotracheal tube touches the lateral wall of the pyriform sinus . this outpouching occurs at the posterolateral aspect of the thyroid cartilage wing and can assist with correct localization of the tube . however , in obese patients or those with neck scars or giant cervical masses , such an outpouching is usually unable to be seen . in these situations , the number of failed intubations , total number of attempts , intubation time , and adverse events during the entire procedure were recorded . the intubation time for a single attempt in each group was defined as the time that had elapsed between insertion of the laryngoscope or endotracheal tube into the oral cavity and complete withdrawal of the core of the endotracheal tube . the spo2 , mean arterial pressure ( map ) , and heart rate ( hr ) were monitored , and the presence of any oropharyngeal mucosal bleeding caused by intubation , sore throat , hoarseness , throat dryness and discomfort , and upper respiratory trauma were recorded . all data were processed using spss 13.0 software ( spss inc . , chicago , il , usa ) . for continuous variables , the test was used for categorical variables , and data are presented as number ( percentage ) after approval by our institution s research ethics committee , informed consent was obtained from all patients . the inclusion criteria were : 1 ) american society of anesthesiologists ( asa ) physical status of i or ii in accordance with the definitions of the asa physical status ( asa ps ) class levels , 2 ) age 18 years , 3 ) no serious cardiovascular or pulmonary disease , 4 ) requirement for general anesthesia and et , and 5 ) no coagulation dysfunction . the exclusion criteria were : 1 ) history of oral , ear , nose , or throat operations ; 2 ) upper respiratory disease ( e.g. , tumors , polyps , severe inflammation or trauma , abscesses , or foreign objects ) ; 3 ) symptoms of sore throat or hoarseness ; 4 ) a mouth opening of < 1.5 cm ; 5 ) inability to tolerate mask ventilation ; and 6 ) anticipated requirement for > 5 h of anesthesia . the modified mallampati scoring system utilizes the following criteria as previously described : class i : visible soft palate , uvula , fauces , and pillars ; class ii : visible soft palate , uvula , and fauces ; class iii : visible soft palate and base of uvula ; and class iv : visible hard palate only . a total of 300 patients who underwent various operations from march 2012 to september 2012 met the inclusion criteria . according to the operation sequence , the patients were randomly divided into the laryngoscope group and blind group in a 1-vs.-2 style in which 1 patient was assigned to the conventional direct laryngoscope et group ( n=100 ; model h33060 shanghai medical devices co. , ltd . , shanghai , china ) and 2 patients were assigned to the pyriform sinus - assisted blind et group ( n=200 ) . all patients were required to fast for at least 8 h before the operation , and no premedication was given . upon arrival in the operating room , preoperative clinical data were recorded ( table 1 ) . standard monitoring was instituted , including noninvasive blood pressure measurement , pulse oximetry , electrocardiography , capnography , and body temperature measurement . after adequate preoxygenation , the patients were induced with 0.1 mg / kg of midazolam , 5.0 g / kg of fentanyl , 1.0 mg / kg of propofol , and 0.1 mg / kg of vecuronium . plastic endotracheal tubes with inside diameter ( i d ) of 7.5 mm ( male patients ) and 7.0 mm ( female patients ) were used . patients in the laryngoscopy group were intubated with conventional direct laryngoscopic orotracheal intubation , as previously described . patients in the blind group were intubated with pyriform sinus localization - assisted blind orotracheal intubation . the key points of this method are : 1 ) shape of endotracheal tube : the proximal end of an endotracheal tube with an ordinary core was bent to an angle of 90135 in a j-shape . the length of the tube at the proximal end was equal to the patient s thyromental distance . the arc of the tube was nearly equal to the angle between the thyromental line and the laryngeal axis ( figure 2 ) . the patient was placed in the supine position without a pillow and with the head slightly extended , or the mandibular angle of the patient was pushed upward to keep the mouth open . 3 ) intubation : the j-shaped tracheal tube was inserted into the mouth from the midline , close to the posterior region of the tongue . when the proximal end of the tube reached the posterior pharyngeal wall , it was swung to one side for placement at a 1015 angle and continuously advanced until it encountered resistance , which indicated that the tube had reached the homolateral pyriform sinus ( figure 3 ) . the tube was then withdrawn 58 mm , and the proximal end of the tube was swung back to midline and continuously advanced as the core was pulled out of the tube . a sense of emptiness indicated that the tube had successfully entered the trachea ( figure 4 ) . 4 ) fixation of the tube : after the endotracheal tube was connected to the anesthesia machine and correct position had been ensured by auscultation and confirmation of the end - tidal carbon dioxide partial pressure , it was fixed using the conventional method as previously described . the following 2 key points should be kept in mind when implementing this technique : 1 ) shape of the endotracheal tube : the angle between the oral and laryngeal axes changes with head movement . additionally , the thyromental line is usually parallel with the oral axis . we usually determine the radius of the endotracheal tube according to the angle between the thyromental line and laryngeal axis . this angle is usually 90135 and j-shaped , and it increases with extension of the head . 2 ) location of pyriform sinus : when the proximal end of the endotracheal tube reaches the posterior pharyngeal wall , the tube should be swung to one side for 1015 angle placement and continuously advanced . in this way if resistance is felt , the tube has most likely reached the homolateral pyriform sinus . at this point , the tube should be slightly withdrawn and swung to the midline , after which the tube can be easily inserted into the trachea . 3 ) a localized outpouching of the neck can be seen when the proximal end of the endotracheal tube touches the lateral wall of the pyriform sinus . this outpouching occurs at the posterolateral aspect of the thyroid cartilage wing and can assist with correct localization of the tube . however , in obese patients or those with neck scars or giant cervical masses , such an outpouching is usually unable to be seen . in these situations , at the end of the operation , any residual neuromuscular blockade was reversed and the patient was extubated . the number of failed intubations , total number of attempts , intubation time , and adverse events during the entire procedure were recorded . the intubation time for a single attempt in each group was defined as the time that had elapsed between insertion of the laryngoscope or endotracheal tube into the oral cavity and complete withdrawal of the core of the endotracheal tube . the spo2 , mean arterial pressure ( map ) , and heart rate ( hr ) were monitored , and the presence of any oropharyngeal mucosal bleeding caused by intubation , sore throat , hoarseness , throat dryness and discomfort , and upper respiratory trauma were recorded . all data were processed using spss 13.0 software ( spss inc . , chicago , il , usa ) . for continuous variables , the data are presented as mean standard deviation . the independent 2-sample t - test the test was used for categorical variables , and data are presented as number ( percentage ) one hundred patients were assigned to the laryngoscopy group and 200 patients were assigned to the blind group . demographic and clinical characteristics of patients included sex , age , height , weight , preoperative evaluation results , asa status , and modified mallampati score . this information is summarized in table 1 ; there were no significant differences in any of these parameters between both groups ( p>0.05 ) . the overall intubation success rate was 100% in the blind group and 99% in the laryngoscopy group ( p=0.33 ) . the first - time success rate was 78.5% and 85.0% ( p=0.18 ) , and the second- and third - time success rate was 21.5% and 14.0% ( p=0.12 ) in the blind and laryngoscopy groups , respectively . no statistically significant differences in the success rates were seen between the 2 groups . in patients with mallampati scores of iii to iv , the success rate was 100% and 90% in the blind and laryngoscopy groups , respectively , with little difference between groups ( p=0.39 ) ; however , 1 failed intubation in the laryngoscopy group was subsequently successfully performed using the blind intubation method . intubation time was significantly shorter in the blind group than in the laryngoscopy group ( 9.73.4 vs. 23.05.8 s , respectively ; p<0.001 ) ( table 2 ) . the patients respiratory and hemodynamic changes were evaluated by monitoring spo2 , map , and hr . spo2 in both groups was maintained at > 98% ; map and hr increased upon completion of intubation , but quickly returned to normal levels . there were no significant differences between groups ( map , p=0.16 ; hr , p=0.21 ) ( table 3 ) . incidence of oral and throat bleeding was significantly lower in the blind group than in the laryngoscopy group ( 1% vs. 6% , respectively ; p=0.03 ) . sore throat was a postoperative complaint in 1.5% of patients in the blind group and 23.0% in the laryngoscopy group ( p<0.001 ) . time required for recovery from these complications was significantly shorter in the blind group than in the laryngoscopy group ( p=0.004 ) . at 12 h after endotracheal extubation , incidences of bleeding and sore throat were 0% and 1% in the blind group and 1% and 8% in the laryngoscopy group , respectively . at 24 h after extubation , incidences of bleeding and sore throat were 0.0% and 0.5% in the blind group and 0% and 2% in the laryngoscopy group , respectively . incidences of hoarseness and throat discomfort were similar in both groups at all time points ( table 4 ) . one hundred patients were assigned to the laryngoscopy group and 200 patients were assigned to the blind group . demographic and clinical characteristics of patients included sex , age , height , weight , preoperative evaluation results , asa status , and modified mallampati score . this information is summarized in table 1 ; there were no significant differences in any of these parameters between both groups ( p>0.05 ) . the overall intubation success rate was 100% in the blind group and 99% in the laryngoscopy group ( p=0.33 ) . the first - time success rate was 78.5% and 85.0% ( p=0.18 ) , and the second- and third - time success rate was 21.5% and 14.0% ( p=0.12 ) in the blind and laryngoscopy groups , respectively . no statistically significant differences in the success rates were seen between the 2 groups . in patients with mallampati scores of iii to iv , the success rate was 100% and 90% in the blind and laryngoscopy groups , respectively , with little difference between groups ( p=0.39 ) ; however , 1 failed intubation in the laryngoscopy group was subsequently successfully performed using the blind intubation method . intubation time was significantly shorter in the blind group than in the laryngoscopy group ( 9.73.4 vs. 23.05.8 s , respectively ; p<0.001 ) ( table 2 ) . the patients respiratory and hemodynamic changes were evaluated by monitoring spo2 , map , and hr . spo2 in both groups was maintained at > 98% ; map and hr increased upon completion of intubation , but quickly returned to normal levels . there were no significant differences between groups ( map , p=0.16 ; hr , p=0.21 ) ( table 3 ) . incidence of oral and throat bleeding was significantly lower in the blind group than in the laryngoscopy group ( 1% vs. 6% , respectively ; p=0.03 ) . sore throat was a postoperative complaint in 1.5% of patients in the blind group and 23.0% in the laryngoscopy group ( p<0.001 ) . time required for recovery from these complications was significantly shorter in the blind group than in the laryngoscopy group ( p=0.004 ) . at 12 h after endotracheal extubation , incidences of bleeding and sore throat were 0% and 1% in the blind group and 1% and 8% in the laryngoscopy group , respectively . at 24 h after extubation , incidences of bleeding and sore throat were 0.0% and 0.5% in the blind group and 0% and 2% in the laryngoscopy group , respectively . incidences of hoarseness and throat discomfort were similar in both groups at all time points ( table 4 ) . results of the present study indicate that pyriform sinus localization - assisted blind et has a success rate similar to that of conventional direct laryngoscopic et . however , intubation time using this method was significantly shorter in the present study . the mean intubation time was 9.7 s for blind intubation and 23.0 s for laryngoscopic intubation . thus , the blind intubation method clearly saves time , which is particularly important for patients in emergency situations . however , we found no statistically significant difference in intubation success rate between the 2 methods . this result was most likely due to the fact that only a small number of patients with difficult airways were enrolled in this study . sixteen patients with mallampati scores of iii underwent blind intubation , and all of them were successfully intubated . one patient underwent failed intubation using the conventional method , but was finally successfully intubated using the blind intubation method . based on the results of the current study , we suggest that the blind intubation method may be more beneficial than the conventional method in management of patients with difficult airways , most likely because conventional laryngoscopic intubation is easily affected by many patient - related factors . these risk factors include degree of mouth opening , neck range of motion , amplitude of jaw protrusion , mallampati score , thyromental distance , obesity , and presence of oropharyngeal secretions [ 2023 ] . however , the relative anatomical position of the pyriform sinus and throat are very stable , and this relatively fixed position is not strongly affected by the above - mentioned factors , causing potential difficulty in laryngoscopic visualization . thus , blind intubation is easier to perform than laryngoscopic intubation , even in patients with difficult airways . j-shaped tracheal tube on one side of the pyriform sinus ; accurate pyriform sinus localization is the key to successful and rapid intubation with this method . thus , pyriform sinus localization - assisted blind intubation is more efficient and time - saving than conventional laryngoscopic intubation , especially in patients with difficult airways . , spo2 levels were maintained at 98% in both groups , suggesting that both methods are able to ensure normal oxygenation during intubation . upon completion of intubation , the map and hr in both groups were higher than their preintubation baseline levels , but these values quickly returned to baseline levels 3 min after intubation . this suggests that although both methods are associated with changes in hemodynamic levels during intubation , these changes are rapidly recovered . our study also revealed that complications associated with the blind method are significantly less severe than those associated with the conventional method . blind intubation allows for the avoidance of complications usually seen with laryngoscopic intubation , such as oral bleeding , injury to surrounding tissue , and tooth loss . complaints of sore throat were more common in patients who underwent laryngoscopic intubation than in those who underwent blind intubation in the present study , and the recovery time from these symptoms was longer in the laryngoscopy group . although we found no statistically significant complications in patients who underwent blind intubation , we are unable to conclude that there are no complications related to this procedure . comprehensive clinical trials are required for precise determination of drawbacks and weaknesses of this method . in addition , we do not recommend application of this method in patients with upper respiratory tract disease , including tumors , cysts , severe infection or trauma , foreign objects , or severely limited degree of mouth opening . a new airway device , the supraglottic airway laryngopharyngeal tube ( s.a.l.t . ) , has been developed to assist with blind orotracheal intubation . evaluated the adjunctive values of the s.a.l.t . in 30 adult patients who underwent blind orotracheal intubation . they reported that only 40% of the patients were successfully intubated and thus suggested that the s.a.l.t has limited value as an adjunctive device in assisting blind orotracheal intubation . pyriform sinus localization - assisted blind orotracheal intubation has shown to be more effective than conventional laryngoscopic et with regard to success rate , intubation time , and postoperative complications .

backgroundconventional endotracheal intubation requires laryngoscopy for a direct view of the glottis . however , laryngoscopy is associated with many potential complications . the aim of the present study was to compare the efficacy and safety of pyriform sinus localization - assisted blind orotracheal intubation with those of conventional laryngoscopic orotracheal intubation.material/methodsa randomized , patient - blind , prospective study of 300 patients who underwent various operations was performed . one hundred patients were assigned to the laryngoscopic intubation group ( laryngoscopy group ) , and 200 patients were assigned to the blind intubation group ( blind group).resultsthe total intubation success rate in the blind group was similar to that in the laryngoscopy group ( 100% vs. 99% , respectively ; p=0.33 ) . oxygen saturation by pulse oximetry in both groups was maintained at > 98% . the intubation time was significantly shorter in the blind group than in the laryngoscopy group ( 9.73.4 s vs. 23.05.8 s , respectively ; p<0.001 ) . postoperative complication rates were significantly lower in the blind group than in the laryngoscopy group . recovery time from these symptoms was significantly shorter in the blind group than in the laryngoscopy group ( p=0.004).conclusionspyriform sinus localization - assisted blind orotracheal intubation was shown to be more effective than conventional laryngoscopic orotracheal intubation in terms of success rate , intubation time , and postoperative complication rate . moreover , it is less affected by common risk factors ; thus , this method may be more beneficial in patients with difficult airways .

Background Material and Methods Patient selection Preoperative evaluation and anesthesia Intubation procedures Postoperative evaluation Statistical analysis Results Preoperative demographic and clinical characteristics Intubation outcomes Respiratory and hemodynamic monitoring Complications Discussion Conclusions

conventional et requires laryngoscopy to clearly visualize the glottis . however , laryngoscopy is associated with many potential complications , including significant hemodynamic changes , tooth loss , oropharyngeal injury , cricoarytenoid joint dislocation , infection , and brain damage . although the use of visualization technology in recent years has significantly reduced complications and difficulties associated with laryngoscopy and et [ 710 ] , the use of such technology has been greatly limited by high costs , low availability of proficient operators , and interference by oropharyngeal secretions . moreover , laryngoscopy may not always be available in a timely manner in urgent situations . it is also easily affected by anesthesia and the patient s state of consciousness . because of the angle between the thyromental line and laryngeal axis , this method is usually contraindicated in clinical practice . due to the aforementioned difficulties encountered during et , a novel blind orotracheal intubation method ( zhong s blind orotracheal intubation ) was created with the assistance of pyriform sinus localization . on either side of the laryngeal orifice the pyriform sinus is a subsite of the hypopharynx and has a fixed anatomical position in relation to the throat and rima glottidis . the aim of the present study was to compare the efficacy and safety of pyriform sinus localization - assisted blind orotracheal intubation with those of conventional laryngoscopic orotracheal intubation . the study revealed that blind orotracheal intubation with the assistance of pyriform sinus localization is equally or slightly more effective than conventional laryngoscopic orotracheal intubation . the inclusion criteria were : 1 ) american society of anesthesiologists ( asa ) physical status of i or ii in accordance with the definitions of the asa physical status ( asa ps ) class levels , 2 ) age 18 years , 3 ) no serious cardiovascular or pulmonary disease , 4 ) requirement for general anesthesia and et , and 5 ) no coagulation dysfunction . a total of 300 patients who underwent various operations from march 2012 to september 2012 met the inclusion criteria . according to the operation sequence , the patients were randomly divided into the laryngoscope group and blind group in a 1-vs.-2 style in which 1 patient was assigned to the conventional direct laryngoscope et group ( n=100 ; model h33060 shanghai medical devices co. , ltd . , shanghai , china ) and 2 patients were assigned to the pyriform sinus - assisted blind et group ( n=200 ) . standard monitoring was instituted , including noninvasive blood pressure measurement , pulse oximetry , electrocardiography , capnography , and body temperature measurement . after adequate preoxygenation , the patients were induced with 0.1 mg / kg of midazolam , 5.0 g / kg of fentanyl , 1.0 mg / kg of propofol , and 0.1 mg / kg of vecuronium . patients in the laryngoscopy group were intubated with conventional direct laryngoscopic orotracheal intubation , as previously described . patients in the blind group were intubated with pyriform sinus localization - assisted blind orotracheal intubation . the length of the tube at the proximal end was equal to the patient s thyromental distance . the patient was placed in the supine position without a pillow and with the head slightly extended , or the mandibular angle of the patient was pushed upward to keep the mouth open . 3 ) intubation : the j-shaped tracheal tube was inserted into the mouth from the midline , close to the posterior region of the tongue . when the proximal end of the tube reached the posterior pharyngeal wall , it was swung to one side for placement at a 1015 angle and continuously advanced until it encountered resistance , which indicated that the tube had reached the homolateral pyriform sinus ( figure 3 ) . the tube was then withdrawn 58 mm , and the proximal end of the tube was swung back to midline and continuously advanced as the core was pulled out of the tube . 4 ) fixation of the tube : after the endotracheal tube was connected to the anesthesia machine and correct position had been ensured by auscultation and confirmation of the end - tidal carbon dioxide partial pressure , it was fixed using the conventional method as previously described . we usually determine the radius of the endotracheal tube according to the angle between the thyromental line and laryngeal axis . this angle is usually 90135 and j-shaped , and it increases with extension of the head . 2 ) location of pyriform sinus : when the proximal end of the endotracheal tube reaches the posterior pharyngeal wall , the tube should be swung to one side for 1015 angle placement and continuously advanced . 3 ) a localized outpouching of the neck can be seen when the proximal end of the endotracheal tube touches the lateral wall of the pyriform sinus . however , in obese patients or those with neck scars or giant cervical masses , such an outpouching is usually unable to be seen . in these situations , the number of failed intubations , total number of attempts , intubation time , and adverse events during the entire procedure were recorded . the intubation time for a single attempt in each group was defined as the time that had elapsed between insertion of the laryngoscope or endotracheal tube into the oral cavity and complete withdrawal of the core of the endotracheal tube . the spo2 , mean arterial pressure ( map ) , and heart rate ( hr ) were monitored , and the presence of any oropharyngeal mucosal bleeding caused by intubation , sore throat , hoarseness , throat dryness and discomfort , and upper respiratory trauma were recorded . the inclusion criteria were : 1 ) american society of anesthesiologists ( asa ) physical status of i or ii in accordance with the definitions of the asa physical status ( asa ps ) class levels , 2 ) age 18 years , 3 ) no serious cardiovascular or pulmonary disease , 4 ) requirement for general anesthesia and et , and 5 ) no coagulation dysfunction . a total of 300 patients who underwent various operations from march 2012 to september 2012 met the inclusion criteria . according to the operation sequence , the patients were randomly divided into the laryngoscope group and blind group in a 1-vs.-2 style in which 1 patient was assigned to the conventional direct laryngoscope et group ( n=100 ; model h33060 shanghai medical devices co. , ltd . , shanghai , china ) and 2 patients were assigned to the pyriform sinus - assisted blind et group ( n=200 ) . all patients were required to fast for at least 8 h before the operation , and no premedication was given . standard monitoring was instituted , including noninvasive blood pressure measurement , pulse oximetry , electrocardiography , capnography , and body temperature measurement . after adequate preoxygenation , the patients were induced with 0.1 mg / kg of midazolam , 5.0 g / kg of fentanyl , 1.0 mg / kg of propofol , and 0.1 mg / kg of vecuronium . patients in the laryngoscopy group were intubated with conventional direct laryngoscopic orotracheal intubation , as previously described . patients in the blind group were intubated with pyriform sinus localization - assisted blind orotracheal intubation . the length of the tube at the proximal end was equal to the patient s thyromental distance . the arc of the tube was nearly equal to the angle between the thyromental line and the laryngeal axis ( figure 2 ) . the patient was placed in the supine position without a pillow and with the head slightly extended , or the mandibular angle of the patient was pushed upward to keep the mouth open . 3 ) intubation : the j-shaped tracheal tube was inserted into the mouth from the midline , close to the posterior region of the tongue . when the proximal end of the tube reached the posterior pharyngeal wall , it was swung to one side for placement at a 1015 angle and continuously advanced until it encountered resistance , which indicated that the tube had reached the homolateral pyriform sinus ( figure 3 ) . the tube was then withdrawn 58 mm , and the proximal end of the tube was swung back to midline and continuously advanced as the core was pulled out of the tube . 4 ) fixation of the tube : after the endotracheal tube was connected to the anesthesia machine and correct position had been ensured by auscultation and confirmation of the end - tidal carbon dioxide partial pressure , it was fixed using the conventional method as previously described . we usually determine the radius of the endotracheal tube according to the angle between the thyromental line and laryngeal axis . this angle is usually 90135 and j-shaped , and it increases with extension of the head . 2 ) location of pyriform sinus : when the proximal end of the endotracheal tube reaches the posterior pharyngeal wall , the tube should be swung to one side for 1015 angle placement and continuously advanced . 3 ) a localized outpouching of the neck can be seen when the proximal end of the endotracheal tube touches the lateral wall of the pyriform sinus . however , in obese patients or those with neck scars or giant cervical masses , such an outpouching is usually unable to be seen . the number of failed intubations , total number of attempts , intubation time , and adverse events during the entire procedure were recorded . the intubation time for a single attempt in each group was defined as the time that had elapsed between insertion of the laryngoscope or endotracheal tube into the oral cavity and complete withdrawal of the core of the endotracheal tube . the spo2 , mean arterial pressure ( map ) , and heart rate ( hr ) were monitored , and the presence of any oropharyngeal mucosal bleeding caused by intubation , sore throat , hoarseness , throat dryness and discomfort , and upper respiratory trauma were recorded . the independent 2-sample t - test the test was used for categorical variables , and data are presented as number ( percentage ) one hundred patients were assigned to the laryngoscopy group and 200 patients were assigned to the blind group . the overall intubation success rate was 100% in the blind group and 99% in the laryngoscopy group ( p=0.33 ) . the first - time success rate was 78.5% and 85.0% ( p=0.18 ) , and the second- and third - time success rate was 21.5% and 14.0% ( p=0.12 ) in the blind and laryngoscopy groups , respectively . no statistically significant differences in the success rates were seen between the 2 groups . in patients with mallampati scores of iii to iv , the success rate was 100% and 90% in the blind and laryngoscopy groups , respectively , with little difference between groups ( p=0.39 ) ; however , 1 failed intubation in the laryngoscopy group was subsequently successfully performed using the blind intubation method . intubation time was significantly shorter in the blind group than in the laryngoscopy group ( 9.73.4 vs. 23.05.8 s , respectively ; p<0.001 ) ( table 2 ) . spo2 in both groups was maintained at > 98% ; map and hr increased upon completion of intubation , but quickly returned to normal levels . incidence of oral and throat bleeding was significantly lower in the blind group than in the laryngoscopy group ( 1% vs. 6% , respectively ; p=0.03 ) . sore throat was a postoperative complaint in 1.5% of patients in the blind group and 23.0% in the laryngoscopy group ( p<0.001 ) . time required for recovery from these complications was significantly shorter in the blind group than in the laryngoscopy group ( p=0.004 ) . at 12 h after endotracheal extubation , incidences of bleeding and sore throat were 0% and 1% in the blind group and 1% and 8% in the laryngoscopy group , respectively . at 24 h after extubation , incidences of bleeding and sore throat were 0.0% and 0.5% in the blind group and 0% and 2% in the laryngoscopy group , respectively . incidences of hoarseness and throat discomfort were similar in both groups at all time points ( table 4 ) . one hundred patients were assigned to the laryngoscopy group and 200 patients were assigned to the blind group . the overall intubation success rate was 100% in the blind group and 99% in the laryngoscopy group ( p=0.33 ) . the first - time success rate was 78.5% and 85.0% ( p=0.18 ) , and the second- and third - time success rate was 21.5% and 14.0% ( p=0.12 ) in the blind and laryngoscopy groups , respectively . no statistically significant differences in the success rates were seen between the 2 groups . in patients with mallampati scores of iii to iv , the success rate was 100% and 90% in the blind and laryngoscopy groups , respectively , with little difference between groups ( p=0.39 ) ; however , 1 failed intubation in the laryngoscopy group was subsequently successfully performed using the blind intubation method . intubation time was significantly shorter in the blind group than in the laryngoscopy group ( 9.73.4 vs. 23.05.8 s , respectively ; p<0.001 ) ( table 2 ) . spo2 in both groups was maintained at > 98% ; map and hr increased upon completion of intubation , but quickly returned to normal levels . incidence of oral and throat bleeding was significantly lower in the blind group than in the laryngoscopy group ( 1% vs. 6% , respectively ; p=0.03 ) . sore throat was a postoperative complaint in 1.5% of patients in the blind group and 23.0% in the laryngoscopy group ( p<0.001 ) . time required for recovery from these complications was significantly shorter in the blind group than in the laryngoscopy group ( p=0.004 ) . at 12 h after endotracheal extubation , incidences of bleeding and sore throat were 0% and 1% in the blind group and 1% and 8% in the laryngoscopy group , respectively . at 24 h after extubation , incidences of bleeding and sore throat were 0.0% and 0.5% in the blind group and 0% and 2% in the laryngoscopy group , respectively . incidences of hoarseness and throat discomfort were similar in both groups at all time points ( table 4 ) . results of the present study indicate that pyriform sinus localization - assisted blind et has a success rate similar to that of conventional direct laryngoscopic et . however , intubation time using this method was significantly shorter in the present study . the mean intubation time was 9.7 s for blind intubation and 23.0 s for laryngoscopic intubation . thus , the blind intubation method clearly saves time , which is particularly important for patients in emergency situations . however , we found no statistically significant difference in intubation success rate between the 2 methods . this result was most likely due to the fact that only a small number of patients with difficult airways were enrolled in this study . sixteen patients with mallampati scores of iii underwent blind intubation , and all of them were successfully intubated . one patient underwent failed intubation using the conventional method , but was finally successfully intubated using the blind intubation method . based on the results of the current study , we suggest that the blind intubation method may be more beneficial than the conventional method in management of patients with difficult airways , most likely because conventional laryngoscopic intubation is easily affected by many patient - related factors . these risk factors include degree of mouth opening , neck range of motion , amplitude of jaw protrusion , mallampati score , thyromental distance , obesity , and presence of oropharyngeal secretions [ 2023 ] . however , the relative anatomical position of the pyriform sinus and throat are very stable , and this relatively fixed position is not strongly affected by the above - mentioned factors , causing potential difficulty in laryngoscopic visualization . thus , blind intubation is easier to perform than laryngoscopic intubation , even in patients with difficult airways . j-shaped tracheal tube on one side of the pyriform sinus ; accurate pyriform sinus localization is the key to successful and rapid intubation with this method . thus , pyriform sinus localization - assisted blind intubation is more efficient and time - saving than conventional laryngoscopic intubation , especially in patients with difficult airways . , spo2 levels were maintained at 98% in both groups , suggesting that both methods are able to ensure normal oxygenation during intubation . upon completion of intubation , the map and hr in both groups were higher than their preintubation baseline levels , but these values quickly returned to baseline levels 3 min after intubation . our study also revealed that complications associated with the blind method are significantly less severe than those associated with the conventional method . blind intubation allows for the avoidance of complications usually seen with laryngoscopic intubation , such as oral bleeding , injury to surrounding tissue , and tooth loss . complaints of sore throat were more common in patients who underwent laryngoscopic intubation than in those who underwent blind intubation in the present study , and the recovery time from these symptoms was longer in the laryngoscopy group . although we found no statistically significant complications in patients who underwent blind intubation , we are unable to conclude that there are no complications related to this procedure . in addition , we do not recommend application of this method in patients with upper respiratory tract disease , including tumors , cysts , severe infection or trauma , foreign objects , or severely limited degree of mouth opening . , has been developed to assist with blind orotracheal intubation . in 30 adult patients who underwent blind orotracheal intubation . they reported that only 40% of the patients were successfully intubated and thus suggested that the s.a.l.t has limited value as an adjunctive device in assisting blind orotracheal intubation . pyriform sinus localization - assisted blind orotracheal intubation has shown to be more effective than conventional laryngoscopic et with regard to success rate , intubation time , and postoperative complications .

neurotensin ( nt ) is a tridecapeptide ( glu - leu - tyr - glu - asn - lys - pro - arg - arg - pro - tyr - ile - leu ) that was identified 40 years ago from bovine hypothalamus . nt functions as a neurotransmitter and neuromodulator demonstrating a range of biological actions . in the cns , nt is colocalized with and regulates the action of mesolimbic and nigrastriatal dopamine as well as mediating nonopioid analgesia and hypothermia . it is believed that nt operates primarily through interaction with two g - protein coupled receptors nts1 and nts2 ( also referred to as ntr1 , ntr2 , ntrh , ntrl ) to regulate these activities . a third receptor , nts3 , also known as sortilin , is a type i receptor bearing a single transmembrane domain . the ability of the nt receptor system to regulate cns dopamine led researchers to postulate that nt might be an endogenous neuroleptic and that drugs acting via the nt system might therefore be useful as antipsychotic agents . methamphetamine also produces profound disruption of dopamine flow in the mesolimbic and nigrastriatal dopamine networks with chronic methamphetamine exposure , eliciting behaviors resembling schizophrenia . it is thus no surprise that compounds acting via the nt network are now being investigated as potential treatments for methamphetamine abuse . the nonopioid analgesia mediated by the nt system has also generated much interest over the years as a potential means of circumventing the side effects produced by opioid analgesics including addiction , constipation , and respiratory depression . nt - mediated analgesia has been demonstrated with compounds selective for both the nts1 and nts2 receptors as well as nonselective compounds . beyond this , there is now substantial evidence that both nts1 and nts2 can mediate relief from chronic or neuropathic pain . this type of pain is difficult to treat with current drugs and does not always respond well to opioid therapy . taken together with their neuroleptic activity , it is easy to understand why the development of compounds acting via the nt network has engendered so much interest . the nts1 receptor has received the most attention since the discovery of the nt system as it binds nt with high affinity , hence the name ntrh ( nt receptor high affinity ) , and is well behaved in heterologous expression systems . the nts2 receptor , on the other hand , has received less attention by comparison , and as a consequence fewer compounds have thus far been identified that interact with this receptor . as is true for nts1 , the most potent and selective nts2 compounds discovered to date have been obtained via modification of the nt(813 ) ( 1 ) fragment . discovery of potent and selective nonpeptide compounds however has proven elusive and remains as a significant challenge . several important milestone compounds from nt receptor research are depicted in chart 1 . the 813 fragment of nt , compound 1 , is as potent as the full length peptide , and several hexapeptide variants of 1 have been reported over the years that either favor nts2 over nts1 or are selective for nts2 versus nts1 . the first of these is jmv-431 ( 2 ) that was produced via reduction of the tyrosine 11 amide bond and shows a clear preference for nts2 . while it is not bioavailable , it is active in several models of chronic pain when delivered intrathecally . the peptide nt79 ( 3 ) , reported more recently , is selective for nts2 ( > 100-fold ) and has provided a wealth of information regarding the role of nts2 in animal models of pain , antipsychotic activity , thermoregulation , and regulation of blood pressure . like peptide 2 , this compound attained nts2 selectivity via modification of the tyrosine 11 residue . the most recent additions to the rolls of nts2-selective compounds are the potent peptide these compounds are ultraselective for nts2 , with selectivity ratios reaching 12000- and 22000-fold , respectively , with 4b also demonstrating excellent plasma stability . although few in number , there are three prominent nonpeptide compounds that interact with nts2 that have been used extensively in the characterization of the nt receptors . these include the two pyrazole - based compounds 5a ( sr48692 ) and sr142948a ( 5b ) and the histamine blocker levocabastine ( 6 ) . pyrazole compound 5a prefers nts1 to nts2 while 5b is nonselective , but they both antagonize the activity of nt at nts1 . compound 6 is selective for nts2 versus nts1 , but it is also a potent antagonist at histamine receptor 1 ( h1 ) . these compounds ( 5a , b and 6 ) highlight the fact that while nts2-selective peptides exist , selective nonpeptide compounds are rare . the sar of pyrazole compounds 5a and 5b has been described at nts1 but , to our knowledge , no systematic survey of sar has ever been described at nts2 . with the goal of discovering novel nts2 selective ligands , we undertook this endeavor by adapting a previously described calcium mobilization assay to our high throughput flipr tetra system and confirmed the functional results of active compounds using a radioligand binding assay . this effort led to the identification of a nonpeptide compound that is selective for nts2 versus nts1 , 1-({[1-(4-fluorophenyl)-5-(2-methoxyphenyl)-1h - pyrazol-3-yl]carbonyl}amino)cyclohexane carboxylic acid ( 7b ) , starting from the nonselective compound 5a . the newly discovered compound ( 7b ) displays neither calcium nor binding activity at nts1 , but is a potent partial agonist at nts2 ( ec50 of 12 6 nm and an emax of 7% relative to 5b ) that gave a ki of 153 10 nm in competition with [ i]nt . the target compounds , depicted in tables 2 and 3 , were synthesized as described in schemes 12 . scheme 1 illustrates the synthesis of the key intermediate pyrazole carboxylic acids ( 11a j ) . these were prepared as described by labeeuw , although improved methods were recently described by jiang et al . and also baxendale et al . this employed a knorr [ 3 + 2]-cyclization reaction between 4-aryl-2,4-diketoesters ( 8a f ) and arylhydrazines 9a c in acetic acid at reflux . the resulting esters 10a j were hydrolyzed using lioh and dioxane to give 11a j . the 4-aryl-2,4-diketoesters were commercially available with the exception of 8b . the 2,6-dimethoxy - butyrophenone used to synthesize compound 8b was prepared exactly as described by lindh . reagents and conditions : ( i ) hoac , hcl , and 9a ( 7-chloroquinolin-4-yl)hydrazinehcl ) ) or 9b ( 1-naphthylhydrazinehcl ) or 9c ( 4-fluorophenylhydrazinehcl ) , reflux 4 h ; ( ii ) lioh 3 equiv , dioxane , rt 16 h. in scheme 2 , the synthetic methods used to produce target compounds 7b , 13 , 14b29b , and 30 are described . thus , a given pyrazole carboxylic acid ( 11a j ) and amino acid ester ( 12a d ) or amine ( 12e ) from chart 2 were coupled using o - benzotriazol-1-yl - n , n , n,n-tetramethyluronium hexafluorophosphate ( hbtu ) and triethylamine to give ester intermediates 7a , and 14a29a and the descarboxy target compound 13 . the ester intermediates 7a , and 14a29a were hydrolyzed to give final products using either basic ( lioh and dioxane ) or acidic ( trifluoroacetic acid ( tfa ) in ch2cl2 ) conditions as shown . we used the tert - butyl rather than the methyl esters of l - cyclohexylglycine ( 12d ) to avoid racemization of the chiral amino acid . the synthesis of target compound 30 was accomplished according to the method of qure , wherein pyrazole acid 11i was converted to its acid chloride using socl2 in toluene , followed by coupling of the acid chloride intermediate with the adamantyl amino acid 12f under schotten bauman conditions . reagents and conditions : ( i ) hbtu , et3n , ch2cl2 , 2-aminoadamantanehcl , rt , 16 h ( 12e ) ; ( ii ) socl2 , toluene , reflux , 3 h ; ( iii ) naoh , thf , 12f , rt , 16 h ; ( iv ) hbtu , et3n , ch2cl2 , amino acid ester 12d , rt , 16 h ; ( v ) tfa , ch2cl2 , rt , 16 h ; ( vi ) hbtu , et3n , ch2cl2 , amino acid ester 12a c , rt , 16 h ; ( vii ) lioh , dioxane , rt , 16 h. the binding affinities of the test compounds for the rnts1 and rnts2 receptors listed in tables 1 through 3 were determined using previously reported competitive binding assays . the cells used in the binding assay were cho - k1 cells ( american type culture collection ) engineered to overexpress either the rnts1 or rnts2 receptor . measures of functional agonist and antagonist activity were obtained by measuring changes in intensity of a calcium - sensitive fluorescent dye as an indirect measure of changes in internal calcium concentrations . these measurements were performed using a flipr tetra ( nts2 ) or a flexstation ii plate reader for nts1 ( molecular devices ) and were analyzed using graphpad prism software . antagonist activity was measured as inhibition of nt - induced calcium release ( nts1 ) or inhibition of 5b - induced calcium release ( nts2 ) . in this article , we provide the details of our effort to identify nonpeptide compounds that are selective for nts2 starting from the nonselective compound 5a . this included developing an assay capable of detecting activity at the nts2 receptor based upon literature reports that compound 5a is an agonist at nts2 ( induced calcium mobilization at the nts2 receptor ) . a summary of the key sar elements of 5a discovered using the nts2 calcium mobilization assay that led to the identification of the potent partial agonist 7b ( ntrc-739 ) are provided in table 2 and includes the nts2 ec50 as well as the nts1 ke and the nts2 binding affinity ( ki ) . each of the appended molecular regions of 5a is discussed herein and includes : the dimethoxyphenyl ring , the amino acid side chain , the 7-chloroquinoline ring , and the 4-position of the pyrazole ring . the data obtained from the testing of nt reference compounds in this assay , including nt , 1 , 5a , b and levocabastine ( 6 ) , are provided in table 1 . ec50 , ke , ic50 , and ki values are all nm sem . in attempting to develop a method of screening for nts2 activity , we observed that the literature reports dealing with functional assays of nts2 receptors yielded contradictory data , exhibiting cell - type expression- and species - dependent pharmacological properties with opposing patterns . indeed , nt has been reported to be an agonist , an inverse agonist , and a neutral antagonist depending upon the cell expression system . similar findings were reported for compounds 5a , 5b , and 6 as well when tested alongside nt in these systems . specifically , the potent nts1 antagonists 5a and 5b were found to be agonists at nts2 as they mobilized calcium release while levocabastine ( 6 ) was found to behave either as an agonist , antagonist , or even as a partial inverse agonist . while the information above illustrated that there was much inconsistency between cell lines and expression systems for nts2 , we believed that each system would produce internally consistent ( reproducible ) data . we moved forward with the working hypothesis that the calcium mobilization described by these various laboratories was indeed nts2-mediated and chose one system to study in greater detail . as we already had the cho - k1-rnts2 cell line available from previous work , this became the obvious choice . we studied the calcium release produced by analogues of the nonpeptide compound 5a by measuring their ability to either stimulate release of calcium or to block the calcium release stimulated by 5b and found that it responded to changes in structure ( sar ) , table 2 . this information was compared with that obtained for ( nt and 1 ) as well as levocabastine , which revealed that this assay was capable of identifying a range of activities from potent agonist ( 5a and 5b ) to partial agonist ( 6 ) to antagonist ( nt and 1 ) , table 1 below , which implied that the structure of the compound made a direct impact on the resulting mobilization of calcium . this indicated that our hypothesis was correct and that we could identify nts2 agonists directly and nts2 antagonists indirectly against the agonist activity of 5b . further support for our hypothesis was obtained by collecting the binding affinity data in competition with [ i]nt for our study set of analogues of 5a , table 2 . these data revealed that compounds that modulated calcium release also competed with nt for binding at nts2 . involvement of nts2 was implicated further by the observation that compounds that mobilized calcium release in the cho - k1-rnts2 cell line did not produce calcium mobilization in the parent cho line . taken together , these data indicated that this assay method could be used to steer sar studies in search of novel nts2 selective compounds . as described above , the baseline for our study was established using the compounds common to nt receptor research . in table 1 , we have summarized the calcium release and binding data obtained for nt , 1 , 5a , and 5b , and the nts2-selective compound levocabastine ( 6 ) in both our rnts1 and rnts2 cell lines . in our cho - k1-nts2 cell line , neither nt nor 1 showed any calcium release in our flipr assay as previously reported . furthermore , we did not observe constitutive activity in this expression system as has been reported by other investigators using alternate expression systems . on the other hand , both 5a and 5b stimulated calcium release with ec50 values of 120 and 20 nm , respectively . compound 5b was more potent than compound 5a in the calcium mobilization assay but equally efficacious , and was designated as our nts2 full agonist standard ( the use of the term agonist here refers to a compound that stimulates calcium release in this assay ) . levocabastine ( 6 ) , a compound used for years to distinguish nts1 from nts2 , was also tested and found to be a potent partial agonist with an ec50 of 28 nm and an emax of 16% relative to 5b . neurotensin and 1 did not stimulate calcium release , but they both blocked the calcium release stimulated by compound 5b in an insurmountable manner as they showed a rightward shift in the dose response curve with an accompanying dose - dependent drop in the emax value of 5b . we thus determined and report here the ic50 for both nt and 1 in competition with 5b and found these to be 18.9 and 5.4 nm , respectively . we found that compound 5a gave a ki of 62 nm , while 5b showed a 10-fold higher affinity with a ki of 6 nm . as in the flipr assay , compound 5b has a higher affinity than 5a . nt gave a ki of 18.5 nm . compound 1 showed similar affinity to nt , while levocabastine ( 6 ) gave a ki of 33 nm . the binding affinities found for nt , 1 , and levocabastine are slightly higher than those found in other laboratories , while those for 5a and b are similar . we attribute this to our use of an in - plate whole cell binding method as opposed to the use of membrane preparations . relating the functional behaviors observed in vitro for these reference compounds to their demonstrated in vivo behaviors is instructive from a drug discovery perspective . nt possesses antipsychotic activity and mediates nonopioid analgesia , while both nt and levocabastine ( 6 ) are active in models of persistent pain . the pyrazole compounds 5a and 5b , on the other hand , are not analgesics in vivo but instead block the analgesic activity of nt in animal models of pain . this is also observed in animal models based on runaway mesolimbic dopamine where the neuroleptic action of nt is blocked by compounds 5a and 5b . according to the functional data in table 1 , the proven analgesic and antipsychotic compounds appeared as either antagonists of calcium mobilization ( nt and peptide 1 ) or potent partial agonists of calcium mobilization ( levocabastine , 6 ) , while the compounds that possess neither analgesic nor antipsychotic activity in vivo , compounds 5a and 5b , appeared as potent agonists . this point of reference provided a means of parsing in vitro active compounds into two categories , those that might possess desirable behaviors in animal models and those that might not . as this data set is small , we set out to buttress these relationships using our functional assay to identify additional examples of novel nts2-selective antagonists and potent partial agonists . the results obtained in our sar study to find a potent partial agonist based on compound 5a were centered on the three pyrazole scaffolds a we began by examining the effect of deleting the carboxylic acid group , compound 13 , as the carboxyl group is known to be a primary attachment point for ligands of the nt receptors . we found that the descarboxy derivative 13 was inactive in both nts1 and nts2 flipr assays and gave a ki > 11 m in the radioligand binding assay . this behavior stands in stark contrast to the activity found for 5a and provided additional evidence that the calcium release observed for 5a ( and 5b ) results from these ligands interacting with nts2 . we chose to use compound 14b as a surrogate for compound 5a because it was easier to produce compound libraries using the l - cyclohexyl glycine amino acid compared with the adamantyl amino acid . compound 14b , like 5a , was previously reported to be a potent nts1 antagonist by qur and thus was expected to be an agonist in the nts2 . the l - isomer was chosen as it is known to be more active than the r isomer . the data from 14b indicated that it would be a suitable stand in for 5a as it provided comparable agonist potency and efficacy for nts2 ( ec50 of 217 nm and emax 86% of 5b ) and nts1 antagonist activity ( ke of 23 nm ) . the 10-fold difference in its relative binding affinity at nts2 illustrated to us that the structure of the amino acid side chain significantly impacts binding affinity . a comparison of the data obtained for compounds 14b18b ( table 2 ) illustrates the sar realized from changing the position of the 6-methoxy group ( 14b , 15b , and 16b ) , elimination of the 6-methoxy group ( 17b ) , and replacing the 2,6-methoxy groups with fluorine atoms ( 18b ) . we found that the nts1 antagonist activity decreased across the series of positional isomers 14b16b with ke values of 23 nm , 1275 nm , and > 10 m , respectively . this trend continued for 17b and the difluoro derivative ( 18b ) , with ke values of 1682 nm and > 10 m . their nts2 efficacy ( emax ) also fell across this series of compounds while the functional potency ( ec50 ) was little varied . the binding affinities showed surprisingly little variation compared with the change seen going between 5a and 14b and reinforced the notion that the structure of the amino acid side chain has a strong impact on binding affinity and appeared to set the range of observed activity within the study group . taken together , we found that the potency of antagonist activity for 14b at the nts1 receptor relied heavily upon the 2,6-dimethoxyphenyl ring for its activity . at nts2 , however , it was the efficacy of calcium release that was most significantly affected by alteration of the 2,6-dimethoxyphenyl ring . the binding affinity data and nts2 potency were much less affected by changes to this molecular region . this in turn suggested that an nts2 potent partial agonist would not possess the 2,6-dimethoxyphenyl ring . the data from compound 19b illustrated the effect on in vitro activity produced by alkylation of the 4-position of the pyrazole ring . the changes seen here resembled the previous set of compounds as 19b showed lower nts1 antagonist relative to 14b and decreased efficacy at nts2 with less effect on nts2 potency and binding affinity . overall , this example illustrated that alkylation of this position provided compounds that favored nts2 activity over nts1 but with only modest levels of affinity at nts2 . collectively , the data suggested that this was not a substitution that would lead in the right direction . the amino acid side chain is known to have a strong influence on ligand behavior , presumably due to its proximity to the carboxyl group , the primary anchoring point of the ligand to the receptor . the data obtained for compounds 5a , 14b , and 20b23b in table 2 illustrate the sar of the large amino acid side chains . this group of compounds demonstrated that large cyclic ( cycloheptyl , 20b ) , large bicyclic ( adamantyl , 5a ) , and large pendant alicyclic ( 14b ) structures are potent nts1 antagonists with ke values of 42 , 4.7 , and 23 nm , respectively . compounds with smaller cyclic rings were somewhat less potent at nts1 , ke = 222 nm for the five - membered cyclic ring ( 22b ) and 157 nm for the six - membered cyclic ring ( 21b ) . according to these data then , the compounds with smaller cyclic rings ( 21b , 22b ) trended toward nts2 selectivity . compounds 20b22b also showed increased nts2 agonist potency ( lower ec50 ) , but this was accompanied by full efficacy ( high emax relative to 5b ) at nts2 , exactly opposite of that found in the potent partial agonist levocabastine ( 6 ) . in their favor , however , these same compounds ( 20b22b ) showed improved binding affinity at nts2 versus 14b ( ki = 170 , 151 , and 102 nm versus 644 nm for 14b ) , more in line with 5a ( ki = 62 nm ) . for these compounds , the binding affinity and nts2 potency improvement worked in concert with the lower nts1 activity to provide enhanced nts2 affinity and potency while maintaining low efficacy and lower nts1 activity . this comparison of nts2 agonist potency to the nts1 antagonist potency for compounds 21b and 23b was an important clue to achieving selectivity in this series of compounds as this data reinforced the data from the previous series , which showed that the nts1 receptor relied much more heavily upon the 2,6-dimethoxyphenyl ring for its activity compared with nts2 and that the amino acid side chain could work in concert with the methoxyphenyl ring to retain these desired properties . this trend was further advanced in compounds that did not possess the chloroquinoline group . two key substitutions for the 7-chloroquinoline group ( a ) were the naphthyl ( b ) and 4-fluorophenyl ( c ) groups , table 2 . these substitutions were included in our compound libraries because the naphthyl compounds were known to yield nts1 antagonists and the 4-fluorophenyl group was found in levocabastine ( 6 ) . like their 7-chloroquinolyl counterparts , the naphthyl - substituted 2,6-dimethoxyphenyl derivatives 24b and 26b possess potent nts1 antagonist activity and also nts2 agonist activity ; however , distinct differences were observed that led us in the right direction . most apparent was that these compounds were inherently less potent at nts1 and less efficacious at nts2 . the ke values for nts1 antagonist activity and the nts2 emax values were roughly half of that found for similarly substituted chloroquinoline - based compounds . this was observed for both the l - cyclohexyl glycine ( 24b ) and the 1-aminocyclohexancarboxylic acid side chains ( 26b ) . comparing the naphthyl ( 24b ) and the 7-chloroquinolyl ( 14b ) compounds , we found ke values of 58 versus 23 nm and nts2 emax values of 45 and 86% of 5b , respectively . comparing compounds 26b and 21b , we found ke values of 230 versus 157 nm and nts2 emax values of 35 and 78% , respectively . as seen in the 7-chloroquinolyl series ( a ) , the naphthyl - substituted 2-methoxyphenyl derivatives 25b and 27b were less potent nts1 antagonists than their 2,6-dimethoxyphenyl substituted counterparts . more surprising was the observation that the nts2 efficacies for these compounds were nearly half again as low as that found in similarly substituted 7-chloroquinolyl compounds 17b and 23b . this is readily revealed in a comparison of the nts2 efficacy data obtained for compound 21b , 23b , and the 2-methoxy naphthyl substituted compound 27b , with nts2 emax values of 78 , 35 , and 18% , respectively . the naphthyl - substituted compounds with the 2,6-dimethoxyphenyl ring were more potent at nts2 than their chloroquinoline - based counterparts ( 14b , 17b , 21b , and 23b ) , but naphthyl compounds bearing the 2-methoxyphenyl ring were equally potent . this was observed for compounds with the l - cyclohexyl glycine as well as those with 1-aminocyclohexancarboxylic acid as in 26b and 27b . the nts2 binding data observed for the naphthyl - substituted compounds 24b27b was found to be in line with that seen for the similarly substituted 7-chloroquinolyl - substituted compounds , with the 2-methoxy derivatives showing lower affinity ( higher ki values ) than the 2,6-dimethoxy counterparts . as before though , the compounds bearing the l - cyclohexyl glycine side chain ( 24b,25b ) were less potent than those with the 1-aminocyclohexancarboxylic side chain ( 26b,27b ) . toward obtaining an nts2 selective potent partial agonist , the calcium data profile for compound 26b looked promising as it moved closer to that of levocabastine ( 6 ) . the binding data was also similar , but in this case , compound 6 was more potent , showing a ki 3.5-fold greater than that for 26b . the similarity between these two compounds ended , however , when comparing receptor selectivity , as compound 26b showed substantial nts1 antagonist activity while levocabastine ( 6 ) displayed no activity at nts1 . the 2-methoxy naphthyl - substituted analogue 27b was not able to compensate for this deficit , for while its nts1 antagonist activity was lowered by 20-fold , the nts2 potency and binding affinity also suffered substantial losses . fortunately , the pyrazole scaffold ( c ) bearing the 4-fluorophenyl - substituent seen in compounds 30 , 28b , 29b , and 7b overcame the deficiencies found in scaffold b to provide selective potent partial agonists . comparison of the adamantyl - substituted compounds 5a and 30 highlights the contribution of the 4-fluorophenyl ring . these data show that the nts1 antagonist activity was diminished by 40-fold for 30 versus 5a , with ke values of 191 and 4.7 nm , respectively . the nts2 potency , on the other hand , was doubled ( ec50 values of 120 versus 68 nm ) while the efficacy was less than half relative to 5a ( emax values of 100 versus 34% of 5b ) . this phenomenon was also observed in the data for compounds 28b , 29b , and 7b . these compounds are all far less active at nts1 compared with their corresponding 7-chloroquinolyl ( 14b , 21b , and 23b ) or naphthyl ( 24b , 26b , and 27b ) analogues and thereby attain enhanced nts2 selectivity with respect to calcium release . the 4-fluorophenyl - substituted compounds ( 29b and 7b ) , bearing the 1-aminocyclohexancarboxylic acid substituent , showed the most levocabastine - like profiles whether they had a 2,6-dimethoxyphenyl ring ( 29b ) or a 2-methoxyphenyl ring ( 7b ) . in keeping with previous observations , the transition to the 2-methoxyphenyl ring ( 7b ) from the 2,6-dimethoxyphenyl ring ( 29b ) led to a nearly 50% reduction in nts2 efficacy , with nts2 emax values of 15 and 8% of 5b , respectively . the nts2 binding affinity observed for 29b and 7b was consistent with similarly substituted 7-chloroquinolyl ( 21b , 23b ) or naphthyl - substituted ( 26b , 27b ) analogues bearing the 1-aminocyclohexanecarboxylic acid , with one important exception ; for 7b , the binding affinity did not decrease in going from the 2,6-dimethoxyphenyl ( 29b ) to the 2-methoxyphenyl ring substitution ( 7b ) as seen in the ki values of 140 and 153 nm , respectively . we imagine that this change in sar for 29b and 7b could result from a change in binding mode at nts2 that is permitted for 4-fluorophenyl but not 7-chloroquinolyl or naphthyl - substituted analogues . in the end , the 4-fluorophenyl - substituted pyrazole scaffold c was able to overcome the deficits found in scaffolds a and b to provide pyrazole - based compounds ( 29b and 7b ) with enhanced nts2 potency and binding affinity with significantly lower efficacy compared to 5a at the nts2 receptor . from the standpoint of calcium mobilization at nts1 and nts2 , compounds ( 29b and 7b ) appear to be selective for the nts2 receptor . however , this was not a fair comparison , as one assay measures calcium mobilization ( nts2 ) while the other blockade of calcium mobilization ( nts1 ) . we therefore acquired the radioligand binding data for both 29b and 7b at nts1 in order to compare like measurements . as seen in table 3 , comparison of the relative binding affinities at nts1 and nts2 showed that compound 7b is 161-fold selective for nts2 versus nts1 while the dimethoxy analogue 29b shows only a 23-fold preference for nts2 over nts1 . these data point out that while the calcium assay described herein is useful for identifying compounds that are active at nts2 , conclusions about selectivity require the use of the binding assay . the identification of 7b as an nts2 selective compound demonstrated that this calcium assay was useful for driving sar studies in the pyrazole carboxamide series of compounds . however , the low efficacy of 7b , less than half of that found for levocabastine , prompted us to revisit mobilization of calcium in the parent cho cell line as a negative control . we thus carried out a final study and examined all of the compounds reported herein that showed nts2 emax values less than 15% of 5b . this was done collectively by comparing all of the low efficacy compounds together in the same assay , under identical conditions , in our cho - k1-nts2 cells and simultaneously in the parent cho line . the results of these experiments confirmed that the calcium mobilization observed was only found in the cho cells stably expressing the nts2 receptor . in summary , we tested the compounds common to neurotensin research and have identified their associated calcium mobilization patterns at nts1 and 2 using a flipr tetra using nt as our agonist standard for nts1 and 5b as our agonist standard for nts2 . we found that compounds known to possess analgesic and antipsychotic activity in vivo appear as antagonists ( nt and 1 ) ) or potent partial agonists ( levocabastine ( 6 ) ) in this nts2 in vitro assay . using the assays described above in combination with binding data , we identified structural changes to compound 5a that led to the discovery of the nts2 selective compound 7b , a pyrazole - based compound with in vitro properties similar to levocabastine in this assay , a compound known to be active against chronic pain . we are now working to identify the relevance of the functional data produced using this method by investigating compound 7b in recognized models of neuropathic pain in an attempt to establish an in vitro / in vivo correlation . reactions were conducted under a nitrogen atmosphere using oven - dried glassware as required . anhydrous tetrahydrofuran ( thf ) , dichloromethane ( dcm ) , and n , n - dimethylformamide ( dmf ) were purchased from vwr and used without further purification . most other reagents were obtained from either aldrich chemical corp . or fischer scientific and used without further purification . flash column chromatography was carried out using a teledyne isco combiflash rf system and redisep rf gold prepacked hp silica columns . characterization of compounds was accomplished by a combination of tlc , combustion analysis , mass spectrometry ( ms ) as well as h and c nmr . chemical shifts are reported in ppm relative to the tetramethylsilane , and coupling constant ( j ) values are reported in hertz ( hz ) . low - resolution mass spectra were obtained using a waters alliance ht / micromass zq system ( esi ) in both positive and negative mode . optical rotations were measured on an auto pol iii polarimeter at the sodium d line . thin layer chromatography ( tlc ) was performed on emd precoated silica gel 60 f254 plates , and spots were visualized with uv light and i2 or phosphomolybdic acid stain . neurotensin was from perkinelmer , calcium 5 dye was from molecular devices , and cell culture reagents were from life technologies . the graphs provided in figures 13 were obtained using graphpad prism software . a magnetically stirred solution of a 4-aryl-2,4-diketoester sodium salt ( 8a f , 5 mmol ) and arylhydrazine hydrochloride ( 9a c , 5 mmol ) in glacial acetic acid ( 35 ml ) and concd hcl ( 1.5 ml ) was heated under reflux for 5 h and then cooled to rt . this was then poured into 300 ml of water and extracted five times with ch2cl2 . the extracts were washed carefully with satd nahco3 and then water and brine then dried over na2so4 and concentrated to give a crude material . this material was purified using flash chromatograph with a gradient of 0100% etoac in hexanes over 30 min collecting peaks only . combination of desired fractions and evaporation provided methyl esters 10a j as foamy solids . pyrazole carboxylic acids 11a j were prepared from the esters ( 10a j ) using the methyl ester hydrolysis method described below . to a magnetically stirred suspension of the appropriate pyrazole carboxylic acid ( 11a j , 0.122 mmol ) in anhydrous ch2cl2 ( 20 ml ) were added successively triethylamine ( 0.366 mmol ) , hbtu ( 0.146 mmol ) , and the appropriate amino acid ester hydrochloride salt ( 12a e , 0.122 mmol ) . after stirring for 16 h , the resulting mixture was concentrated and the residue purified by flash chromatography using a gradient of 0100% etoac in hexanes over 30 min collecting peaks only . this gave an intermediate ester that was taken directly to the hydrolysis step . to a magnetically stirred solution of methyl ester ( 7a,2023a , 26a , 27a , and 29a ) from the coupling reaction ( 0.1 mmol ) in 1,4-dioxane ( 5 ml ) was added 1n lioh ( 2 ml ) , followed by stirring at room temperature overnight . the mixture was then concentrated , and the residue was taken up in water ( 2 ml ) and extracted with ethyl acetate ( 15 ml ) . after this , 2 n hcl was added to the aqueous layer to precipitate the carboxylic acid final product . this was extracted twice with ch2cl2 , and the combined extracts were dried over na2so4 and concentrated to give solid final products . to a magnetically stirred solution of the tert - butyl ester intermediate ( 1419a,24a,25a , and 28a ) obtained from the coupling reaction ( 0.08 mmol ) in ch2cl2 ( 10 ml ) was added excess tfa ( 10 ml ) at room temp . after stirring for 16 h , the mixture was concentrated and then triturated with ethyl ether to give a solid product that was isolated by vacuum filtration , washed with ether , and then dried under high vacuum overnight . following the general amide coupling procedure , 7a was prepared from 11j and 12b as an amorphous off - white solid ( 55% yield ) . h nmr ( cdcl3 ) 7.36 ( t , j = 7.8 hz , 1h ) 7.237.31 ( m , 3h ) , 7.18 ( s , 1h ) , 6.947.04 ( m , 3h ) , 6.80 ( d , j = 8.5 hz , 1h ) , 3.75 ( s , 3h ) , 3.48 ( s , 3h ) , 2.152.25 ( m , 2h ) , 1.882.02 ( m , 2h ) , 1.461.56 ( m , 6h ) . following the general methyl ester hydrolysis procedure , 7b was obtained from 7a as a white solid ( 85% ) . h nmr ( 300 mhz , cdcl3 ) 7.337.42 ( m , 1h ) , 7.187.31 ( m , 4h ) , 6.947.05 ( m , 4h ) , 6.81 ( d , j = 8.2 hz , 1h ) , 3.44 ( s , 3h ) , 2.28 ( d , j = 13.9 hz , 2h ) , 1.942.08 ( m , 2h ) , 1.331.83 ( m , 6h ) . c nmr ( cdcl3 ) 175.00 , 163.76 , 163.45 , 160.16 , 156.36 , 145.66 , 142.48 , 136.54 , 136.50 , 131.24 , 131.16 , 125.81 , 125.70 , 120.87 , 118.66 , 115.71 , 115.41 , 111.24 , 109.45 , 60.25 , 54.94 , 32.20 , 25.13 , 21.30 . ms ( esi ) m / z : 436.7 ( m h ) . ( c24h24fn3o4 ) c , h , n. ester 10a was prepared via the general procedure starting from methyl 4-(2,6-dimethoxyphenyl)-2,4-dioxobutanoate sodium salt ( 8a ) and 7-chloro-4-hydrazinylquinoline hydrochloride ( 9a ) to give 10a as a yellow foam ( 68% ) . h nmr ( cdcl3 ) 8.98 ( d , j = 5.2 hz , 1h ) , 8.88 ( d , j = 1.8 hz , 1h ) , 8.37 ( d , j = 9.2 hz , 1h ) , 7.82 ( dd , j = 1.8 , 9.2 hz , 1h ) , 7.45 ( d , j = 5.2 hz , 1h ) , 7.34 ( t , j = 8.4 hz , 1h ) , 7.157.24 ( m , 1h ) , 6.50 ( d , j = 8.4 hz , 2h ) , 4.01 ( s , 3h ) , 3.48 ( s , 6h ) . ester 10b was prepared from methyl 3-[(2,6-dimethoxyphenyl)carbonyl]-2-oxopentanoate , sodium salt ( 8b ) , and 9a according to the general method ( pale - yellow solid , 45% ) . h nmr ( cdcl3 ) 8.87 ( d , j = 4.8 hz , 1h ) , 8.18 ( d , j = 1.9 hz , 1h ) , 7.95 ( d , j = 9.1 hz , 1h ) , 7.50 ( d , j = 2.0 hz , 1h ) , 7.127.27 ( m , 2h ) , 6.42 ( d , j = 8.5 hz , 2h ) , 3.76 ( s , 3h ) , 3.50 ( s , 6h ) , 2.69 ( q , j = 7.4 hz , 2h ) , 1.17 ( t , j = 7.4 hz , 3h ) . ester 10c was prepared from methyl 4-(2,5-dimethoxyphenyl)-2,4-dioxobutanoate sodium salt ( 8c ) and 9a according to the general method ( pale - yellow solid , 57% ) . h nmr ( cdcl3 ) 8.77 ( d , j = 4.5 hz , 1h ) , 8.14 ( d , j = 2.1 hz , 1h ) , 7.91 ( d , j = 9.0 hz , 1h ) , 7.54 ( dd , j = 2.1 , 8.95 hz , 1h ) , 7.14 ( s , 1h ) , 7.01 ( d , j = 4.5 hz , 1h ) , 6.786.91 ( m , 2h ) , 6.57 ( d , j = 8.9 hz , 1h ) , 3.99 ( s , 3h ) , 3.73 ( s , 3h ) , 2.91 ( s , 3h ) . ester 10d was prepared from methyl 4-(2,4-dimethoxyphenyl)-2,4-dioxobutanoate sodium salt ( 8d ) and 7-chloro-4-hydrazinylquinoline hydrochloride ( 9a ) according to the general method ( yellow amorphous solid , 45% ) . h nmr ( cdcl3 ) 8.78 ( d , j = 4.6 hz , 1h ) , 8.14 ( d , j = 1.8 hz , 1h ) , 7.87 ( d , j = 9.0 hz , 1h ) , 7.52 ( dd , j = 2.0 , 9.0 hz , 1h ) , 7.20 ( d , j = 8.4 hz , 1h ) , 7.09 ( s , 1h ) , 7.02 ( d , j = 4.6 hz , 1h ) , 6.47 ( dd , j = 2.2 , 8.43 hz , 1h ) , 6.19 ( d , j = 2.1 hz , 1h ) , 3.98 ( s , 3h ) , 3.77 ( s , 3h ) , 2.99 ( s , 3h ) . ester 10e was prepared from methyl 4-(2,6-difluorophenyl)-2,4-dioxobutanoate sodium salt ( 8e ) and 9a according to the general method ( yellow amorphous solid , 52% ) . h nmr ( cdcl3 ) 8.87 ( d , j = 4.5 hz , 1h ) , 8.14 ( d , j = 1.9 hz , 1h ) , 7.617.75 ( m , 1h ) , 7.51 ( dd , j = 1.9 , 9.0 hz , 1h ) , 7.41 ( dd , j = 0.9 , 8.7 hz , 1h ) , 7.257.35 ( m , 1h ) , 7.22 ( d , j = 4.5 hz , 1h ) , 6.776.89 ( m , 2h ) , 4.01 ( s , 3h ) . ester 10f was prepared from methyl 4-(2-methoxyphenyl)-2,4-dioxo - butanoate sodium salt ( 8f ) and 9a according to the general method ( yellow solid , 43% ) . h nmr ( cdcl3 ) 8.78 ( d , j = 4.7 hz , 1h ) , 8.15 ( s , 1h ) , 7.91 ( d , j = 9.0 hz , 1h ) , 7.50 ( td , j = 1.0 , 9.0 hz , 1h ) , 7.277.32 ( m , 2h ) , 6.967.01 ( m , 2h ) , 6.65 ( d , j = 8.5 hz , 2h ) , 4.0 ( s , 3h ) , 4.0 ( s , 3h ) . ester 10 g was prepared from 8a and 1-naphthylhydrazine hydrochloride ( 9b ) according to the general method ( colorless foam , 55% ) . h nmr ( cdcl3 ) 7.677.83 ( m , 3h ) , 7.387.49 ( m , 2h ) , 7.227.33 ( m , 2h ) , 7.057.17 ( m , 2h ) , 6.34 ( d , j = 8.3 hz , 2h ) , 3.96 ( s , 3h ) , 3.41 ( br s , 6h ) . ester 10h was prepared from methyl 4-(2-methoxyphenyl)-2,4-dioxobutanoate sodium salt ( 8f ) and 9b according to the general method ( off - white solid , 40% ) . h nmr ( cdcl3 ) 7.787.88 ( m , 2h ) , 7.627.71 ( m , 1h ) , 7.447.53 ( m , 2h ) , 7.297.36 ( m , 1h ) , 7.247.29 ( m , 1h ) , 7.107.24 ( m , 3h ) , 6.81 ( dt , j = 0.8 , 7.5 hz , 1h ) , 6.64 ( d , j = 8.2 hz , 1h ) , 3.97 ( s , 3h ) , 3.13 ( s , 3h ) . ester 10i was prepared from 8a and 4-fluorophenyl hydrazine hydrochloride ( 9c ) according to the general method ( white amorphous powder , 55% ) . h nmr ( cdcl3 ) 7.227.33 ( m , 3h ) , 6.897.00 ( m , 3h ) , 6.51 ( d , j = 8.3 hz , 2h ) , 3.96 ( s , 3h ) , 3.59 ( s , 6h ) . ester 10j was prepared from 8f and 9c according to the general method ( off - white powder , 45% ) . h nmr ( cdcl3 ) 7.327.43 ( m , 1h ) , 7.237.32 ( m , 3h ) , 6.927.04 ( m , 4h ) , 6.81 ( d , j = 8.5 hz , 1h ) , 3.97 ( s , 3h ) , 3.44 ( s , 3h ) . pyrazole acid 11a was prepared from ester 10a according to the general methyl ester hydrolysis method ( pale - yellow powder , 78% ) . h nmr ( 300 mhz , dmso - d6 ) 8.90 ( d , j = 4.7 hz , 1h ) , 8.17 ( s , 1h ) , 7.73 ( s , 2h ) , 7.26 ( t , j = 8.4 hz , 1h ) , 7.20 ( d , j = 4.5 hz , 1h ) , 6.99 ( s , 1h ) , 6.54 ( d , j = 8.5 hz , 2h ) , 3.39 ( s , 6h ) . pyrazole acid 11b was prepared from ester 10b according to the general methyl ester hydrolysis method ( pale - yellow solid , 80% ) . h nmr ( cdcl3 ) 10.3611.51 ( m , 2h ) , 8.87 ( d , j = 4.8 hz , 1h ) , 8.19 ( d , j = 2.0 hz , 1h ) , 7.94 ( d , j = 9.0 hz , 1h ) , 7.49 ( dd , j = 2.0 , 9.0 hz , 1h ) , 7.197.32 ( m , 1h ) , 7.17 ( d , j = 4.7 hz , 1h ) , 6.42 ( d , j = 8.4 hz , 2h ) , 3.50 ( s , 6h ) , 2.68 ( q , j = 7.4 hz , 2h ) , 1.16 ( t , j = 7.4 hz , 3h ) . ms ( esi ) m / z : 436.5 ( m h ) . pyrazole acid 11c was prepared from ester 10c according to the general methyl ester hydrolysis method ( amorphous , pale - yellow solid , 94% ) . h nmr ( cdcl3 ) 8.80 ( d , j = 4.7 hz , 1h ) , 8.18 ( d , j = 1.9 hz , 1h ) , 7.91 ( d , j = 9.2 hz , 1h ) , 7.56 ( dd , j = 1.9 , 9.0 hz , 1h ) , 7.19 ( s , 1h ) , 7.02 ( d , j = 4.7 hz , 1h ) , 6.916.82 ( m , 2h ) , 6.57 ( d , j = 9.0 hz , 1h ) , 3.75 ( s , 3h ) , 2.92 ( m , 3h ) . pyrazole acid 11d was prepared from ester 10d according to the general methyl ester hydrolysis method ( pale - yellow solid , 78% ) . h nmr ( dmso - d6 ) 8.91 ( d , j = 4.7 hz , 1h ) , 8.22 ( d , j = 1.7 hz , 1h ) , 7.78 ( s , 1h ) , 7.76 ( d , j = 2.0 hz , 1h ) , 7.32 ( d , j = 8.4 hz , 1h ) , 7.24 ( d , j = 4.7 hz , 1h ) , 7.04 ( s , 1h ) , 6.56 ( dd , j = 2.2 , 8.4 hz , 1h ) , 6.34 ( d , j = 2.2 hz , 1h ) , 3.72 ( s , 3h ) , 2.92 ( s , 3h ) . pyrazole acid 11e was prepared from ester 10e according to the general methyl ester hydrolysis method ( pale - yellow solid , 94% ) . h nmr ( cd3od ) 8.90 ( d , j = 4.7 hz , 1h ) , 8.10 ( s , 1h ) , 7.80 ( d , j = 9.0 hz , 1h ) , 7.62 ( d , j = 9.0 hz , 1h ) , 7.237.45 ( m , 2h ) , 7.04 ( s , 1h ) , 6.54 ( d , j = 8.5 hz , 2h ) . pyrazole acid 11f was prepared from ester 10f according to the general methyl ester hydrolysis method ( pale - yellow solid , 92% ) . h nmr ( dmso - d6 ) 8.80 ( d , j = 4.7 hz , 1h ) , 8.15 ( d , j = 2.1 hz , 1h ) , 7.98 ( d , j = 9.0 hz , 1h ) , 7.69 ( dd , j = 2.1 , 9.0 hz , 1h ) , 7.257.39 ( m , 2h ) , 7.04 ( d , j = 4.7 hz , 1h ) , 6.97 ( t , j = 7.4 hz , 1h ) , 6.79 ( d , j = 8.1 hz , 1h ) , 6.70 ( s , 1h ) , 2.91 ( s , 3h ) . pyrazole acid 11 g was prepared from ester 10 g according to the general methyl ester hydrolysis method ( off - white solid , 70% ) . h nmr ( cdcl3 ) 7.787.86 ( m , 2h ) , 7.72 ( dd , j = 3.5 , 6.3 hz , 1h ) , 7.447.52 ( m , 2h ) , 7.227.35 ( m , 2h ) , 7.107.19 ( m , 2h ) , 6.35 ( d , j = 8.3 hz , 2h ) , 3.41 ( s , 6h ) . pyrazole acid 11h was prepared from ester 11h according to the general methyl ester hydrolysis method ( off - white powder , 92% ) . h nmr ( cdcl3 ) 7.817.91 ( m , 2h ) , 7.68 ( dd , j = 3.4 , 6.2 hz , 1h ) , 7.467.56 ( m , 2h ) , 7.287.37 ( m , 1h ) , 7.147.24 ( m , 3h ) , 6.83 ( t , j = 7.4 hz , 1h ) , 6.65 ( d , j = 8.3 hz , 1h ) , 3.12 ( s , 3h ) . ms ( esi ) m / z : 343.2 ( m h ) . pyrazole acid 11i was prepared from ester 10i according to the general methyl ester hydrolysis method ( off - white powder , 93% ) . h nmr ( cdcl3 ) 7.237.36 ( m , 3h ) , 6.917.03 ( m , 3h ) , 6.52 ( d , j = 8.5 hz , 2h ) , 3.60 ( s , 6h ) . pyrazole acid 11j was prepared from ester 10j according to the general methyl ester hydrolysis method ( off - white powder , 91% ) . h nmr ( cdcl3 ) 7.347.44 ( m , 1h ) , 7.227.33 ( m , 3h ) , 6.957.07 ( m , 4h ) , 6.82 ( d , j = 8.3 hz , 1h ) , 3.44 ( s , 3h ) . compound 13 was prepared from 11a and 2-aminoadamantane hydrochloride ( 12e ) following the general amide coupling method ( off - white powder , 65% ) . h nmr ( cdcl3 ) 8.77 ( d , j = 4.7 hz , 1h ) , 8.16 ( d , j = 1.9 hz , 1h ) , 7.96 ( d , j = 9.0 hz , 1h ) , 7.55 ( dd , j = 1.9 , 9.0 hz , 1h ) , 7.247.37 ( m , 2h ) , 7.13 ( s , 1h ) , 6.927.02 ( m , 2h ) , 6.64 ( d , j = 8.3 hz , 1h ) , 4.28 ( d , j = 8.1 hz , 1h ) , 2.98 ( s , 6h ) , 2.022.11 ( m , 2h ) , 1.801.95 ( m , 9h ) , 1.701.79 ( m , 2h ) , 1.591.70 ( m , 2h ) . ( c31h31cln4o3 ) c , h , n. following the general amide coupling procedures , 14a was obtained from 11a and tert - butyl ( 2s)-amino(cyclohexyl)ethanoate hydrochloride ( 12d ) . the material was purified by flash chromatography ( 0100% etoac / hexanes ) to afford 14a ( 84% ) as a pale - yellow film . h nmr ( cdcl3 ) 8.78 ( d , j = 4.7 hz , 1h ) , 8.12 ( d , j = 2.0 hz , 1h ) , 7.97 ( d , j = 9.0 hz , 1h ) , 7.50 ( dd , j = 2.1 , 9.0 hz , 1h ) , 7.42 ( d , j = 9.0 hz , 1h ) , 7.21 ( t , j = 8.4 hz , 1h ) , 7.017.12 ( m , 2h ) , 6.39 ( d , j = 8.2 hz , 2h ) , 4.66 ( dd , j = 5.1 , 9.1 hz , 1h ) , 3.40 ( br s , 6h ) , 1.541.99 ( m , 6h ) , 1.441.53 ( m , 9h ) , 1.011.32 ( m , 5h ) . following the general tert - butyl ester hydrolysis procedure , 14b was obtained as a pale - yellow solid in 91% yield from 14a . h nmr ( cdcl3 ) 9.10 ( br s , 1 h ) , 8.74 ( br s , 1 h ) , 8.31 ( d , j = 8.9 hz , 1 h ) , 7.78 ( d , j = 8.9 hz , 1 h ) , 7.607.41 ( m , 2 h ) , 7.397.25 ( m , 1 h ) , 6.48 ( d , j = 7.5 hz , 2 h ) , 4.80 ( dd , j = 4.7 , 7.9 hz , 1 h ) , 3.47 ( br s , 6 h ) , 2.101.92 ( m , 1 h ) , 1.901.60 ( m , 5 h ) , 1.321.09 ( m , 5 h ) . ms ( esi ) m / z : 548.4 ( m h ) . ( c29h29cln4o5 ) c , h , n. following the general amide coupling procedure , 15a was obtained from 11c and 12d as a pale - yellow film ( 70% ) . h nmr ( cdcl3 ) 8.78 ( d , j = 4.7 hz , 1h ) , 8.16 ( d , j = 1.9 hz , 1h ) , 8.01 ( d , j = 9.2 hz , 1h ) , 7.55 ( dd , j = 2.3 , 9.0 hz , 1h ) , 7.40 ( d , j = 9.0 hz , 1h ) , 7.147.11 ( m , 1h ) , 7.00 ( d , j = 4.7 hz , 1h ) , 6.90 ( d , j = 3.0 hz , 1h ) , 6.83 ( dd , j = 3.2 , 9.0 hz , 1h ) , 6.55 ( d , j = 9.0 hz , 1h ) , 4.694.62 ( m , 1h ) , 3.72 ( s , 3h ) , 2.89 ( m , 3h ) , 2.051.60 ( m , 6h ) , 1.50 ( s , 9h ) , 1.391.11 ( m , 5h ) . following the general tert - butyl ester hydrolysis procedure , h nmr ( cdcl3 ) 9.00 ( d , j = 5.3 hz , 1h ) , 8.39 ( d , j = 1.8 hz , 1h ) , 8.17 ( d , j = 9.2 hz , 1h ) , 7.7 ( dd , j = 1.9 , 9.0 hz , 1h ) , 7.30 ( d , j = 9.0 hz , 1h ) , 7.277.16 ( m , 2h ) , 6.90 ( d , j = 3.0 hz , 1h ) , 6.83 ( dd , j = 3.2 , 9.0 hz , 1h ) , 6.58 ( d , j = 9.0 hz , 1h ) , 4.814.72 ( m , 1h ) , 3.78 ( s , 3h ) , 2.90 ( m , 3h ) , 2.051.60 ( m , 6h ) , 1.371.03 ( m , 5h ) . ms ( esi ) m / z : 547.8 ( m h ) . ( c29h29cln4o5 ) c , h , n. following the general amide coupling procedure , 16a was obtained as a pale - yellow film ( 86% ) from 11d and 12d . h nmr ( cdcl3 ) 8.79 ( d , j = 4.7 hz , 1h ) , 8.16 ( d , j = 2.0 hz , 1h ) , 7.96 ( d , j = 9.0 hz , 1h ) , 7.54 ( dd , j = 2.1 , 9.0 hz , 1h ) , 7.40 ( d , j = 9.0 hz , 1h ) , 7.21 ( d , j = 8.4 hz , 1h ) , 7.07 ( s , 1h ) , 7.01 ( d , j = 4.6 hz , 1h ) , 6.47 ( dd , j = 2.3 , 8.4 hz , 1h ) , 6.18 ( d , j = 2.3 hz , 1h ) , 4.66 ( dd , j = 5.1 , 9.0 hz , 1h ) , 3.77 ( s , 3h ) , 2.96 ( s , 3h ) , 1.561.81 ( m , 6h ) , 1.48 ( d , j = 4.5 hz , 9h ) , 1.001.34 ( m , 5h ) . following the general tert - butyl ester hydrolysis procedure , 16b was obtained as a yellow solid in 86% yield from 16a . h nmr ( cdcl3 ) 9.11 ( br s , 1 h ) , 8.48 ( s , 1 h ) , 8.27 ( d , j = 9.1 hz , 1 h ) , 7.80 ( d , j = 8.9 hz , 1 h ) , 7.46 ( d , j = 8.4 hz , 1 h ) , 7.427.35 ( m , 1 h ) , 7.31 ( d , j = 8.4 hz , 1 h ) , 7.12 ( s , 1 h ) , 6.59 ( d , j = 8.4 hz , 1 h ) , 6.23 ( d , j = 1.7 hz , 1 h ) , 4.73 ( dd , j = 5.4 , 8.4 hz , 1 h ) , 3.81 ( s , 3 h ) , 3.02 ( s , 3 h ) , 1.871.61 ( m , 5 h ) , 1.371.04 ( m , 6 h ) . ms ( esi ) m / z : 548.5 ( m h ) . ( c29h29cln4o5 ) c , h , n. following the general amide coupling procedure , 17a was obtained from 11f and 12d in 94% yield as a pale - yellow solid . h nmr ( cdcl3 ) 8.77 ( d , j = 4.7 hz , 1h ) , 8.16 ( d , j = 1.9 hz , 1h ) , 7.98 ( d , j = 9.0 hz , 1h ) , 7.55 ( dd , j = 2.0 , 9.1 hz , 1h ) , 7.41 ( d , j = 9.1 hz , 1h ) , 7.237.35 ( m , 2h ) , 7.097.16 ( m , 1h ) , 6.897.03 ( m , 2h ) , 6.64 ( d , j = 8.1 hz , 1h ) , 4.66 ( dd , j = 5.0 , 9.1 hz , 1h ) , 2.98 ( s , 3h ) , 1.531.99 ( m , 7h ) , 1.49 ( s , 9h ) , 1.011.35 ( m , 6h ) . following the general tert - butyl ester hydrolysis procedure , 17b was obtained as a pale - yellow film in 74% yield from 17a . h nmr ( cdcl3 ) 9.09 ( br s , 1 h ) , 8.76 ( br s , 1 h ) , 8.33 ( d , j = 8.9 hz , 1 h ) , 7.82 ( d , j = 8.9 hz , 1 h ) , 7.577.32 ( m , 4 h ) , 7.22 ( s , 1 h ) , 7.10 ( t , j = 6.9 hz , 1 h ) , 6.70 ( d , j = 7.7 hz , 1 h ) , 4.78 ( dd , j = 4.8 , 8.2 hz , 1 h ) , 3.04 ( br s , 3 h ) , 2.101.90 ( m , 1 h ) , 1.891.60 ( m , 5 h ) , 1.251.07 ( m , 5 h ) . ms ( esi ) m / z : 518.0 ( m h ) . ( c28h27cln4o4 ) c , h , n. following the general amide coupling procedure , 18a was obtained from 11e and 12d as a pale - yellow foam in 53% yield . h nmr ( cdcl3 ) 8.89 ( d , j = 4.7 hz , 1h ) , 8.15 ( d , j = 4.7 hz , 1h ) , 7.75 ( d , j = 9.0 hz , 1h ) , 7.75 ( d , j = 9.0 hz , 1h ) , 7.52 ( dd , j = 1.9 , 9.0 hz , 1h ) , 7.4 ( d , j = 9.0 hz , 1h ) , 7.327.25 ( m , 3h ) , 7.2 ( d , j = 9.0 hz , 1h ) , 6.80 ( t , j = 7.6 hz , 1h ) , 4.694.63 ( m , 1h ) , 1.901.85 ( m , 1h ) , 1.841.60 ( m , 5h ) , 1.50 ( s , 9h ) , 1.321.06 ( m , 5h ) . following the general tert - butyl ester hydrolysis procedure , 18b was obtained as a white foam ( 90% ) from 18a . h nmr ( cdcl3 ) 8.91 ( d , j = 4.7 hz , 1h ) , 8.18 ( s , 1h ) , 8.17 ( d , j = 9.2 hz , 1h ) , 7.74 ( d , j = 9.0 hz , 1h ) , 7.53 ( dd , j = 1.9 , j = 9.0 hz , 1h ) , 7.40 ( d , j = 9.0 hz , 1h ) , 7.207.05 ( m , 4h ) , 6.80 ( t , j = 7.6 hz , 1h ) , 4.824.74 ( m , 1h ) , 2.081.94 ( m , 1h ) , 1.891.60 ( m , 5h ) , 1.371.05 ( m , 5h ) . ( c27h23clf2n4o3 ) c , h , n. following the general amide coupling procedures , 19a was obtained as a pale - yellow film ( 86% ) 11b and 12d . h nmr ( cdcl3 ) 8.76 ( d , j = 4.6 hz , 1h ) , 8.09 ( d , j = 2.1 hz , 1h ) , 7.98 ( d , j = 9.1 hz , 1h ) , 7.48 ( td , j = 2.2 , 9.1 hz , 2h ) , 7.21 ( t , j = 8.4 hz , 1h ) , 7.09 ( d , j = 4.6 hz , 1h ) , 6.40 ( dd , j = 8.4 , 15.6 hz , 2h ) , 4.64 ( dd , j = 4.9 , 9.0 hz , 1h ) , 3.53 ( s , 3h ) , 3.42 ( s , 3h ) , 2.542.82 ( m , 2h ) , 1.611.95 ( m , 6h ) , 1.49 ( s , 9h ) , 1.171.36 ( m , 5h ) , 1.061.15 ( m , 3h ) . ms ( esi ) m / z : 633.7 ( m + h ) . following the general tert - butyl ester hydrolysis procedure , 19b was obtained as a pale - yellow solid in 96% yield from 19a . h nmr ( cdcl3 ) 9.12 ( d , j = 5.7 hz , 1 h ) , 8.43 ( s , 1 h ) , 8.38 ( d , j = 9.2 hz , 1 h ) , 7.77 ( dd , j = 1.3 , 9.3 hz , 1 h ) , 7.56 ( d , j = 8.7 hz , 1 h ) , 7.48 ( d , j = 5.7 hz , 1 h ) , 7.34 ( t , j = 8.4 hz , 1 h ) , 6.51 ( dd , j = 8.5 , 15.5 hz , 2 h ) , 4.77 ( dd , j = 5.2 , 8.7 hz , 1 h ) , 3.673.45 ( m , 6 h ) , 2.66 ( dd , j = 3.2 , 7.3 hz , 2 h ) , 2.121.91 ( m , 1 h ) , 1.901.60 ( m , 6 h ) , 1.381.16 ( m , 4 h ) , 1.151.01 ( m , 3 h ) . ms ( esi ) m / z : 575.8 ( m h ) . ( c31h33cln4o5 ) c , h , n. following the general amide coupling procedure , 20a was obtained from 11a and methyl 1-aminocycloheptanecarboxylate hydrochloride ( 12c ) as an off - white powder in 65% yield . h nmr ( cdcl3 ) 8.77 ( d , j = 4.7 hz , 1h ) , 8.16 ( d , j = 1.9 hz , 1h ) , 7.96 ( d , j = 9.0 hz , 1h ) , 7.55 ( dd , j = 1.9 , 9.0 hz , 1h ) , 7.247.37 ( m , 2h ) , 7.13 ( s , 1h ) , 6.927.02 ( m , 2h ) , 6.64 ( d , j = 8.3 hz , 1h ) , 4.28 ( d , j = 8.1 hz , 1h ) , 2.98 ( s , 6h ) , 2.022.11 ( m , 2h ) , 1.801.95 ( m , 9h ) , 1.701.79 ( m , 2h ) , 1.591.70 ( m , 2h ) . following the general methyl ester hydrolysis procedure , 20b was obtained from 20a as a white powder in 86% yield . h nmr ( cdcl3 ) 8.80 ( d , j = 4.7 hz , 1h ) , 8.14 ( d , j = 2.1 hz , 1h ) , 7.90 ( d , j = 9.0 hz , 1h ) , 7.50 ( dd , j = 2.1 , 9.0 hz , 1h ) , 7.247.17 ( m , 1h ) , 7.11 ( s , 1h ) , 7.07 ( d , j = 4.7 hz , 1h ) 6.40 ( d , j = 8.7 hz , 2h ) , 3.42 ( s , 6h ) , 2.422.28 ( m , 2h ) , 2.242.13 ( m , 2h ) , 1.741.53 ( m , 10h ) . ( c29h29cln4o5 ) c , h , n. following the general amide coupling procedures , 21a was obtained from 11a and ethyl 1-aminocyclohexanecarboxylate hydrochloride ( 12b ) as a white solid in 87% yield . h nmr ( cdcl3 ) 8.748.84 ( m , 1h ) , 8.14 ( d , j = 2.1 hz , 1h ) , 7.96 ( d , j = 9.0 hz , 1h ) , 7.52 ( dd , j = 2.1 , 9.0 hz , 1h ) , 7.157.25 ( m , 1h ) , 7.047.15 ( m , 2h ) , 6.39 ( d , j = 8.5 hz , 2h ) , 4.25 ( q , j = 7.2 hz , 2h ) , 3.41 ( s , 6h ) , 2.18 ( d , j = 13.6 hz , 2h ) , 1.862.02 ( m , 2h ) , 1.431.74 ( m , 6h ) , 1.231.34 ( m , 3h ) . following the general methyl ester hydrolysis procedure , 21b was obtained from 21a in 87% yield as a white solid . h nmr ( dmso - d6 ) 8.92 ( d , j = 4.7 hz , 1h ) , 8.15 ( d , j = 2.1 hz , 1h ) , 7.867.75 ( m , 2h ) , 7.69 ( dd , j = 2.1 , 9.0 hz , 1h ) , 7.307.21 ( m , 2h ) , 6.95 ( s , 1h ) , 6.53 ( d , j = 8.7 hz , 1h ) , 3.43 ( s , 6h ) , 2.172.06 ( m , 2h ) , 1.851.72 ( m , 2h ) , 1.621.25 ( m , 6h ) . ( c28h27cln4o5 ) c , h , n. following the general amide coupling procedures , 22a was obtained from 11a and methyl 1-aminocyclopentanecarboxylate hydrochloride ( 12a ) as a white powder in 85% yield . h nmr ( cdcl3 ) 8.78 ( d , j = 4.7 hz , 1h ) , 8.12 ( d , j = 2.1 hz , 1h ) , 7.94 ( d , j = 9.0 hz , 1h ) , 7.52 ( dd , j = 2.1 , 9.0 hz , 1h ) , 7.297.22 ( m , 1h ) , 7.217.16 ( m , 1h ) , 7.117.05 ( m , 1h ) , 6.39 ( d , j = 8.5 hz , 2h ) , 3.78 ( s , 3h ) , 3.41 ( s , 6h ) , 2.422.28 ( m , 2h ) , 2.172.05 ( m , 2h ) , 1.901.80 ( m , 4h ) . following the general methyl ester hydrolysis procedure , 22b was obtained from 22a as a white powder in 72% yield . h nmr ( cdcl3 ) 8.80 ( d , j = 4.7 hz , 1h ) , 8.14 ( d , j = 2.1 hz , 1h ) , 7.90 ( d , j = 9.0 hz , 1h ) , 7.53 ( dd , j = 2.1 , j = 9.0 hz , 1h ) , 7.307.18 ( m , 1h ) , 7.11 ( s , 1h ) , 7.07 ( d , j = 4.7 hz , 1h ) 6.40 ( d , j = 8.7 hz , 2h ) , 3.42 ( s , 6h ) , 2.542.40 ( m , 2h ) , 2.202.07 ( m , 2h ) , 1.901.77 ( m , 4h ) . ms ( esi ) m / z : 547.8 ( m h ) . ( c28h27cln4o5 ) c , h , n. following the general amide coupling procedure , 23a was obtained from 11f and ethyl 1-aminocyclohexanecarboxylate hydrochloride ( 12b ) as a white solid in 63% yield . h nmr ( cdcl3 ) 8.78 ( d , j = 4.7 hz , 1h ) , 8.17 ( d , j = 1.9 hz , 1h ) , 7.96 ( d , j = 9.0 hz , 1h ) , 7.56 ( dd , j = 2.2 , 9.1 hz , 1h ) , 7.247.34 ( m , 2h ) , 7.10 ( s , 1h ) , 6.907.01 ( m , 1h ) , 6.64 ( d , j = 8.7 hz , 2h ) , 3.78 ( s , 3h ) , 2.99 ( s , 3h ) , 2.18 ( d , j = 13.8 hz , 2h ) , 1.882.01 ( m , 2h ) , 1.631.74 ( m , 6h ) . following the general methyl ester hydrolysis procedure , 23b was obtained from 23a as a white solid in 77% yield . h nmr ( cdcl3 ) 8.80 ( d , j = 4.7 hz , 1h ) , 8.17 ( d , j = 2.1 hz , 1h ) , 7.90 ( d , j = 9.0 hz , 1h ) , 7.5 ( dd , j = 2.1 , j = 9.0 hz , 1h ) , 7.357.21 ( m , 1h ) , 7.207.11 ( m , 2h ) , 6.63 ( d , j = 8.7 hz , 1h ) , 2.98 ( s , 3h ) , 2.302.16 ( m , 2h ) , 2.101.96 ( m , 2h ) , 1.761.53 ( m , 6h ) . ( c28h27cln4o4 ) c , h , n. following the general amide coupling procedures , 24a was obtained from 11 g and 12d as a white solid in 53% yield . h nmr ( cdcl3 ) 7.887.72 ( m , 3h ) , 7.517.38 ( m , 3h ) , 7.367.21 ( m , 2h ) , 7.11 ( t , j = 8.4 hz , 1h ) , 7.077.03 ( m , 1h ) , 6.33 ( d , j = 5.9 hz , 2h ) , 4.66 ( dd , j = 5.4 , 9.1 hz , 1h ) , 3.40 ( br s , 6h ) , 1.961.52 ( m , 8h ) , 1.47 ( s , 9h ) , 1.331.02 ( m , 6h ) . following the general tert - butyl ester hydrolysis procedure 24b was obtained as a slightly yellow solid in 53% yield from 24a . h nmr ( cdcl3 ) 7.82 ( d , j = 6.8 hz , 2 h ) , 7.73 ( d , j = 6.4 hz , 2 h ) , 7.48 ( dd , j = 3.2 , 6.2 hz , 2 h ) , 7.387.21 ( m , 2 h ) , 7.197.01 ( m , 2 h ) , 6.33 ( d , j = 8.1 hz , 2 h ) , 4.73 ( dd , j = 5.8 , 8.3 hz , 1 h ) , 3.40 ( br s , 6 h ) , 1.93 ( br s , 1 h ) , 1.831.55 ( m , 5 h ) , 1.230.99 ( m , 5 h ) . ms ( esi ) m / z : 512.4 ( m h ) . ( c30h31n3o5 ) c , h , n. following the general amide coupling procedure , 25a was obtained from 11h and 12d in 81% yield as a colorless film . h nmr ( cdcl3 ) 7.84 ( s , 1h ) , 7.81 ( s , 1h ) , 7.75 ( d , j = 3.5 hz , 1h ) , 7.557.46 ( m , 2h ) , 7.43 ( d , j = 9.1 hz , 1h ) , 7.367.28 ( m , 1h ) , 7.237.14 ( m , 3h ) , 7.12 ( s , 1h ) , 6.856.75 ( m , 1h ) , 6.61 ( d , j = 8.6 hz , 1h ) , 4.66 ( dd , j = 5.4 , 9.1 hz , 1h ) , 3.07 ( s , 3h ) , 1.961.57 ( m , 7h ) , 1.47 ( s , 9h ) , 1.321.05 ( m , 6h ) . following the general tert - butyl ester hydrolysis procedure , 25b was obtained from 25a as a white foam ( 43% ) . h nmr ( cdcl3 ) 7.917.81 ( m , 2 h ) , 7.737.61 ( m , 2 h ) , 7.567.47 ( m , 2 h ) , 7.397.29 ( m , 1 h ) , 7.257.18 ( m , 2 h ) , 7.15 ( s , 2 h ) , 6.80 ( t , j = 7.4 hz , 1 h ) , 6.63 ( d , j = 8.3 hz , 1 h ) , 4.74 ( dd , j = 5.7 , 8.8 hz , 1 h ) , 3.12 ( s , 3 h ) , 1.93 ( br s , 1 h ) , 1.831.53 ( m , 5 h ) , 1.241.02 ( m , 5 h ) . ( c29h29n3o4 ) c , h , n. following the general amide coupling procedure , 26a was obtained from 11 g and 12b as a white solid in 98% yield . h nmr ( cdcl3 ) 7.737.89 ( m , 3h ) , 7.457.52 ( m , 2h ) , 7.277.37 ( m , 2h ) , 7.067.16 ( m , 2h ) , 7.02 ( s , 1h ) , 6.32 ( d , j = 8.3 hz , 2h ) , 3.78 ( s , 3h ) , 3.233.63 ( m , 6h ) , 2.082.32 ( m , 4h ) , 1.56 ( br s , 8h ) . following the general ethyl ester hydrolysis procedure , 26b was obtained from 26a as an off - white powder in 35% yield . h nmr ( cdcl3 ) 7.887.78 ( m , 2 h ) , 7.777.67 ( m , 1 h ) , 7.547.45 ( m , 2 h ) , 7.377.29 ( m , 1 h ) , 7.287.22 ( m , 4 h ) , 7.15 ( t , j = 8.4 hz , 1 h ) , 7.08 ( s , 1 h ) , 6.34 ( d , j = 8.5 hz , 2 h ) , 3.41 ( br s , 6 h ) , 2.24 ( d , j = 14.1 hz , 2 h ) , 2.101.94 ( m , 2 h ) , 1.791.48 ( m , 6 h ) . ( c30h31n3o5 ) c , h , n. following the general amide coupling procedures , 27a was obtained from 11h and 12b as a colorless film in 73% yield . h nmr ( cdcl3 ) 7.937.80 ( m , 2h ) , 7.807.71 ( m , 1h ) , 7.597.46 ( m , 2h ) , 7.407.29 ( m , 1h ) , 7.257.12 ( m , 4h ) , 7.10 ( s , 1h ) , 6.876.74 ( m , 1h ) , 6.62 ( d , j = 8.2 hz , 1h ) , 3.77 ( s , 3h ) , 3.09 ( s , 3h ) , 2.13 ( br s , 2h ) , 1.96 ( br s , 3h ) , 1.711.30 ( m , 10h ) . following the general ethyl ester hydrolysis procedure , h nmr ( cdcl3 ) 7.927.82 ( m , 2 h ) , 7.68 ( d , j = 3.4 hz , 1 h ) , 7.53 ( dd , j = 3.3 , 6.3 hz , 2 h ) , 7.387.30 ( m , 1 h ) , 7.257.09 ( m , 5 h ) , 6.82 ( t , j = 7.4 hz , 1 h ) , 6.63 ( d , j = 8.2 hz , 1 h ) , 3.10 ( s , 3 h ) , 2.22 ( d , j = 13.9 hz , 2 h ) , 2.01 ( t , j = 11.2 hz , 2 h ) , 1.771.19 ( m , 8 h ) , 0.97 ( br s , 1 h ) . ms ( esi ) m / z : 503.6 ( m h ) . anal . ( c28h27n3o4 ) c , h , n. following the general amide coupling procedure , 28a was obtained from 11i and 12d as an off - white powder in 94% yield . h nmr ( cdcl3 ) 7.427.54 ( m , 1h ) , 7.217.35 ( m , 3h ) , 6.907.03 ( m , 3h ) , 6.50 ( dd , j = 8.5 , 13.9 hz , 2h ) , 4.67 ( dd , j = 5.09 , 9.14 hz , 1h ) , 3.62 ( s , 3h ) , 3.52 ( s , 3h ) , 1.601.92 ( m , 7h ) , 1.481.52 ( m , 9h ) , 1.161.33 ( m , 5h ) . ms ( esi ) m / z : 536.5 ( m h ) . following the general tert - butyl ester hydrolysis procedure , h nmr ( cd3od ) 7.237.42 ( m , 3h ) , 6.997.15 ( m , 2h ) , 6.766.86 ( m , 1h ) , 6.62 ( d , j = 8.29 hz , 2h ) , 4.57 ( d , j = 6.03 hz , 1h ) , 3.60 ( s , 6h ) , 1.742.10 ( m , 7h ) , 1.211.44 ( m , 4h ) . ( c26h26fn3o5 ) c , h , n. following the general amide coupling procedure , 29a was prepared from 11i and 12b as a colorless solid in 50% yield . h nmr ( cdcl3 ) 7.337.25 ( m , 4h ) , 7.006.90 ( m , 2h ) , 6.50 ( d , j = 8.5 hz , 2h ) , 3.75 ( s , 3h ) , 3.58 ( s , 6h ) , 2.252.13 ( m , 2h ) , 2.011.89 ( m , 2h ) , 1.741.52 ( m , 6h ) . following the general methyl ester hydrolysis procedure , 29b was obtained from 29a as a white solid ( 89% ) . h nmr ( cdcl3 ) 7.367.21 ( m , 4h ) , 7.036.95 ( m , 2h ) , 6.50 ( d , j = 8.5 hz , 2h ) , 3.60 ( s , 6h ) , 2.342.20 ( m , 2h ) , 2.101.96 ( m , 2h ) , 1.791.47 ( m , 6h ) . ( c25h26fn3o5 ) c , h , n. following the method of qur,30 was obtained from 11i and 9-aminobicyclo[3.3.1]nonane-9-carboxylic acid ( 12f ) . briefly , 11i was heated under reflux in socl2 for 2 h , followed by cooling and concentration on a rotavap . the residue was then taken up in dry thf and added to a well agitated mixture of 5:1 thf : water ( 15 ml / g of acid chloride ) containing 1.1 equiv of 2-aminoadamantane - carboxylic acid and 2.2 equiv of naoh at 5 c . following the addition , the stirring was continued for 18 h at ambient temp . after this time , the mixture is made acidic with 1 n hcl and extracted 3 with ch2cl2 , dried over sodium sulfate , and evaporated . evaporation provided the desired compound as an off - white solid ( 71% ) . h nmr ( cdcl3 ) 7.207.36 ( m , 4h ) , 6.907.04 ( m , 3h ) , 6.51 ( d , j = 8.5 hz , 2h ) , 3.543.63 ( m , 6h ) , 2.712.81 ( m , 2h ) , 2.23 ( d , j = 12.8 hz , 2h ) , 2.07 ( d , j = 12.2 hz , 2h ) , 1.681.95 ( m , 8h ) . ms ( esi ) m / z : 518.9 ( m h ) . ( c29h30fn3o5 ) c , h , n. cho - k1-rnts1 cells were maintained in dmem / f12 medium supplemented with 10% fetal bovine serum ( gibco ) , 100 u / ml penicillin/100 g / ml streptomycin , 100 g / ml normocin ( invivogen ) , and 250 g / ml geneticin . for calcium mobilization assays , cells were plated at 30000 cells / well in black , clear - bottom 96-well plates the day of the assay and incubated at 37 c , 5% co2 . prior to the assay , calcium 5 dye ( molecular devices ) the reconstituted dye was diluted 1:40 in assay buffer ( 1 hbss , 20 mm hepes , and 2.5 mm probenicid ( sigma ) , ph 7.4 ) . growth media was removed , and 200 l of this diluted dye was added to each well . plates were incubated for 45 min at 37 c , 5% co2 after dye addition . for agonist assays , cells were pretreated with 1:10 addition ( 20 l ) of 10% dmso in assay buffer , and the plates were returned to 37 c , 5% co2 for 15 min . full - log serial dilutions of the test compounds were made at 10 the desired final concentration in 1% dmso assay buffer and warmed to 37 c . after the pretreatment incubation , fluorescence intensity was measured on a flexstation ii fluorometric imaging plate reader ( molecular devices ) . relative fluorescence units ( rfu ) were measured before ( 20 readings ) and after ( 40 readings ) the agonist compound addition for a total 60 s read time ( excitation = 485 nm , emission = 525 nm , cutoff = 515 nm ) . for antagonist assays , cells were treated with calcium 5 dye for 45 min at 37 c , 5% co2 as with the agonist assays . then 1/10th volume of 10 final concentration of the test antagonist compounds were added in 1% dmso in assay buffer , and the plate was incubated for 15 min at 37 c , 5% co2 . for ke assays , the cells were pretreated with a single concentration of test compound and the flexstation ii added serial dilutions of the control agonist nt . for ic50 assays , cells were pretreated with serial dilutions of test compound and the flexstation ii added 1 nm nt . cho - k1-rnts2 cells were maintained in dmem / f12 supplemented with 10% fbs , pen / strep , 100 g / ml normocin , and 400 g / ml geneticin . for calcium mobilization assays , cells were plated at 25000 cells / well in black , clear - bottom 96-well plates the day before the assay and incubated at 37 c , 5% co2 . then 100 l of reconstituted calcium 5 dye ( molecular devices , diluted 1:20 in assay buffer ( 1 hbss , 20 mm hepes , 2.5 mm probenicid , ph 7.4 ) ) was added per well and plates were incubated for 45 min at 37 c , 5% co2 . for agonist assays , cells were pretreated with 1:10 addition of 10% dmso and the plates were returned to 37 c , 5% co2 . full - log serial dilutions of the test compounds were made at 10 the desired final concentration in 1% dmso assay buffer and warmed to 37 c . after the pretreatment incubation , fluorescence intensity was measured on a flipr tetra fluorometric imaging plate reader ( molecular devices ) . relative fluorescence units ( rfus ) were measured every second for 100 s ( 14 readings before compound addition to establish baseline fluorescence , 85 after ) , exposure time 0.53 s , gain = 2000 , excitation intensity = 30% , gate open = 10% . for antagonist assays , 1/10 volume of 10 concentration of the test compound dilutions in 10% dmso in assay buffer was added to cells in lieu of the 10% dmso only in assay buffer for the pretreatment . ke assays were run against dose response curves of the control agonist 5b , and ic50 assays were run against the ec80 of 5b ( 73 nm final concentration ) . relative binding affinity was evaluated using i labeled neurotensin and cho - k1 cell lines overexpressing either the rnts1 or rnts2 receptor essentially as described by gendron . briefly , cells were plated at 100000 cells / well in 24-well plates in complete dmem / f12 medium supplemented with 10% fetal bovine serum , 100 units of penicillin / ml , 100 mg of streptomycin / ml , 0.1 mg / ml of normocin , and 250 mg / ml geneticin . cells were incubated at 37 c with 5% co2 and 95% humidity for 48 h. cells were then equilibrated for 10 min in earle s buffer ( 130 mm nacl , 5 mm kcl , 1.8 mm cacl2 , 0.8 mm mgcl2 , and 20 mm hepes , ph 7.4 ) supplemented with 0.1% bsa and 0.1% glucose and then incubated with or without 10 nm test compound in 0.1 nm [ i]nt at 37 c for 30 min . cells were washed twice in earle s buffer , extracted in 1 ml of 0.1n naoh and counted in a packard cobra ii gamma counter for 1 min . total binding was determined in the absence of test compound , and nonspecific binding was determined in the presence of 1.0 m nonradiolabeled neurotensin . to determine ec50 , ic50 , and ke values , data were fit to a three - parameter logistic equation using graphpad prism software . ki values for radioligand binding assays were determined from ic50 values using the equation of cheng and prusoff .

compounds active at neurotensin receptors ( nts1 and nts2 ) exert analgesic effects on different types of nociceptive modalities , including thermal , mechanical , and chemical stimuli . the nts2 preferring peptide jmv-431 ( 2 ) and the nts2 selective nonpeptide compound levocabastine ( 6 ) have been shown to be effective in relieving the pain associated with peripheral neuropathies . with the aim of identifying novel nonpeptide compounds selective for nts2 , we examined analogues of sr48692 ( 5a ) using a flipr calcium assay in cho cells stably expressing rat nts2 . this led to the discovery of the nts2 selective nonpeptide compound 1-({[1-(4-fluorophenyl)-5-(2-methoxyphenyl)-1h - pyrazol-3-yl]carbonyl}amino)cyclohexane carboxylic acid ( ntrc-739 , 7b ) starting from the nonselective compound 5a .

Introduction Chemistry Biological Results Results and Discussion Conclusions Experimental Section

nt - mediated analgesia has been demonstrated with compounds selective for both the nts1 and nts2 receptors as well as nonselective compounds . beyond this , there is now substantial evidence that both nts1 and nts2 can mediate relief from chronic or neuropathic pain . the nts1 receptor has received the most attention since the discovery of the nt system as it binds nt with high affinity , hence the name ntrh ( nt receptor high affinity ) , and is well behaved in heterologous expression systems . as is true for nts1 , the most potent and selective nts2 compounds discovered to date have been obtained via modification of the nt(813 ) ( 1 ) fragment . discovery of potent and selective nonpeptide compounds however has proven elusive and remains as a significant challenge . the 813 fragment of nt , compound 1 , is as potent as the full length peptide , and several hexapeptide variants of 1 have been reported over the years that either favor nts2 over nts1 or are selective for nts2 versus nts1 . the first of these is jmv-431 ( 2 ) that was produced via reduction of the tyrosine 11 amide bond and shows a clear preference for nts2 . the peptide nt79 ( 3 ) , reported more recently , is selective for nts2 ( > 100-fold ) and has provided a wealth of information regarding the role of nts2 in animal models of pain , antipsychotic activity , thermoregulation , and regulation of blood pressure . the most recent additions to the rolls of nts2-selective compounds are the potent peptide these compounds are ultraselective for nts2 , with selectivity ratios reaching 12000- and 22000-fold , respectively , with 4b also demonstrating excellent plasma stability . although few in number , there are three prominent nonpeptide compounds that interact with nts2 that have been used extensively in the characterization of the nt receptors . these include the two pyrazole - based compounds 5a ( sr48692 ) and sr142948a ( 5b ) and the histamine blocker levocabastine ( 6 ) . these compounds ( 5a , b and 6 ) highlight the fact that while nts2-selective peptides exist , selective nonpeptide compounds are rare . with the goal of discovering novel nts2 selective ligands , we undertook this endeavor by adapting a previously described calcium mobilization assay to our high throughput flipr tetra system and confirmed the functional results of active compounds using a radioligand binding assay . this effort led to the identification of a nonpeptide compound that is selective for nts2 versus nts1 , 1-({[1-(4-fluorophenyl)-5-(2-methoxyphenyl)-1h - pyrazol-3-yl]carbonyl}amino)cyclohexane carboxylic acid ( 7b ) , starting from the nonselective compound 5a . thus , a given pyrazole carboxylic acid ( 11a j ) and amino acid ester ( 12a d ) or amine ( 12e ) from chart 2 were coupled using o - benzotriazol-1-yl - n , n , n,n-tetramethyluronium hexafluorophosphate ( hbtu ) and triethylamine to give ester intermediates 7a , and 14a29a and the descarboxy target compound 13 . the ester intermediates 7a , and 14a29a were hydrolyzed to give final products using either basic ( lioh and dioxane ) or acidic ( trifluoroacetic acid ( tfa ) in ch2cl2 ) conditions as shown . the synthesis of target compound 30 was accomplished according to the method of qure , wherein pyrazole acid 11i was converted to its acid chloride using socl2 in toluene , followed by coupling of the acid chloride intermediate with the adamantyl amino acid 12f under schotten bauman conditions . these measurements were performed using a flipr tetra ( nts2 ) or a flexstation ii plate reader for nts1 ( molecular devices ) and were analyzed using graphpad prism software . in this article , we provide the details of our effort to identify nonpeptide compounds that are selective for nts2 starting from the nonselective compound 5a . a summary of the key sar elements of 5a discovered using the nts2 calcium mobilization assay that led to the identification of the potent partial agonist 7b ( ntrc-739 ) are provided in table 2 and includes the nts2 ec50 as well as the nts1 ke and the nts2 binding affinity ( ki ) . each of the appended molecular regions of 5a is discussed herein and includes : the dimethoxyphenyl ring , the amino acid side chain , the 7-chloroquinoline ring , and the 4-position of the pyrazole ring . the data obtained from the testing of nt reference compounds in this assay , including nt , 1 , 5a , b and levocabastine ( 6 ) , are provided in table 1 . in attempting to develop a method of screening for nts2 activity , we observed that the literature reports dealing with functional assays of nts2 receptors yielded contradictory data , exhibiting cell - type expression- and species - dependent pharmacological properties with opposing patterns . specifically , the potent nts1 antagonists 5a and 5b were found to be agonists at nts2 as they mobilized calcium release while levocabastine ( 6 ) was found to behave either as an agonist , antagonist , or even as a partial inverse agonist . while the information above illustrated that there was much inconsistency between cell lines and expression systems for nts2 , we believed that each system would produce internally consistent ( reproducible ) data . we studied the calcium release produced by analogues of the nonpeptide compound 5a by measuring their ability to either stimulate release of calcium or to block the calcium release stimulated by 5b and found that it responded to changes in structure ( sar ) , table 2 . this information was compared with that obtained for ( nt and 1 ) as well as levocabastine , which revealed that this assay was capable of identifying a range of activities from potent agonist ( 5a and 5b ) to partial agonist ( 6 ) to antagonist ( nt and 1 ) , table 1 below , which implied that the structure of the compound made a direct impact on the resulting mobilization of calcium . in table 1 , we have summarized the calcium release and binding data obtained for nt , 1 , 5a , and 5b , and the nts2-selective compound levocabastine ( 6 ) in both our rnts1 and rnts2 cell lines . compound 5b was more potent than compound 5a in the calcium mobilization assay but equally efficacious , and was designated as our nts2 full agonist standard ( the use of the term agonist here refers to a compound that stimulates calcium release in this assay ) . levocabastine ( 6 ) , a compound used for years to distinguish nts1 from nts2 , was also tested and found to be a potent partial agonist with an ec50 of 28 nm and an emax of 16% relative to 5b . compound 1 showed similar affinity to nt , while levocabastine ( 6 ) gave a ki of 33 nm . nt possesses antipsychotic activity and mediates nonopioid analgesia , while both nt and levocabastine ( 6 ) are active in models of persistent pain . the results obtained in our sar study to find a potent partial agonist based on compound 5a were centered on the three pyrazole scaffolds a we began by examining the effect of deleting the carboxylic acid group , compound 13 , as the carboxyl group is known to be a primary attachment point for ligands of the nt receptors . the data from 14b indicated that it would be a suitable stand in for 5a as it provided comparable agonist potency and efficacy for nts2 ( ec50 of 217 nm and emax 86% of 5b ) and nts1 antagonist activity ( ke of 23 nm ) . a comparison of the data obtained for compounds 14b18b ( table 2 ) illustrates the sar realized from changing the position of the 6-methoxy group ( 14b , 15b , and 16b ) , elimination of the 6-methoxy group ( 17b ) , and replacing the 2,6-methoxy groups with fluorine atoms ( 18b ) . the data obtained for compounds 5a , 14b , and 20b23b in table 2 illustrate the sar of the large amino acid side chains . compounds 20b22b also showed increased nts2 agonist potency ( lower ec50 ) , but this was accompanied by full efficacy ( high emax relative to 5b ) at nts2 , exactly opposite of that found in the potent partial agonist levocabastine ( 6 ) . this comparison of nts2 agonist potency to the nts1 antagonist potency for compounds 21b and 23b was an important clue to achieving selectivity in this series of compounds as this data reinforced the data from the previous series , which showed that the nts1 receptor relied much more heavily upon the 2,6-dimethoxyphenyl ring for its activity compared with nts2 and that the amino acid side chain could work in concert with the methoxyphenyl ring to retain these desired properties . these substitutions were included in our compound libraries because the naphthyl compounds were known to yield nts1 antagonists and the 4-fluorophenyl group was found in levocabastine ( 6 ) . comparing the naphthyl ( 24b ) and the 7-chloroquinolyl ( 14b ) compounds , we found ke values of 58 versus 23 nm and nts2 emax values of 45 and 86% of 5b , respectively . comparing compounds 26b and 21b , we found ke values of 230 versus 157 nm and nts2 emax values of 35 and 78% , respectively . this is readily revealed in a comparison of the nts2 efficacy data obtained for compound 21b , 23b , and the 2-methoxy naphthyl substituted compound 27b , with nts2 emax values of 78 , 35 , and 18% , respectively . the naphthyl - substituted compounds with the 2,6-dimethoxyphenyl ring were more potent at nts2 than their chloroquinoline - based counterparts ( 14b , 17b , 21b , and 23b ) , but naphthyl compounds bearing the 2-methoxyphenyl ring were equally potent . the nts2 binding data observed for the naphthyl - substituted compounds 24b27b was found to be in line with that seen for the similarly substituted 7-chloroquinolyl - substituted compounds , with the 2-methoxy derivatives showing lower affinity ( higher ki values ) than the 2,6-dimethoxy counterparts . toward obtaining an nts2 selective potent partial agonist , the calcium data profile for compound 26b looked promising as it moved closer to that of levocabastine ( 6 ) . the similarity between these two compounds ended , however , when comparing receptor selectivity , as compound 26b showed substantial nts1 antagonist activity while levocabastine ( 6 ) displayed no activity at nts1 . in keeping with previous observations , the transition to the 2-methoxyphenyl ring ( 7b ) from the 2,6-dimethoxyphenyl ring ( 29b ) led to a nearly 50% reduction in nts2 efficacy , with nts2 emax values of 15 and 8% of 5b , respectively . the nts2 binding affinity observed for 29b and 7b was consistent with similarly substituted 7-chloroquinolyl ( 21b , 23b ) or naphthyl - substituted ( 26b , 27b ) analogues bearing the 1-aminocyclohexanecarboxylic acid , with one important exception ; for 7b , the binding affinity did not decrease in going from the 2,6-dimethoxyphenyl ( 29b ) to the 2-methoxyphenyl ring substitution ( 7b ) as seen in the ki values of 140 and 153 nm , respectively . from the standpoint of calcium mobilization at nts1 and nts2 , compounds ( 29b and 7b ) appear to be selective for the nts2 receptor . as seen in table 3 , comparison of the relative binding affinities at nts1 and nts2 showed that compound 7b is 161-fold selective for nts2 versus nts1 while the dimethoxy analogue 29b shows only a 23-fold preference for nts2 over nts1 . these data point out that while the calcium assay described herein is useful for identifying compounds that are active at nts2 , conclusions about selectivity require the use of the binding assay . the identification of 7b as an nts2 selective compound demonstrated that this calcium assay was useful for driving sar studies in the pyrazole carboxamide series of compounds . the results of these experiments confirmed that the calcium mobilization observed was only found in the cho cells stably expressing the nts2 receptor . in summary , we tested the compounds common to neurotensin research and have identified their associated calcium mobilization patterns at nts1 and 2 using a flipr tetra using nt as our agonist standard for nts1 and 5b as our agonist standard for nts2 . we found that compounds known to possess analgesic and antipsychotic activity in vivo appear as antagonists ( nt and 1 ) ) or potent partial agonists ( levocabastine ( 6 ) ) in this nts2 in vitro assay . using the assays described above in combination with binding data , we identified structural changes to compound 5a that led to the discovery of the nts2 selective compound 7b , a pyrazole - based compound with in vitro properties similar to levocabastine in this assay , a compound known to be active against chronic pain . chemical shifts are reported in ppm relative to the tetramethylsilane , and coupling constant ( j ) values are reported in hertz ( hz ) . a magnetically stirred solution of a 4-aryl-2,4-diketoester sodium salt ( 8a f , 5 mmol ) and arylhydrazine hydrochloride ( 9a c , 5 mmol ) in glacial acetic acid ( 35 ml ) and concd hcl ( 1.5 ml ) was heated under reflux for 5 h and then cooled to rt . pyrazole carboxylic acids 11a j were prepared from the esters ( 10a j ) using the methyl ester hydrolysis method described below . to a magnetically stirred suspension of the appropriate pyrazole carboxylic acid ( 11a j , 0.122 mmol ) in anhydrous ch2cl2 ( 20 ml ) were added successively triethylamine ( 0.366 mmol ) , hbtu ( 0.146 mmol ) , and the appropriate amino acid ester hydrochloride salt ( 12a e , 0.122 mmol ) . after stirring for 16 h , the resulting mixture was concentrated and the residue purified by flash chromatography using a gradient of 0100% etoac in hexanes over 30 min collecting peaks only . to a magnetically stirred solution of methyl ester ( 7a,2023a , 26a , 27a , and 29a ) from the coupling reaction ( 0.1 mmol ) in 1,4-dioxane ( 5 ml ) was added 1n lioh ( 2 ml ) , followed by stirring at room temperature overnight . the mixture was then concentrated , and the residue was taken up in water ( 2 ml ) and extracted with ethyl acetate ( 15 ml ) . this was extracted twice with ch2cl2 , and the combined extracts were dried over na2so4 and concentrated to give solid final products . to a magnetically stirred solution of the tert - butyl ester intermediate ( 1419a,24a,25a , and 28a ) obtained from the coupling reaction ( 0.08 mmol ) in ch2cl2 ( 10 ml ) was added excess tfa ( 10 ml ) at room temp . ester 10b was prepared from methyl 3-[(2,6-dimethoxyphenyl)carbonyl]-2-oxopentanoate , sodium salt ( 8b ) , and 9a according to the general method ( pale - yellow solid , 45% ) . ester 10e was prepared from methyl 4-(2,6-difluorophenyl)-2,4-dioxobutanoate sodium salt ( 8e ) and 9a according to the general method ( yellow amorphous solid , 52% ) . ester 10f was prepared from methyl 4-(2-methoxyphenyl)-2,4-dioxo - butanoate sodium salt ( 8f ) and 9a according to the general method ( yellow solid , 43% ) . ester 10h was prepared from methyl 4-(2-methoxyphenyl)-2,4-dioxobutanoate sodium salt ( 8f ) and 9b according to the general method ( off - white solid , 40% ) . then 1/10th volume of 10 final concentration of the test antagonist compounds were added in 1% dmso in assay buffer , and the plate was incubated for 15 min at 37 c , 5% co2 .

the integrated microbial genomes ( img ) system integrates genomes from all three domains of life , as well as viruses , plasmids and genome fragments ( partial sequences of genomic regions of interest , such as biosynthetic clusters ) . until 2012 , img used ncbi s refseq resource ( 1 ) as its main source of public genome sequence data and annotations consisting of predicted genes and protein products , with a refseq - specific pipeline used for retrieving new genomes from refseq s ftp site . for non - public ( i.e. private ) datasets , the img er submission system allowed scientists to select their sequencing projects in gold ( 2 ) and then submit their genome sequence data for annotation and integration into the expert review version of img , img / er ( http://img.jgi.doe.gov/er ) . public and private genomes were processed using different annotation and data integration pipelines , and recorded in different databases . in an effort to improve the efficiency of data processing and tracking , img s genome submission , annotation and integration pipelines were consolidated in november 2012 . the img er submission system ( http://img.jgi.doe.gov/submit ) and associated ( submission , gene prediction , functional annotation and data integration ) data processing pipelines were extended to handle both public and private genomes in a uniform manner . the pipelines use a common mechanism for tracking the processing status of genome datasets , gold provides the information needed for retrieving new public genomes from refseq or genbank ( 3 ) and both public and private genomes are recorded in a common img data warehouse . for every genome , the img data warehouse records primary genome sequence information including its organization into chromosomal replicons ( for finished genomes ) and scaffolds and/or contigs ( for draft genomes ) , together with predicted protein - coding sequences , some rna - coding genes and protein product names that are provided by the genome sequence centers or generated by img s functional annotation pipeline . public and private genomes submitted for annotation and integration by img s pipelines are first associated with sequencing projects in gold . custom tools and metadata about the topology of contigs and scaffolds are used to identify the origin of replication of circular replicons and permute the corresponding scaffold or contig if necessary . to ensure accurate identification of partial genes bordering the gaps , gene models and other features are initially predicted on individual contigs and combined thereafter to generate scaffold - level structural annotation . predictions from both methods are concatenated , and in case of overlapping elements , the shorter one is removed . ribosomal rna genes ( 5s , 16s and 23s ) are predicted using hmmsearch against the custom models generated for each type of rrna in bacteria and archaea ( 7,8 ) . with the exception of trna and rrna , sequences are first compared with a database containing all the non - coding rna genes in the rfam database using blast , then sequences that have hits to genes belonging to an rfam model are searched using the program infernal ( 10 ) . signal peptides are computed using signalp ( 11 ) , whereas transmembrane helices are computed using tmhmm ( 12 ) . protein - coding genes are predicted using prodigal ( 13 ) ; models overlapping with crisprs and certain types of rnas ( e.g. rrnas ) are removed . after a new genome is processed , protein - coding genes are compared with protein families and the proteome of selected publicly available first , protein sequences are compared with cog ( 14 ) using rps - blast , pfam - a ( 15 ) using hmmer 3.0b2 executed inside sanger s pfam_scan.pl wrapper script and tigrfam ( 16 ) databases using hmmer 3.0 ( 8) , and associated with kegg orthology ( ko ) terms ( 17 ) using usearch ( 18 ) . genomes in img are associated with kegg pathways using the assignment of ko terms to protein - coding genes , while their association with metacyc pathways ( 19 ) is based on correlating enzyme ec numbers in metacyc reactions with ec numbers associated with protein - coding genes via ko terms . genes are further characterized using an img native collection of generic ( protein cluster - independent ) functional roles called img terms that are defined by their association with generic ( organism - independent ) functional hierarchies , called img pathways ( 20 ) . img terms and pathways are specified by domain experts at doe - jgi as part of the process of annotating specific genomes of interest , and are subsequently propagated to all the genomes in img using a rule - based methodology . transporter genes are linked to the transport classification database ( 21 ) based on their assignment to cog , pfam or tigrfam domains or img terms that correspond to transporter families . the integration of new genomes into img involves computing protein sequence similarities between their genes and genes of all other ( new or existing ) genomes in the system , assigning img terms and protein product names to the genes of the new genomes , identifying fusions and computing conserved gene cassettes ( putative operons ) . for each gene , img provides lists of related ( e.g. homolog , paralog and ortholog ) genes that are based on sequence similarities computed using usearch for protein - coding and rna genes . a fused gene ( fusion ) is defined as a gene that is formed from the composition ( fusion ) of two or more previously separate genes ( 22 ) . fusions are identified based on computing usearch similarities between genes . only genes from finished genomes are considered as putative components to avoid false predictions from fragmented genes in draft genomes . furthermore , genes that frequently appear as fragmented in finished genomes , such as transposases and integrases , as well as the phylogenetic distribution of best hits against a set of reference isolate genomes also provides additional information on possible horizontal gene transfers for isolates . chromosomal cassette is defined as a stretch of genes with intergenic distance 300 bp , whereby the genes can be on the same or different strands of the chromosome . chromosomal cassettes with a minimum size of two genes common in at least two separate genomes are defined as conserved chromosomal cassettes. the identification of common genes across organisms is based on two gene clustering methods , namely , participation in cog and pfam clusters ( 23 ) . note that for public and private genomes that are already associated with genes and/or protein product names , the native gene and/or product names are preserved in img unless their replacement is explicitly requested at the time they are submitted for annotation and integration into img . the content of img has grown steadily since the first version released in march 2005 , with the current version of img ( as on 10 september 2013 ) containing 11 568 bacterial , archaeal and eukaryotic genomes , an increase of > 300% since august 2011 ( 24 ) . img also includes 2848 viral genomes , 1198 plasmids that did not come from a specific microbial genome sequencing project and 581 genome fragments , bringing its total content to 16 195 genome datasets with > 42 million protein - coding genes . the number of single cell genomes included into img has increased substantially : there are 1341 single cell genomes in the current version of img compared with only 21 in august 2011 . approximately 240 single cell genomes are part of the microbial dark matter project that aims to expand the genomic encyclopedia of bacteria and archaea by targeting 100 single cell representatives of uncultured candidate phyla ( 25 ) . img has 13 342 genome datasets that are publicly available to all users without restrictions via the img / w datamart ( http://img.jgi.doe.gov/w ) . genomes that have not been yet published ( also known as private ) are password - protected and available only to the scientists who study ( own ) them through the img / er ( expert review ) datamart ( http://img.jgi.doe.gov/er ) . private genomes are usually publicly released 6 months after the dataset becomes available in img . img / er allows individual scientists or groups of scientists to review and curate the functional annotation of microbial genomes in the context of img s public genomes ( 26 ) . since august 2011 , hundreds of private genomes have been reviewed and curated using img / er , a relatively small fraction of the 9000 genomes that were processed by img s data annotation and integration pipelines , as genome curation is a time - consuming process . genome curation is usually carried out for identifying missing genes or for correcting functional annotations , e.g. as part of the process of curating img native terms and pathways . , 64 new protein expression datasets ( samples ) that are part of two studies were included into img , bringing the total to 90 samples across five studies . the organization and analysis of proteomic data in img are discussed in ( 24 ) . the first rnaseq ( transcriptomic ) datasets included into img in 2011 are part of the synechococcus pcc study consisting of 40 samples ( billis , k . , billini , m . , kyrpides , n.c . , and mavromatis , k . , submitted for publication ) . as of august 2013 , img contains 99 samples across 10 rnaseq studies . a typical rnaseq study involves the sequencing of cdna from a genome under different experimental conditions , with the effect of each experimental condition being captured by a sample . as part of rnaseq sequencing analysis , reads are mapped to the reference genome involved in the study , and the expressed genes in each sample are recorded with their observed read counts , mean , median and strand . the scope of mapping is determined by the type of cdna sample ( sscdna / dscdna ) and the directionality of the libraries , whereby reads may map to a single strand or both strands of the reference sequence . expression levels are normalized by computing rpkm ( reads per kilobase per million ) , quantile or affine transformations and may need to be interpreted based on the type of cdna in the sample . for genomes involved in rnaseq studies , the experiments / samples are recorded in img together with experimental conditions , and the read counts are organized per expressed gene , as illustrated in figure 1 . img statistics on img s front page , following the experiments link available on the ( ii ) an rna - seq study is associated with samples and the number of genes expressed across all samples . ( iii ) each sample is associated with the number of expressed genes , the total number of reads and the average number of reads per gene . ( iv ) an expressed gene is associated with a read count ( total number of reads divided by the size of the gene ) and normalized coverage ( coverage for a gene in the experiment divided by the total number of reads in that experiment ) . img statistics on img s front page , following the experiments link available on the ( ii ) an rna - seq study is associated with samples and the number of genes expressed across all samples . ( iii ) each sample is associated with the number of expressed genes , the total number of reads and the average number of reads per gene . ( iv ) an expressed gene is associated with a read count ( total number of reads divided by the size of the gene ) and normalized coverage ( coverage for a gene in the experiment divided by the total number of reads in that experiment ) . img contains biosynthetic clusters of genes associated with pathways involved in the generation of secondary metabolites in isolate prokaryotic genomes . experimentally validated biosynthetic clusters were identified by searching ncbi 's nucleotide database for genome fragments ( partially sequenced genomes ) containing gene clusters associated with secondary metabolites / natural products ( 28 ) . biosynthetic clusters in img are associated with img , metacyc and kegg pathways as well as information available in gold on their natural products . ( i ) genomes associated with biosynthetic clusters can be retrieved and examined using the genome browser. ( ii ) the number of biosynthetic clusters is provided in the genome statistics section of the organism detail page of a genome , together with a hyperlink to ( iii ) the list of biosynthetic clusters , whereby for each cluster the number of associated genes , the evidence type and the corresponding natural product are provided . ( i ) genomes associated with biosynthetic clusters can be retrieved and examined using the genome browser. ( ii ) the number of biosynthetic clusters is provided in the genome statistics section of the organism detail page of a genome , together with a hyperlink to ( iii ) the list of biosynthetic clusters , whereby for each cluster the number of associated genes , the evidence type and the corresponding natural product are provided . genomes associated with biosynthetic clusters can be examined as illustrated in figure 2 , where these genomes are listed in descending order of the number of biosynthetic clusters present in them . alternatively , img can be used to find genomes associated with natural products associated with genome fragments but not with biosynthetic clusters , as illustrated in figure 2(v ) . natural products are small metabolites found in nature , and although the biosynthetic clusters associated with the generation of natural products have been identified , there are still natural products whose production mechanisms in prokaryotes remain unknown . figure 3.rna-seq data exploration . ( i ) the list of rna - seq studies associated with a genome can be accessed from its organism details , with each study associated with ( ii ) a list of rna - seq experiments ( samples ) . individual samples can be selected for further analysis , such as ( iii ) examining its expressed genes as a list or using the ( iv ) chromosome viewer . a sample can be also examined in the context of ( v ) pathways that have at least one enzyme associated with an expressed gene in the sample , whereby for each pathway ( vi ) enzymes are displayed with colors representing the level of expression for the associated genes ; mousing over an enzyme shows the number of expressed genes associated with the enzyme . ( i ) rna - seq sample comparison starts with the selection of samples of interest . ( ii ) pairwise sample analysis supports comparing samples in terms of up / downregulated genes , with ( iii ) a histogram preview helping setting the thresholds for comparison . ( iv ) the result of the comparison can be examined in terms of functions , whereby genes associated with kegg pathways or cog functions are grouped together . ( v ) the strength of the association of gene expression between pairs of samples can be examined using rank correlation. ( vi ) linear regression analysis helps estimate whether two samples are technical replicates . ( vii ) multiple sample analysis consists of clustering samples based on the abundance of expressed genes , using a variety of clustering methods . ( viii ) clusters of samples can be examined in the context of pathways , whereby enzymes are displayed with colors representing the cluster . ( i ) the list of rna - seq studies associated with a genome can be accessed from its organism details , with each study associated with ( ii ) a list of rna - seq experiments ( samples ) . individual samples can be selected for further analysis , such as ( iii ) examining its expressed genes as a list or using the ( iv ) chromosome viewer . a sample can be also examined in the context of ( v ) pathways that have at least one enzyme associated with an expressed gene in the sample , whereby for each pathway ( vi ) enzymes are displayed with colors representing the level of expression for the associated genes ; mousing over an enzyme shows the number of expressed genes associated with the enzyme . ( i ) rna - seq sample comparison starts with the selection of samples of interest . ( ii ) pairwise sample analysis supports comparing samples in terms of up / downregulated genes , with ( iii ) a histogram preview helping setting the thresholds for comparison . ( iv ) the result of the comparison can be examined in terms of functions , whereby genes associated with kegg pathways or cog functions are grouped together . ( v ) the strength of the association of gene expression between pairs of samples can be examined using rank correlation. ( vi ) linear regression analysis helps estimate whether two samples are technical replicates . ( vii ) multiple sample analysis consists of clustering samples based on the abundance of expressed genes , using a variety of clustering methods . ( viii ) clusters of samples can be examined in the context of pathways , whereby enzymes are displayed with colors representing the cluster . the content of img has grown steadily since the first version released in march 2005 , with the current version of img ( as on 10 september 2013 ) containing 11 568 bacterial , archaeal and eukaryotic genomes , an increase of > 300% since august 2011 ( 24 ) . img also includes 2848 viral genomes , 1198 plasmids that did not come from a specific microbial genome sequencing project and 581 genome fragments , bringing its total content to 16 195 genome datasets with > 42 million protein - coding genes . the number of single cell genomes included into img has increased substantially : there are 1341 single cell genomes in the current version of img compared with only 21 in august 2011 . approximately 240 single cell genomes are part of the microbial dark matter project that aims to expand the genomic encyclopedia of bacteria and archaea by targeting 100 single cell representatives of uncultured candidate phyla ( 25 ) . img has 13 342 genome datasets that are publicly available to all users without restrictions via the img / w datamart ( http://img.jgi.doe.gov/w ) . genomes that have not been yet published ( also known as private ) are password - protected and available only to the scientists who study ( own ) them through the img / er ( expert review ) datamart ( http://img.jgi.doe.gov/er ) . private genomes are usually publicly released 6 months after the dataset becomes available in img . img / er allows individual scientists or groups of scientists to review and curate the functional annotation of microbial genomes in the context of img s public genomes ( 26 ) . since august 2011 , hundreds of private genomes have been reviewed and curated using img / er , a relatively small fraction of the 9000 genomes that were processed by img s data annotation and integration pipelines , as genome curation is a time - consuming process . genome curation is usually carried out for identifying missing genes or for correcting functional annotations , e.g. as part of the process of curating img native terms and pathways . , 64 new protein expression datasets ( samples ) that are part of two studies were included into img , bringing the total to 90 samples across five studies . the organization and analysis of proteomic data in img are discussed in ( 24 ) . the first rnaseq ( transcriptomic ) datasets included into img in 2011 are part of the synechococcus pcc study consisting of 40 samples ( billis , k . , billini , m . , kyrpides , n.c . , and mavromatis , k . , a typical rnaseq study involves the sequencing of cdna from a genome under different experimental conditions , with the effect of each experimental condition being captured by a sample . as part of rnaseq sequencing analysis , reads are mapped to the reference genome involved in the study , and the expressed genes in each sample are recorded with their observed read counts , mean , median and strand . the scope of mapping is determined by the type of cdna sample ( sscdna / dscdna ) and the directionality of the libraries , whereby reads may map to a single strand or both strands of the reference sequence . expression levels are normalized by computing rpkm ( reads per kilobase per million ) , quantile or affine transformations and may need to be interpreted based on the type of cdna in the sample . for genomes involved in rnaseq studies , the experiments / samples are recorded in img together with experimental conditions , and the read counts are organized per expressed gene , as illustrated in figure 1 . img statistics on img s front page , following the experiments link available on the ( ii ) an rna - seq study is associated with samples and the number of genes expressed across all samples . ( iii ) each sample is associated with the number of expressed genes , the total number of reads and the average number of reads per gene . ( iv ) an expressed gene is associated with a read count ( total number of reads divided by the size of the gene ) and normalized coverage ( coverage for a gene in the experiment divided by the total number of reads in that experiment ) . img statistics on img s front page , following the experiments link available on the ( ii ) an rna - seq study is associated with samples and the number of genes expressed across all samples . ( iii ) each sample is associated with the number of expressed genes , the total number of reads and the average number of reads per gene . ( iv ) an expressed gene is associated with a read count ( total number of reads divided by the size of the gene ) and normalized coverage ( coverage for a gene in the experiment divided by the total number of reads in that experiment ) . img contains biosynthetic clusters of genes associated with pathways involved in the generation of secondary metabolites in isolate prokaryotic genomes . experimentally validated biosynthetic clusters were identified by searching ncbi 's nucleotide database for genome fragments ( partially sequenced genomes ) containing gene clusters associated with secondary metabolites / natural products ( 28 ) . biosynthetic clusters in img are associated with img , metacyc and kegg pathways as well as information available in gold on their natural products . figure 2.biosynthetic clusters . ( i ) genomes associated with biosynthetic clusters can be retrieved and examined using the genome browser. ( ii ) the number of biosynthetic clusters is provided in the genome statistics section of the organism detail page of a genome , together with a hyperlink to ( iii ) the list of biosynthetic clusters , whereby for each cluster the number of associated genes , the evidence type and the corresponding natural product are provided . ( i ) genomes associated with biosynthetic clusters can be retrieved and examined using the genome browser. ( ii ) the number of biosynthetic clusters is provided in the genome statistics section of the organism detail page of a genome , together with a hyperlink to ( iii ) the list of biosynthetic clusters , whereby for each cluster the number of associated genes , the evidence type and the corresponding natural product are provided . genomes associated with biosynthetic clusters can be examined as illustrated in figure 2 , where these genomes are listed in descending order of the number of biosynthetic clusters present in them . alternatively , img can be used to find genomes associated with natural products associated with genome fragments but not with biosynthetic clusters , as illustrated in figure 2(v ) . natural products are small metabolites found in nature , and although the biosynthetic clusters associated with the generation of natural products have been identified , there are still natural products whose production mechanisms in prokaryotes remain unknown . figure 3.rna-seq data exploration . ( i ) the list of rna - seq studies associated with a genome can be accessed from its organism details , with each study associated with ( ii ) a list of rna - seq experiments ( samples ) . individual samples can be selected for further analysis , such as ( iii ) examining its expressed genes as a list or using the ( iv ) chromosome viewer . a sample can be also examined in the context of ( v ) pathways that have at least one enzyme associated with an expressed gene in the sample , whereby for each pathway ( vi ) enzymes are displayed with colors representing the level of expression for the associated genes ; mousing over an enzyme shows the number of expressed genes associated with the enzyme . ( i ) rna - seq sample comparison starts with the selection of samples of interest . ( ii ) pairwise sample analysis supports comparing samples in terms of up / downregulated genes , with ( iii ) a histogram preview helping setting the thresholds for comparison . ( iv ) the result of the comparison can be examined in terms of functions , whereby genes associated with kegg pathways or cog functions are grouped together . ( v ) the strength of the association of gene expression between pairs of samples can be examined using rank correlation. ( vi ) linear regression analysis helps estimate whether two samples are technical replicates . ( vii ) multiple sample analysis consists of clustering samples based on the abundance of expressed genes , using a variety of clustering methods . ( viii ) clusters of samples can be examined in the context of pathways , whereby enzymes are displayed with colors representing the cluster . ( i ) the list of rna - seq studies associated with a genome can be accessed from its organism details , with each study associated with ( ii ) a list of rna - seq experiments ( samples ) . individual samples can be selected for further analysis , such as ( iii ) examining its expressed genes as a list or using the ( iv ) chromosome viewer . a sample can be also examined in the context of ( v ) pathways that have at least one enzyme associated with an expressed gene in the sample , whereby for each pathway ( vi ) enzymes are displayed with colors representing the level of expression for the associated genes ; mousing over an enzyme shows the number of expressed genes associated with the enzyme . ( i ) rna - seq sample comparison starts with the selection of samples of interest . ( ii ) pairwise sample analysis supports comparing samples in terms of up / downregulated genes , with ( iii ) a histogram preview helping setting the thresholds for comparison . ( iv ) the result of the comparison can be examined in terms of functions , whereby genes associated with kegg pathways or cog functions are grouped together . ( v ) the strength of the association of gene expression between pairs of samples can be examined using rank correlation. ( vi ) linear regression analysis helps estimate whether two samples are technical replicates . ( vii ) multiple sample analysis consists of clustering samples based on the abundance of expressed genes , using a variety of clustering methods . ( viii ) clusters of samples can be examined in the context of pathways , whereby enzymes are displayed with colors representing the cluster . browsers and search tools allow finding and selecting genomes , genes and functions of interest , which can then be examined individually or analyzed in a comparative context . gene content - based comparison of genomes is provided by the phylogenetic profiler and the phylogenetic profiler for gene cassettes tools that allow identifying genes in a query genome in terms of presence or absence of homologs in other genomes , or participation in conserved gene cassettes across other genomes ( 29,30 ) . function - based comparison of genomes is provided by the abundance profile overview and function profile tools that allow comparing the relative abundance of protein families ( cogs , pfams , tigrfams ) and functional families ( enzymes ) across genomes . the composition of analysis operations is facilitated by genome , scaffold , gene and function carts that handle lists of genomes , scaffolds , genes and functions , respectively . tools for identifying and correcting annotation anomalies , such as dubious protein product names , and for filling annotation gaps , such as genes that may have been missed by gene prediction tools or genes without predicted functions , are discussed in ( 26 ) . img analysis tool extensions have addressed performance ( 30 ) , data quality control , such as single cell data decontamination ( 31 ) , and new data types , such as rna - seq and biosynthetic cluster data . rna - seq studies can be accessed from the experiments statistics section of the for example , the expression studies link in the organism details page , such as that shown in figure 3(i ) , leads to the list of associated rna - seq samples , as the list shown in figure 3(ii ) . rna - seq studies associated with a genome can be compared using pairwise or multiple sample analysis tools as illustrated in figure 4 . after samples are selected for comparison ( figure 4(i ) ) , pairs of samples can be compared in terms of up- or downregulation of genes , as illustrated in figure 4(ii ) , with a threshold specified for the difference in gene expression . the difference in expression is computed using the logr = log2(query / reference ) or the reldiff = 2(query - reference)/(query + reference ) metric . the comparison can be first previewed using a histogram , as illustrated in figure 4(iii ) , which can help set the thresholds for the search of over - expressed or under - expressed genes between a pair of samples . the result of the comparison can be examined at the level of individual up- and downregulated genes , which can be selected for inclusion in the gene cart for further analysis . alternatively , the result of the comparison can be examined in terms of functions , as illustrated in figure 4(iv ) , with genes associated with kegg pathways or cog functions grouped together . genes associated with a specific kegg pathway can be examined in the context of the pathway , similar to the example shown in figure 3(vi ) earlier . rank correlation , as illustrated in figure 4(v ) , whereas linear regression analysis , illustrated in figure 4(vi ) , helps determine whether two samples are technical replicates . multiple rna - seq sample analysis usually involves clustering based on the abundance of expressed genes , where the proximity of grouping indicates the relative degree of similarity of samples to each other . there is a choice of clustering methods , such as pairwise complete linkage and pairwise single linkage , and distance measure , such as pearson correlation , spearman s rank correlation and euclidean distance , as illustrated in figure 4(vii ) . the result of clustering is displayed as a hierarchical tree of samples and a normalized heat map of coverage values for each gene for each sample . clusters of multiple samples can be also examined in the context of pathways , as illustrated in figure 4(viii ) , whereby enzymes are displayed with colors representing the cluster . img s genome sequence data content is maintained through regular updates managed by the img submission system and involving new genomes sequenced at jgi , genomes sequenced at other organizations and submitted for inclusion into img by scientists worldwide and genomes from genbank . for genomes with multiple submissions , img genome data are distributed through genome data portals available at : http://genome.jgi.doe.gov/. img s data annotation and integration pipelines have been automated , thus improving their ability to keep pace with the rapidly increasing number of sequenced genomes . thus , the quality of gene models for genomes available in public resources is known to vary greatly depending on the quality of sequence and the software used for annotation . an analysis conducted at jgi of the protein - coding genes of microbial genes in genbank indicates that 10% ( > 1 million ) of predicted protein - coding are erroneous : they are false - positive genes , unidentified pseudogene fragments or genes with translational exceptions or have incorrectly predicted start sites . to improve the consistency of annotation and the quality of predicted genes , all public microbial genomes in img will be re - annotated using img s annotation pipeline . a rapidly increasing number of single cell genomes are included into img . typically , the first version of a single cell genome is analyzed for identifying contigs that may come from contaminant ( e.g. pseudomonas , ralstonia ) organisms . the sequence of analysis steps needed to identify and remove contaminated contigs is described in ( 31 ) . the importance of functional genomics in validating gene function in an integrated comparative genomics context is also being underscored , pushing experimental data from methylomics and transposon mutagenesis experiments into img . systematic paradigms for associating computationally predicted gene structural and functional information with experimental functional genomics are being constructed . tools are being developed for mining and visualizing different types of omics datasets in an integrated genomic context . img s users are faced with the increasing burden of analyzing a rapidly growing number of genomic datasets . this analytical challenge can be alleviated by synthesizing genomic data using the pangenome conceptual abstraction ( 32 ) . a pangenome consists of the core part of a species ( i.e. the genes present in all of the sequenced strains or of all samples of a microbial community ) and the variable part ( the genes present in some but not all of the strains or samples ) . an experimental version of img has been extended with five pangenomes , as well as analysis tools and viewers that allow users to explore individual pangenomes and compare pangenomes and genomes . a public version of img containing pangenome data and analysis tools is expected to be released in the near future . the director , office of science , office of biological and environmental research , life sciences division , u.s . [ de - ac02 - 05ch11231 ] ; this research used resources of the national energy research scientific computing center , which is supported by the office of science of the u.s .

the integrated microbial genomes ( img ) data warehouse integrates genomes from all three domains of life , as well as plasmids , viruses and genome fragments . img provides tools for analyzing and reviewing the structural and functional annotations of genomes in a comparative context . img s data content and analytical capabilities have increased continuously since its first version released in 2005 . since the last report published in the 2012 nar database issue , img s annotation and data integration pipelines have evolved while new tools have been added for recording and analyzing single cell genomes , rna seq and biosynthetic cluster data . different img datamarts provide support for the analysis of publicly available genomes ( img / w : http://img.jgi.doe.gov/w ) , expert review of genome annotations ( img / er : http://img.jgi.doe.gov/er ) and teaching and training in the area of microbial genome analysis ( img / edu : http://img.jgi.doe.gov/edu ) .

DATA SOURCES AND PROCESSING DATA CONTENT Genomics data Omics data Biosynthetic clusters ANALYSIS TOOLS FUTURE PLANS FUNDING

the integrated microbial genomes ( img ) system integrates genomes from all three domains of life , as well as viruses , plasmids and genome fragments ( partial sequences of genomic regions of interest , such as biosynthetic clusters ) . until 2012 , img used ncbi s refseq resource ( 1 ) as its main source of public genome sequence data and annotations consisting of predicted genes and protein products , with a refseq - specific pipeline used for retrieving new genomes from refseq s ftp site . private ) datasets , the img er submission system allowed scientists to select their sequencing projects in gold ( 2 ) and then submit their genome sequence data for annotation and integration into the expert review version of img , img / er ( http://img.jgi.doe.gov/er ) . public and private genomes were processed using different annotation and data integration pipelines , and recorded in different databases . in an effort to improve the efficiency of data processing and tracking , img s genome submission , annotation and integration pipelines were consolidated in november 2012 . the img er submission system ( http://img.jgi.doe.gov/submit ) and associated ( submission , gene prediction , functional annotation and data integration ) data processing pipelines were extended to handle both public and private genomes in a uniform manner . the pipelines use a common mechanism for tracking the processing status of genome datasets , gold provides the information needed for retrieving new public genomes from refseq or genbank ( 3 ) and both public and private genomes are recorded in a common img data warehouse . for every genome , the img data warehouse records primary genome sequence information including its organization into chromosomal replicons ( for finished genomes ) and scaffolds and/or contigs ( for draft genomes ) , together with predicted protein - coding sequences , some rna - coding genes and protein product names that are provided by the genome sequence centers or generated by img s functional annotation pipeline . public and private genomes submitted for annotation and integration by img s pipelines are first associated with sequencing projects in gold . with the exception of trna and rrna , sequences are first compared with a database containing all the non - coding rna genes in the rfam database using blast , then sequences that have hits to genes belonging to an rfam model are searched using the program infernal ( 10 ) . signal peptides are computed using signalp ( 11 ) , whereas transmembrane helices are computed using tmhmm ( 12 ) . after a new genome is processed , protein - coding genes are compared with protein families and the proteome of selected publicly available first , protein sequences are compared with cog ( 14 ) using rps - blast , pfam - a ( 15 ) using hmmer 3.0b2 executed inside sanger s pfam_scan.pl wrapper script and tigrfam ( 16 ) databases using hmmer 3.0 ( 8) , and associated with kegg orthology ( ko ) terms ( 17 ) using usearch ( 18 ) . genomes in img are associated with kegg pathways using the assignment of ko terms to protein - coding genes , while their association with metacyc pathways ( 19 ) is based on correlating enzyme ec numbers in metacyc reactions with ec numbers associated with protein - coding genes via ko terms . img terms and pathways are specified by domain experts at doe - jgi as part of the process of annotating specific genomes of interest , and are subsequently propagated to all the genomes in img using a rule - based methodology . the integration of new genomes into img involves computing protein sequence similarities between their genes and genes of all other ( new or existing ) genomes in the system , assigning img terms and protein product names to the genes of the new genomes , identifying fusions and computing conserved gene cassettes ( putative operons ) . for each gene , img provides lists of related ( e.g. furthermore , genes that frequently appear as fragmented in finished genomes , such as transposases and integrases , as well as the phylogenetic distribution of best hits against a set of reference isolate genomes also provides additional information on possible horizontal gene transfers for isolates . note that for public and private genomes that are already associated with genes and/or protein product names , the native gene and/or product names are preserved in img unless their replacement is explicitly requested at the time they are submitted for annotation and integration into img . the content of img has grown steadily since the first version released in march 2005 , with the current version of img ( as on 10 september 2013 ) containing 11 568 bacterial , archaeal and eukaryotic genomes , an increase of > 300% since august 2011 ( 24 ) . img also includes 2848 viral genomes , 1198 plasmids that did not come from a specific microbial genome sequencing project and 581 genome fragments , bringing its total content to 16 195 genome datasets with > 42 million protein - coding genes . the number of single cell genomes included into img has increased substantially : there are 1341 single cell genomes in the current version of img compared with only 21 in august 2011 . approximately 240 single cell genomes are part of the microbial dark matter project that aims to expand the genomic encyclopedia of bacteria and archaea by targeting 100 single cell representatives of uncultured candidate phyla ( 25 ) . img has 13 342 genome datasets that are publicly available to all users without restrictions via the img / w datamart ( http://img.jgi.doe.gov/w ) . genomes that have not been yet published ( also known as private ) are password - protected and available only to the scientists who study ( own ) them through the img / er ( expert review ) datamart ( http://img.jgi.doe.gov/er ) . img / er allows individual scientists or groups of scientists to review and curate the functional annotation of microbial genomes in the context of img s public genomes ( 26 ) . since august 2011 , hundreds of private genomes have been reviewed and curated using img / er , a relatively small fraction of the 9000 genomes that were processed by img s data annotation and integration pipelines , as genome curation is a time - consuming process . genome curation is usually carried out for identifying missing genes or for correcting functional annotations , e.g. as of august 2013 , img contains 99 samples across 10 rnaseq studies . as part of rnaseq sequencing analysis , reads are mapped to the reference genome involved in the study , and the expressed genes in each sample are recorded with their observed read counts , mean , median and strand . the scope of mapping is determined by the type of cdna sample ( sscdna / dscdna ) and the directionality of the libraries , whereby reads may map to a single strand or both strands of the reference sequence . expression levels are normalized by computing rpkm ( reads per kilobase per million ) , quantile or affine transformations and may need to be interpreted based on the type of cdna in the sample . for genomes involved in rnaseq studies , the experiments / samples are recorded in img together with experimental conditions , and the read counts are organized per expressed gene , as illustrated in figure 1 . img statistics on img s front page , following the experiments link available on the ( ii ) an rna - seq study is associated with samples and the number of genes expressed across all samples . ( iv ) an expressed gene is associated with a read count ( total number of reads divided by the size of the gene ) and normalized coverage ( coverage for a gene in the experiment divided by the total number of reads in that experiment ) . img statistics on img s front page , following the experiments link available on the ( ii ) an rna - seq study is associated with samples and the number of genes expressed across all samples . ( iv ) an expressed gene is associated with a read count ( total number of reads divided by the size of the gene ) and normalized coverage ( coverage for a gene in the experiment divided by the total number of reads in that experiment ) . img contains biosynthetic clusters of genes associated with pathways involved in the generation of secondary metabolites in isolate prokaryotic genomes . biosynthetic clusters in img are associated with img , metacyc and kegg pathways as well as information available in gold on their natural products . ( ii ) the number of biosynthetic clusters is provided in the genome statistics section of the organism detail page of a genome , together with a hyperlink to ( iii ) the list of biosynthetic clusters , whereby for each cluster the number of associated genes , the evidence type and the corresponding natural product are provided . alternatively , img can be used to find genomes associated with natural products associated with genome fragments but not with biosynthetic clusters , as illustrated in figure 2(v ) . natural products are small metabolites found in nature , and although the biosynthetic clusters associated with the generation of natural products have been identified , there are still natural products whose production mechanisms in prokaryotes remain unknown . a sample can be also examined in the context of ( v ) pathways that have at least one enzyme associated with an expressed gene in the sample , whereby for each pathway ( vi ) enzymes are displayed with colors representing the level of expression for the associated genes ; mousing over an enzyme shows the number of expressed genes associated with the enzyme . ( viii ) clusters of samples can be examined in the context of pathways , whereby enzymes are displayed with colors representing the cluster . a sample can be also examined in the context of ( v ) pathways that have at least one enzyme associated with an expressed gene in the sample , whereby for each pathway ( vi ) enzymes are displayed with colors representing the level of expression for the associated genes ; mousing over an enzyme shows the number of expressed genes associated with the enzyme . ( viii ) clusters of samples can be examined in the context of pathways , whereby enzymes are displayed with colors representing the cluster . the content of img has grown steadily since the first version released in march 2005 , with the current version of img ( as on 10 september 2013 ) containing 11 568 bacterial , archaeal and eukaryotic genomes , an increase of > 300% since august 2011 ( 24 ) . img also includes 2848 viral genomes , 1198 plasmids that did not come from a specific microbial genome sequencing project and 581 genome fragments , bringing its total content to 16 195 genome datasets with > 42 million protein - coding genes . the number of single cell genomes included into img has increased substantially : there are 1341 single cell genomes in the current version of img compared with only 21 in august 2011 . approximately 240 single cell genomes are part of the microbial dark matter project that aims to expand the genomic encyclopedia of bacteria and archaea by targeting 100 single cell representatives of uncultured candidate phyla ( 25 ) . img has 13 342 genome datasets that are publicly available to all users without restrictions via the img / w datamart ( http://img.jgi.doe.gov/w ) . genomes that have not been yet published ( also known as private ) are password - protected and available only to the scientists who study ( own ) them through the img / er ( expert review ) datamart ( http://img.jgi.doe.gov/er ) . img / er allows individual scientists or groups of scientists to review and curate the functional annotation of microbial genomes in the context of img s public genomes ( 26 ) . since august 2011 , hundreds of private genomes have been reviewed and curated using img / er , a relatively small fraction of the 9000 genomes that were processed by img s data annotation and integration pipelines , as genome curation is a time - consuming process . genome curation is usually carried out for identifying missing genes or for correcting functional annotations , e.g. the organization and analysis of proteomic data in img are discussed in ( 24 ) . as part of rnaseq sequencing analysis , reads are mapped to the reference genome involved in the study , and the expressed genes in each sample are recorded with their observed read counts , mean , median and strand . the scope of mapping is determined by the type of cdna sample ( sscdna / dscdna ) and the directionality of the libraries , whereby reads may map to a single strand or both strands of the reference sequence . expression levels are normalized by computing rpkm ( reads per kilobase per million ) , quantile or affine transformations and may need to be interpreted based on the type of cdna in the sample . img statistics on img s front page , following the experiments link available on the ( ii ) an rna - seq study is associated with samples and the number of genes expressed across all samples . ( iv ) an expressed gene is associated with a read count ( total number of reads divided by the size of the gene ) and normalized coverage ( coverage for a gene in the experiment divided by the total number of reads in that experiment ) . img statistics on img s front page , following the experiments link available on the ( ii ) an rna - seq study is associated with samples and the number of genes expressed across all samples . ( iv ) an expressed gene is associated with a read count ( total number of reads divided by the size of the gene ) and normalized coverage ( coverage for a gene in the experiment divided by the total number of reads in that experiment ) . img contains biosynthetic clusters of genes associated with pathways involved in the generation of secondary metabolites in isolate prokaryotic genomes . biosynthetic clusters in img are associated with img , metacyc and kegg pathways as well as information available in gold on their natural products . ( ii ) the number of biosynthetic clusters is provided in the genome statistics section of the organism detail page of a genome , together with a hyperlink to ( iii ) the list of biosynthetic clusters , whereby for each cluster the number of associated genes , the evidence type and the corresponding natural product are provided . ( ii ) the number of biosynthetic clusters is provided in the genome statistics section of the organism detail page of a genome , together with a hyperlink to ( iii ) the list of biosynthetic clusters , whereby for each cluster the number of associated genes , the evidence type and the corresponding natural product are provided . alternatively , img can be used to find genomes associated with natural products associated with genome fragments but not with biosynthetic clusters , as illustrated in figure 2(v ) . natural products are small metabolites found in nature , and although the biosynthetic clusters associated with the generation of natural products have been identified , there are still natural products whose production mechanisms in prokaryotes remain unknown . a sample can be also examined in the context of ( v ) pathways that have at least one enzyme associated with an expressed gene in the sample , whereby for each pathway ( vi ) enzymes are displayed with colors representing the level of expression for the associated genes ; mousing over an enzyme shows the number of expressed genes associated with the enzyme . ( viii ) clusters of samples can be examined in the context of pathways , whereby enzymes are displayed with colors representing the cluster . a sample can be also examined in the context of ( v ) pathways that have at least one enzyme associated with an expressed gene in the sample , whereby for each pathway ( vi ) enzymes are displayed with colors representing the level of expression for the associated genes ; mousing over an enzyme shows the number of expressed genes associated with the enzyme . browsers and search tools allow finding and selecting genomes , genes and functions of interest , which can then be examined individually or analyzed in a comparative context . gene content - based comparison of genomes is provided by the phylogenetic profiler and the phylogenetic profiler for gene cassettes tools that allow identifying genes in a query genome in terms of presence or absence of homologs in other genomes , or participation in conserved gene cassettes across other genomes ( 29,30 ) . function - based comparison of genomes is provided by the abundance profile overview and function profile tools that allow comparing the relative abundance of protein families ( cogs , pfams , tigrfams ) and functional families ( enzymes ) across genomes . the composition of analysis operations is facilitated by genome , scaffold , gene and function carts that handle lists of genomes , scaffolds , genes and functions , respectively . tools for identifying and correcting annotation anomalies , such as dubious protein product names , and for filling annotation gaps , such as genes that may have been missed by gene prediction tools or genes without predicted functions , are discussed in ( 26 ) . img analysis tool extensions have addressed performance ( 30 ) , data quality control , such as single cell data decontamination ( 31 ) , and new data types , such as rna - seq and biosynthetic cluster data . rna - seq studies can be accessed from the experiments statistics section of the for example , the expression studies link in the organism details page , such as that shown in figure 3(i ) , leads to the list of associated rna - seq samples , as the list shown in figure 3(ii ) . after samples are selected for comparison ( figure 4(i ) ) , pairs of samples can be compared in terms of up- or downregulation of genes , as illustrated in figure 4(ii ) , with a threshold specified for the difference in gene expression . the comparison can be first previewed using a histogram , as illustrated in figure 4(iii ) , which can help set the thresholds for the search of over - expressed or under - expressed genes between a pair of samples . alternatively , the result of the comparison can be examined in terms of functions , as illustrated in figure 4(iv ) , with genes associated with kegg pathways or cog functions grouped together . rank correlation , as illustrated in figure 4(v ) , whereas linear regression analysis , illustrated in figure 4(vi ) , helps determine whether two samples are technical replicates . there is a choice of clustering methods , such as pairwise complete linkage and pairwise single linkage , and distance measure , such as pearson correlation , spearman s rank correlation and euclidean distance , as illustrated in figure 4(vii ) . clusters of multiple samples can be also examined in the context of pathways , as illustrated in figure 4(viii ) , whereby enzymes are displayed with colors representing the cluster . img s genome sequence data content is maintained through regular updates managed by the img submission system and involving new genomes sequenced at jgi , genomes sequenced at other organizations and submitted for inclusion into img by scientists worldwide and genomes from genbank . for genomes with multiple submissions , img genome data are distributed through genome data portals available at : http://genome.jgi.doe.gov/. img s data annotation and integration pipelines have been automated , thus improving their ability to keep pace with the rapidly increasing number of sequenced genomes . an analysis conducted at jgi of the protein - coding genes of microbial genes in genbank indicates that 10% ( > 1 million ) of predicted protein - coding are erroneous : they are false - positive genes , unidentified pseudogene fragments or genes with translational exceptions or have incorrectly predicted start sites . to improve the consistency of annotation and the quality of predicted genes , all public microbial genomes in img will be re - annotated using img s annotation pipeline . a rapidly increasing number of single cell genomes are included into img . typically , the first version of a single cell genome is analyzed for identifying contigs that may come from contaminant ( e.g. systematic paradigms for associating computationally predicted gene structural and functional information with experimental functional genomics are being constructed . an experimental version of img has been extended with five pangenomes , as well as analysis tools and viewers that allow users to explore individual pangenomes and compare pangenomes and genomes . a public version of img containing pangenome data and analysis tools is expected to be released in the near future .

systemic lupus erythematosus ( sle ) is a multisystemic autoimmune disease caused by immune system mediated tissue damage . manifestations of sle can involve the skin , joints , kidney , central nervous system , cardiovascular system , serosal membranes and hematologic and immune systems . the disease is highly heterogeneous , with patients manifesting variable combinations of clinical features . in most patients with sle , the disease is characterized by a waxing and waning clinical course , although some demonstrate a pattern of chronic activity . the molecular triggers of disease are not known , but the pathogenesis is understood including production of autoantibodies exhibiting multiple specificities , with reactivity with nucleic acid - binding proteins as a common feature . immune complexes , along with immune system cells and soluble mediators , generate inflammation and tissue damage . like hashimoto s thyroiditis and sjgren s syndrome , female - to - male ratio is approximately 8:1 to 9:1 in adults , and most cases are diagnosed between the ages of 15 and 44 years ( 1 , 2 ) . autoimmune thyroiditis ( at ) is an organ - specific disease associated with production of a variety of antibodies such as antinuclear antibodies , anti - double - stranded dna , anti - ro antibodies , anti - cardiolipin antibodies and others ( 3 ) . autoimmune thyroiditis typically produces a modest goiter as a result of glandular infiltration with lymphocytes , inflammatory changes in thyrocytes and fibrosis . the hypothyroid state caused by autoimmune thyroiditis is associated with increased tsh levels that further stimulates thyroid enlargement . graves disease is characterized by diffuse thyroid enlargement due to the action of thyroid stimulating immunoglobulins . other forms of thyroiditis can also present with goitrous enlargement of the thyroid gland , including subacute , lymphocytic and acute ( suppurative ) thyroiditis ( 4 ) . the association between at , thyroid dysfunction and sle has been reported in several studies with conflicting conclusions ( 5 - 12 ) . while some studies reported a higher prevalence of hyperthyroidism in sle patients ( 5 - 7 ) , others reported a higher prevalence of hypothyroidism ( 8 - 12 ) . one controlled study could not demonstrate a significantly higher prevalence of hypothyroidism in patients with sle ( 13 ) . the prevalence of anti - thyroid antibodies ( ata ) , namely anti - thyroglobulin antibodies ( atg ) and anti - thyroid peroxidase antibodies ( tpo ) was reported to be higher in patients with sle ( 8 - 13 ) . in most studies , the results were compared to the prevalence of these disorders in the general population and were independent from sle disease activity . the present study was performed to evaluate the prevalence of thyroid dysfunction and thyroid autoantibodies in iranian patients with sle and to investigate its relation with sle disease and other autoantibodies . this was a cross - sectional observational , case - controlled study performed in lupus clinic of hafez hospital , a specialized , referral , teaching hospital affiliated to shiraz university of medical sciences in islamic republic of iran . we included a total of 88 concecutive lupus ( sle ) patients diagnosed according to the acrcriteria published in 1997 and 88 age- and sex - matched healthy volunteers as control group . the study protocol was approved by the institutional review board ( irb ) of shiraz university of medical sciences and the ethics committee with document no . inclusion criterion was lupus diagnosis according to the acr criteria with at least one year of disease duration ( 14 ) . all lupus patients selected by one rheumatologist and if he or she had criteria for selection referred for study inclusion . sle patients were not in a flare of their disease based on the sle disease activity index ( less than 3 ) . the exclusion criteria were patients with other immune - suppressed conditions such as diabetes mellitus , kidney or bone marrow recipients , those infected with human immunodeficiency virus ( hiv ) and those with primary immunodeficiency diseases such as x - linked agammaglobulinemia , severe combined immunodeficiency ( scid ) and common variable immune deficiency ( cvid ) , simultaneous rheumatic disease such as sjogren s syndrome , rheumatoid arthritis ( ra ) and systemic sclerosis ( ss ) and those with malignancies . none of the healthy volunteers in control group had history of immunologic or rheumatic disease as well as allergy and thyroid diseases . all patients underwent clinical and laboratory evaluations at diagnosis and quarterly during the follow - up and followed and examined by one rheumatologist throughout the year . demographic and clinical information such as disease duration , disease manifestations at any time and laboratory data , were collected from all patients . for this study , we analyzed symptoms and clinical findings that could be attributed to both autoimmune thyroid disease and sle activity ( or treatment related ) , including fatigue , nervousness , trembling , heat or cold intolerance , weight change , hyper- or hyporeflexia , tachy- or bradycardia , menstrual abnormalities , muscle atrophy and edema . the levels of tsh , total triiodothyronine ( t3 ) , total thyroxine ( t4 ) , antinuclear antibody ( ana ) , antibodies to double - stranded dna ( dsdna ) , anti- thyroglobulin antibodies ( anti- tg ) and anti - thyroid peroxidase ( anti - tpo ) were determined in all patients and controls . standard enzyme - linked immunosorbent assay ( elisa ) was used to screen ana ( elisa , eia-2395 ; drg diagnostic , marburg , germany ) and dsdna ( elisa , eia-3356 ; drg diagnostic , marburg , germany ) . total t3 ( total t3 ria - kit , immunotech , prague , czech republic ) , total t4 ( total t4 ria - kit , immunotech , prague , czech republic ) and anti - tpo ( anti - tpo ria - kit , immunotech , prague , czech republic ) were measured by radioimmunoassay method , while serum tsh ( tsh iria - kit , immunotech , prague , czech republic ) and anti - tg ( anti - htg iria - kit , immunotech , prague , czech republic ) were evaluated by immunoradiometric assay methods . all hormonal tests were performed in endocrine and metabolism research center with gamma counter instrument ( kontron made by austria ) , which is calibrated regularly by local representative company in ir iran . all statistical analyses were performed using statistical package for social sciences version 17.0 ( spss inc . , all of the qualitative variables were checked for normal distribution in the case and control groups . this was a cross - sectional observational , case - controlled study performed in lupus clinic of hafez hospital , a specialized , referral , teaching hospital affiliated to shiraz university of medical sciences in islamic republic of iran . we included a total of 88 concecutive lupus ( sle ) patients diagnosed according to the acrcriteria published in 1997 and 88 age- and sex - matched healthy volunteers as control group . the study protocol was approved by the institutional review board ( irb ) of shiraz university of medical sciences and the ethics committee with document no . inclusion criterion was lupus diagnosis according to the acr criteria with at least one year of disease duration ( 14 ) . all lupus patients selected by one rheumatologist and if he or she had criteria for selection referred for study inclusion . sle patients were not in a flare of their disease based on the sle disease activity index ( less than 3 ) . the exclusion criteria were patients with other immune - suppressed conditions such as diabetes mellitus , kidney or bone marrow recipients , those infected with human immunodeficiency virus ( hiv ) and those with primary immunodeficiency diseases such as x - linked agammaglobulinemia , severe combined immunodeficiency ( scid ) and common variable immune deficiency ( cvid ) , simultaneous rheumatic disease such as sjogren s syndrome , rheumatoid arthritis ( ra ) and systemic sclerosis ( ss ) and those with malignancies . none of the healthy volunteers in control group had history of immunologic or rheumatic disease as well as allergy and thyroid diseases . all patients underwent clinical and laboratory evaluations at diagnosis and quarterly during the follow - up and followed and examined by one rheumatologist throughout the year . demographic and clinical information such as disease duration , disease manifestations at any time and laboratory data , were collected from all patients . for this study , we analyzed symptoms and clinical findings that could be attributed to both autoimmune thyroid disease and sle activity ( or treatment related ) , including fatigue , nervousness , trembling , heat or cold intolerance , weight change , hyper- or hyporeflexia , tachy- or bradycardia , menstrual abnormalities , muscle atrophy and edema . the levels of tsh , total triiodothyronine ( t3 ) , total thyroxine ( t4 ) , antinuclear antibody ( ana ) , antibodies to double - stranded dna ( dsdna ) , anti- thyroglobulin antibodies ( anti- tg ) and anti - thyroid peroxidase ( anti - tpo ) were determined in all patients and controls . standard enzyme - linked immunosorbent assay ( elisa ) was used to screen ana ( elisa , eia-2395 ; drg diagnostic , marburg , germany ) and dsdna ( elisa , eia-3356 ; drg diagnostic , marburg , germany ) . total t3 ( total t3 ria - kit , immunotech , prague , czech republic ) , total t4 ( total t4 ria - kit , immunotech , prague , czech republic ) and anti - tpo ( anti - tpo ria - kit , immunotech , prague , czech republic ) were measured by radioimmunoassay method , while serum tsh ( tsh iria - kit , immunotech , prague , czech republic ) and anti - tg ( anti - htg iria - kit , immunotech , prague , czech republic ) were evaluated by immunoradiometric assay methods . the results were compared between cases and controls . all hormonal tests were performed in endocrine and metabolism research center with gamma counter instrument ( kontron made by austria ) , which is calibrated regularly by local representative company in ir iran . all statistical analyses were performed using statistical package for social sciences version 17.0 ( spss inc . , chicago , il , usa ) . chi - square test was used for comparison of qualitative variables between both groups . all of the qualitative variables were checked for normal distribution in the case and control groups . a total of 88 sle patients and 88 healthy age- and sex - matched individuals entered the study . six ( 6.8% ) of our study population were men and 82 ( 93.2% ) women . the mean age of sle patients was 32.16 9.19 years ranged from 18 to 62 years . there was not any significant difference between both study groups regarding age ( p = 0.821 ) and sex ( p = 0.783 ) . about eighty one percent ( 81.8% ) of patients group had positive results for ana and 70.4 % for anti - dsdna . forty three lupus patients had positive findings for anti - tg antibody compared to 23.9% in control group with significant p - value ( p = 0.015 ) . no significant difference between the two groups was found regarding anti - tpo antibody ( 31.8% vs. 22.7% ; p = 0.236 ) . abbreviations : ana , antinuclear antibody ; anti dsdna , anti double stranded dna ; anti - tg ab , anti - thyroglobulin antibodies ; anti - tpo ab , anti - thyroid peroxidase antibodies ; tsh , thyroid stimulating hormone . data are reported as no ( % ) or means sd for 95% ci with 5% degree of freedom . tsh levels were normal in 65.9% and 76.1% , high in 31.8% and 23.9% and low in 2.3% and 0% of patients and control groups , respectively with non - significant p - value ( p = 0.161 ) . t3 levels were normal in 85.2% and 80.7% , high in 13.6% and 19.3% and low in 1.1% and 0.0% of patients and control group , respectively with no significant p - value ( p = 0.373 ) . the titers of t4 did not differ significantly between the two study groups ( 7.63 2.1 vs. 8.19 1.82 ; p = 0.054 ) . we performed correlation analysis between the autoantibodies and thyroid function test in those with sle ( table 2 ) . ana was positively correlated with dsdna ( r = 0.320 , p = 0.002 ) . in the same way , anti - tpo was positively correlated to anti - tg ( r = 0.187 , p = 0.018 ) . anti - tpo antibodies were correlated positively with t3 ( r = 0.390 , p < 0.0001 ) and t4 ( r = 0.251 , p = 0.018 ) . t3 and t4 were also correlated with each other positively ( r = 0.610 , p < 0.0001 ) . abbreviations : ana , antinuclear antibody ; anti dsdna , anti double stranded dna ; anti - tg ab , anti - thyroglobulin antibodies ; anti - tpo ab , anti - thyroid peroxidase antibodies ; tsh , thyroid stimulating hormone . sle is a multisystemic disease and can involve any organ in the body including the thyroid gland ( 15 - 18 ) . in the other hand , because of its autoimmune nature , other autoantibodies such as anti - tpo and anti - tg can be produced during the natural course of disease ( 8 , 9 ) . several studies demonstrated that patients with sle have increased prevalence of thyroid dysfunction and autoimmune thyroiditis ( 5 - 12 ) . while some studies reported a higher prevalence of hyperthyroidism in sle patients ( 5 - 7 ) , others reported a higher prevalence of hypothyroidism ( 8 - 12 ) . in this study , we investigated the prevalence of thyroid dysfunction in patients with sle and compared it to healthy individuals . our sle series had higher titers of anti - tg antibodies accompanied by lower titers of t3 . this shows that patients with sle are at increased risk of developing thyroid dysfunction , especially hypothyroidism . our results are consistent with some previous studies ( 7 - 9 ) and are contrary to some others ( 5 , 10 , 12 ) . a large scale study analyzed the association between sle and thyroid disease . in this study , data collected during 2000 - 2009 and concluded that sle patients had a lower rate of thyroid disease than matched control group ( 19 , 20 ) . one controlled study that addressed this issue could not demonstrate a significantly higher prevalence of hypothyroidism in patients with sle ( 13 ) . the prevalence of anti - thyroid antibodies ( ata ) , namely anti - thyroglobulin antibodies ( atg ) and anti - thyroid peroxidase antibodies ( tpo ) was reported to be higher in patients with sle ( 8 - 13 ) . in most studies , the results were compared to the prevalence of these disorders in the general population and were independent from sle disease activity . in this ( 10 ) evaluated the prevalence of clinical and subclinical thyroid disorders in patients with sle compared with sex- and age - matched controls . thyroid hormones and antithyroid antibodies were tested and thyroid ultrasonography was performed in 213 patients with sle compared with 426 sex- and age - matched controls , from the same geographic area , with a well - defined status of iodine intake . the odds ratio for subclinical hypothyroidism for female patients with sle with respect to controls was 4.5 ( 95% confidence interval [ ci ] , 2.5 - 8.4 ) , for antithyroid peroxidase antibody ( abtpo ) positivity was 2.6 ( 95% ci , 1.7 - 4.1 ) and for thyroid autoimmunity 2.9 ( 95% ci , 2.0 - 4.4 ) . the mean values of thyroid stimulating hormone and abtpo were higher in female sle patients than controls ( p < 0.01 ) . a significantly ( p < 0.01 ) higher prevalence of clinical hypothyroidism and grave s disease was observed in female sle patients than controls . no significant difference between sle patients and controls was detected regarding free triiodothyronine and thyroxine . it is suggested to assess thyroid function and abtpos and perform ultrasonography as part of the clinical profile in sle patients . subjects at high risk ( women , positive abtpos , hypoechoic and small thyroid ) should have thyroid function follow - up and appropriate treatment in due course ( 10 ) . many systemic autoimmune disorders have been reported to be associated with autoimmune thyroiditis in up to 50% of cases . these disorders include sle , rheumatoid arthritis , sjogren s disease , mixed connective tissue disease and others . in particular , the incidence of sle in hashimoto thyroiditis was reported to be 6.5% compared to 0.05% - 0.1% in the general population ( 21 ) . in this ( 12 ) examined the frequencies of abnormal thyroid function tests and serum thyroid autoantibodies in healthy kuwaitis and those with autoimmune diseases . serum concentrations of sensitive thyrotropin and free thyroxine were measured in 577 apparently healthy controls , 177 patients with rheumatoid arthritis ( ra ) , 60 with systemic lupus erythematosus ( sle ) and 25 with primary sjogren s syndrome ( pss ) . serum microsomal and thyroglobulin autoantibodies were also measured . for analysis of thyroid function tests , the subjects were classified into five categories of normal , subclinical hypothyroidism , overt hypothyroidism , euthyroid sick syndrome and biochemical hyperthyroidism . subclinical hypothyroidism was seen in 1.7% of healthy controls , 10.2% of ra , 13.3% of sle and 16% of pss patients . among ra patients , the frequency of 9 subclinical hypothyroidism in females ( 11.4% ) was significantly higher than males ( 5.4% ; p < 0.01 ) . in sle and pss patients , overt hypothyroidism was seen in 1.4% of controls , 10.2% of ra , 8.3% of sle and 4% of pss patients . biochemical hyperthyroidism was seen in 0.2% of controls , 4.5% of ra , 5% of sle and none of pss patients . the euthyroid sick syndrome was seen in 0.4% of controls , 13.6% of ra , 16.7% of sle and none of pss patients . thyroid autoantibodies were present in 3.1% of controls , 12.4% of ra , 18.3% of sle and 12% of pss patients . these data showed that abnormal thyroid function tests and thyroid autoantibodies occur frequently in kuwaitis with autoimmune diseases . therefore , ordering these tests in these diseases is recommended ( 12 ) . however , in most studies , higher prevalence of hypothyroidism , either clinical or sub - clinical , was reported in patients with sle . many of these cases were also accompanied by elevated ata suggestive of autoimmune thyroiditis ( 13 ) . it was also found that the ata levels correlated with serum levels of other sle characteristic antibodies such as anti - sm , ribonucleoprotein ( rnp ) and double stranded dna antibodies ( 6 ) . this notion can explain the results of previous studies showing that ata was often elevated in sle patients , and these patients had a higher prevalence of thyroid dysfunction , mainly clinical or subclinical hypothyroidism ( 22 , 23 ) . in most studies , thyroid function tests and ata in sle patients were not measured in a control group and were compared to existing data in the general population ( 8) . in this study , we compared the results of the study group to an age- and sex - matched control group . the prevalence of thyroid dysfunction did not differ significantly between the groups in our series . this can be due to this fact that all of our patients were in remission and none had disease activity score more than 3 . the prevalence of ata in the study group was similar to the previously reported prevalence in sle ( 7 ) . the prevalence of ata in a small control group and in an autopsy study was reported to be 27% in female patients without overt thyroid disease ( 13 , 24 , 25 ) . however , a large welsh study reported the prevalence of ata as 10% in healthy individuals aged 18 - 45 years ( 26 ) . this is similar to the data obtained in both our study and control groups and highlights the need for an appropriate control group . as autoimmune thyroiditis affects more often females than males , the absence of difference in the prevalence of ata between our study group and the control group can not be ascribed to the gender differences between the groups . it could presumably reduce the production of antibodies , which might explain the paucity of ata production in sle patients ( 27 ) . the two main autoantibodies are atg and tpo , with the latter being the most important and representative for autoimmune thyroiditis ( 14 ) . it was shown that some patients with positive results for ata at a certain point during the course of their disease had negative results for one or more of the antibodies tested during follow - up visits . however , patients with persistently elevated ata were more likely to develop thyroid diseases ( 23 ) . the time when the tests are performed is therefore important when sle patients are evaluated for ata . however , this objective is beyond the scope of cross - sectional studies , like reported studies to date . in a 20-year follow - up study on the incidence of thyroid disease , it was found that the odds ratios for development of hypothyroidism in ata - positive individuals were 8 for women and 25 for men ( 28 ) . showed that free t3 and free t4 concentrations were lower in lupus patients compared to control group ( 29 ) . ( 30 ) showed that primary and subclinical hypothyroidism is the most common thyroid disease in lupus patients and rarely hyperthyroidism occurs in these patients . franco et al . indicated that autoimmune thyroid disease in sle is frequent and has no correlation with severity of disease ( 31 ) . one meta - analysis by pan et al . suggested that autoimmune thyroid disease is more prevalent in patients with lupus ( 20 ) . ( 9 ) showed that ata levels did not correlate with the degree of disease activity measured by the sledai score or with any component of the sledai . therefore , it can be assumed that production of ata is unrelated to the disease activity . in our study , we performed thyroid tests in lupus patients and compared with normal population as control group , which is representative of our population as strength point . in contrast , our study had a small sample size and cross - sectional study as a weak point . more longitudinal prospective cohort studies are needed for more evaluation of the prevalence of thyroid dysfunction in sle patients . in conclusion , this cross - sectional study showed that thyroid dysfunction was not more prevalent in sle patients compared to healthy individuals . it has not yet been established that thyroid function tests should be performed routinely in sle patients . however , testing ata in euthyroid sle patients seems unjustified . larger controlled longitudinal studies are needed to elucidate the causal relationships between thyroid dysfunction , ata , sle and sle disease activity . larger sample size , presence of control group or a meta - analysis are needed for obtaining better results and be able to generalize them .

background : systemic lupus erythematosus ( sle ) is a multisystemic autoimmune disease caused by immune system - mediated tissue damage . autoimmune thyroiditis ( at ) is an organ - specific disease associated with production of a variety of antibodies such as antinuclear antibodies , anti - double - stranded dna , anti - ro antibodies and anti - cardiolipin antibodies.objectives:the aim of this study was to evaluate the prevalence of thyroid dysfunction and thyroid auto - antibodies in patients with sle and its relation to sle disease and other autoantibodies.patients and methods : this was a case - control study . the study included a total of 88 patients with sle and 88 age- and sex - matched healthy volunteers as control group . two study groups were compared regarding thyroid function test , antinuclear antibody ( ana ) , antibodies to double - stranded dna ( dsdna ) , anti- thyroglobulin antibody ( anti - tg ) , and anti - thyroid peroxidase ( anti - tpo ) antibody.results:the mean age of sle patients and controls were 32.16 9.19 and 32.48 9.47 years , respectively ( p = 0.821 ) . patients had significantly higher prevalence ( 43.2% vs. 23.9% ; p = 0.015 ) and titers ( 221.8 570.5 vs. 78.2 277.2 ; p = 0.036 ) of antibodies to tg compared to controls . the patients had significantly lower titers of t3 compared to controls ( 125.2 35.6 vs. 136.2 26.5 ; p = 0.021 ) . the titers of t4 , tsh and anti - tpo antibody did not differ significantly between the two study groups.conclusions:thyroid dysfunction was not higher in sle patients compared to healthy individuals . however , anti - tg antibodies were higher in sle patients . it has not yet been established that thyroid function tests should be performed routinely in sle patients .

1. Background 2. Objectives 3. Patients and Methods 3.1. Patients 3.2. Study Protocol 3.3. Statistical Analysis 4. Results 5. Discussion

systemic lupus erythematosus ( sle ) is a multisystemic autoimmune disease caused by immune system mediated tissue damage . in most patients with sle , the disease is characterized by a waxing and waning clinical course , although some demonstrate a pattern of chronic activity . immune complexes , along with immune system cells and soluble mediators , generate inflammation and tissue damage . like hashimoto s thyroiditis and sjgren s syndrome , female - to - male ratio is approximately 8:1 to 9:1 in adults , and most cases are diagnosed between the ages of 15 and 44 years ( 1 , 2 ) . autoimmune thyroiditis ( at ) is an organ - specific disease associated with production of a variety of antibodies such as antinuclear antibodies , anti - double - stranded dna , anti - ro antibodies , anti - cardiolipin antibodies and others ( 3 ) . the hypothyroid state caused by autoimmune thyroiditis is associated with increased tsh levels that further stimulates thyroid enlargement . graves disease is characterized by diffuse thyroid enlargement due to the action of thyroid stimulating immunoglobulins . the association between at , thyroid dysfunction and sle has been reported in several studies with conflicting conclusions ( 5 - 12 ) . while some studies reported a higher prevalence of hyperthyroidism in sle patients ( 5 - 7 ) , others reported a higher prevalence of hypothyroidism ( 8 - 12 ) . one controlled study could not demonstrate a significantly higher prevalence of hypothyroidism in patients with sle ( 13 ) . the prevalence of anti - thyroid antibodies ( ata ) , namely anti - thyroglobulin antibodies ( atg ) and anti - thyroid peroxidase antibodies ( tpo ) was reported to be higher in patients with sle ( 8 - 13 ) . in most studies , the results were compared to the prevalence of these disorders in the general population and were independent from sle disease activity . the present study was performed to evaluate the prevalence of thyroid dysfunction and thyroid autoantibodies in iranian patients with sle and to investigate its relation with sle disease and other autoantibodies . this was a cross - sectional observational , case - controlled study performed in lupus clinic of hafez hospital , a specialized , referral , teaching hospital affiliated to shiraz university of medical sciences in islamic republic of iran . we included a total of 88 concecutive lupus ( sle ) patients diagnosed according to the acrcriteria published in 1997 and 88 age- and sex - matched healthy volunteers as control group . the study protocol was approved by the institutional review board ( irb ) of shiraz university of medical sciences and the ethics committee with document no . sle patients were not in a flare of their disease based on the sle disease activity index ( less than 3 ) . the exclusion criteria were patients with other immune - suppressed conditions such as diabetes mellitus , kidney or bone marrow recipients , those infected with human immunodeficiency virus ( hiv ) and those with primary immunodeficiency diseases such as x - linked agammaglobulinemia , severe combined immunodeficiency ( scid ) and common variable immune deficiency ( cvid ) , simultaneous rheumatic disease such as sjogren s syndrome , rheumatoid arthritis ( ra ) and systemic sclerosis ( ss ) and those with malignancies . none of the healthy volunteers in control group had history of immunologic or rheumatic disease as well as allergy and thyroid diseases . for this study , we analyzed symptoms and clinical findings that could be attributed to both autoimmune thyroid disease and sle activity ( or treatment related ) , including fatigue , nervousness , trembling , heat or cold intolerance , weight change , hyper- or hyporeflexia , tachy- or bradycardia , menstrual abnormalities , muscle atrophy and edema . the levels of tsh , total triiodothyronine ( t3 ) , total thyroxine ( t4 ) , antinuclear antibody ( ana ) , antibodies to double - stranded dna ( dsdna ) , anti- thyroglobulin antibodies ( anti- tg ) and anti - thyroid peroxidase ( anti - tpo ) were determined in all patients and controls . standard enzyme - linked immunosorbent assay ( elisa ) was used to screen ana ( elisa , eia-2395 ; drg diagnostic , marburg , germany ) and dsdna ( elisa , eia-3356 ; drg diagnostic , marburg , germany ) . total t3 ( total t3 ria - kit , immunotech , prague , czech republic ) , total t4 ( total t4 ria - kit , immunotech , prague , czech republic ) and anti - tpo ( anti - tpo ria - kit , immunotech , prague , czech republic ) were measured by radioimmunoassay method , while serum tsh ( tsh iria - kit , immunotech , prague , czech republic ) and anti - tg ( anti - htg iria - kit , immunotech , prague , czech republic ) were evaluated by immunoradiometric assay methods . this was a cross - sectional observational , case - controlled study performed in lupus clinic of hafez hospital , a specialized , referral , teaching hospital affiliated to shiraz university of medical sciences in islamic republic of iran . we included a total of 88 concecutive lupus ( sle ) patients diagnosed according to the acrcriteria published in 1997 and 88 age- and sex - matched healthy volunteers as control group . the study protocol was approved by the institutional review board ( irb ) of shiraz university of medical sciences and the ethics committee with document no . sle patients were not in a flare of their disease based on the sle disease activity index ( less than 3 ) . the exclusion criteria were patients with other immune - suppressed conditions such as diabetes mellitus , kidney or bone marrow recipients , those infected with human immunodeficiency virus ( hiv ) and those with primary immunodeficiency diseases such as x - linked agammaglobulinemia , severe combined immunodeficiency ( scid ) and common variable immune deficiency ( cvid ) , simultaneous rheumatic disease such as sjogren s syndrome , rheumatoid arthritis ( ra ) and systemic sclerosis ( ss ) and those with malignancies . none of the healthy volunteers in control group had history of immunologic or rheumatic disease as well as allergy and thyroid diseases . for this study , we analyzed symptoms and clinical findings that could be attributed to both autoimmune thyroid disease and sle activity ( or treatment related ) , including fatigue , nervousness , trembling , heat or cold intolerance , weight change , hyper- or hyporeflexia , tachy- or bradycardia , menstrual abnormalities , muscle atrophy and edema . the levels of tsh , total triiodothyronine ( t3 ) , total thyroxine ( t4 ) , antinuclear antibody ( ana ) , antibodies to double - stranded dna ( dsdna ) , anti- thyroglobulin antibodies ( anti- tg ) and anti - thyroid peroxidase ( anti - tpo ) were determined in all patients and controls . standard enzyme - linked immunosorbent assay ( elisa ) was used to screen ana ( elisa , eia-2395 ; drg diagnostic , marburg , germany ) and dsdna ( elisa , eia-3356 ; drg diagnostic , marburg , germany ) . total t3 ( total t3 ria - kit , immunotech , prague , czech republic ) , total t4 ( total t4 ria - kit , immunotech , prague , czech republic ) and anti - tpo ( anti - tpo ria - kit , immunotech , prague , czech republic ) were measured by radioimmunoassay method , while serum tsh ( tsh iria - kit , immunotech , prague , czech republic ) and anti - tg ( anti - htg iria - kit , immunotech , prague , czech republic ) were evaluated by immunoradiometric assay methods . the results were compared between cases and controls . a total of 88 sle patients and 88 healthy age- and sex - matched individuals entered the study . the mean age of sle patients was 32.16 9.19 years ranged from 18 to 62 years . there was not any significant difference between both study groups regarding age ( p = 0.821 ) and sex ( p = 0.783 ) . about eighty one percent ( 81.8% ) of patients group had positive results for ana and 70.4 % for anti - dsdna . forty three lupus patients had positive findings for anti - tg antibody compared to 23.9% in control group with significant p - value ( p = 0.015 ) . no significant difference between the two groups was found regarding anti - tpo antibody ( 31.8% vs. 22.7% ; p = 0.236 ) . abbreviations : ana , antinuclear antibody ; anti dsdna , anti double stranded dna ; anti - tg ab , anti - thyroglobulin antibodies ; anti - tpo ab , anti - thyroid peroxidase antibodies ; tsh , thyroid stimulating hormone . tsh levels were normal in 65.9% and 76.1% , high in 31.8% and 23.9% and low in 2.3% and 0% of patients and control groups , respectively with non - significant p - value ( p = 0.161 ) . t3 levels were normal in 85.2% and 80.7% , high in 13.6% and 19.3% and low in 1.1% and 0.0% of patients and control group , respectively with no significant p - value ( p = 0.373 ) . the titers of t4 did not differ significantly between the two study groups ( 7.63 2.1 vs. 8.19 1.82 ; p = 0.054 ) . we performed correlation analysis between the autoantibodies and thyroid function test in those with sle ( table 2 ) . ana was positively correlated with dsdna ( r = 0.320 , p = 0.002 ) . in the same way , anti - tpo was positively correlated to anti - tg ( r = 0.187 , p = 0.018 ) . anti - tpo antibodies were correlated positively with t3 ( r = 0.390 , p < 0.0001 ) and t4 ( r = 0.251 , p = 0.018 ) . t3 and t4 were also correlated with each other positively ( r = 0.610 , p < 0.0001 ) . abbreviations : ana , antinuclear antibody ; anti dsdna , anti double stranded dna ; anti - tg ab , anti - thyroglobulin antibodies ; anti - tpo ab , anti - thyroid peroxidase antibodies ; tsh , thyroid stimulating hormone . sle is a multisystemic disease and can involve any organ in the body including the thyroid gland ( 15 - 18 ) . in the other hand , because of its autoimmune nature , other autoantibodies such as anti - tpo and anti - tg can be produced during the natural course of disease ( 8 , 9 ) . several studies demonstrated that patients with sle have increased prevalence of thyroid dysfunction and autoimmune thyroiditis ( 5 - 12 ) . while some studies reported a higher prevalence of hyperthyroidism in sle patients ( 5 - 7 ) , others reported a higher prevalence of hypothyroidism ( 8 - 12 ) . in this study , we investigated the prevalence of thyroid dysfunction in patients with sle and compared it to healthy individuals . our sle series had higher titers of anti - tg antibodies accompanied by lower titers of t3 . this shows that patients with sle are at increased risk of developing thyroid dysfunction , especially hypothyroidism . a large scale study analyzed the association between sle and thyroid disease . in this study , data collected during 2000 - 2009 and concluded that sle patients had a lower rate of thyroid disease than matched control group ( 19 , 20 ) . one controlled study that addressed this issue could not demonstrate a significantly higher prevalence of hypothyroidism in patients with sle ( 13 ) . the prevalence of anti - thyroid antibodies ( ata ) , namely anti - thyroglobulin antibodies ( atg ) and anti - thyroid peroxidase antibodies ( tpo ) was reported to be higher in patients with sle ( 8 - 13 ) . in most studies , the results were compared to the prevalence of these disorders in the general population and were independent from sle disease activity . in this ( 10 ) evaluated the prevalence of clinical and subclinical thyroid disorders in patients with sle compared with sex- and age - matched controls . thyroid hormones and antithyroid antibodies were tested and thyroid ultrasonography was performed in 213 patients with sle compared with 426 sex- and age - matched controls , from the same geographic area , with a well - defined status of iodine intake . the odds ratio for subclinical hypothyroidism for female patients with sle with respect to controls was 4.5 ( 95% confidence interval [ ci ] , 2.5 - 8.4 ) , for antithyroid peroxidase antibody ( abtpo ) positivity was 2.6 ( 95% ci , 1.7 - 4.1 ) and for thyroid autoimmunity 2.9 ( 95% ci , 2.0 - 4.4 ) . the mean values of thyroid stimulating hormone and abtpo were higher in female sle patients than controls ( p < 0.01 ) . a significantly ( p < 0.01 ) higher prevalence of clinical hypothyroidism and grave s disease was observed in female sle patients than controls . no significant difference between sle patients and controls was detected regarding free triiodothyronine and thyroxine . it is suggested to assess thyroid function and abtpos and perform ultrasonography as part of the clinical profile in sle patients . many systemic autoimmune disorders have been reported to be associated with autoimmune thyroiditis in up to 50% of cases . in particular , the incidence of sle in hashimoto thyroiditis was reported to be 6.5% compared to 0.05% - 0.1% in the general population ( 21 ) . in this ( 12 ) examined the frequencies of abnormal thyroid function tests and serum thyroid autoantibodies in healthy kuwaitis and those with autoimmune diseases . serum concentrations of sensitive thyrotropin and free thyroxine were measured in 577 apparently healthy controls , 177 patients with rheumatoid arthritis ( ra ) , 60 with systemic lupus erythematosus ( sle ) and 25 with primary sjogren s syndrome ( pss ) . for analysis of thyroid function tests , the subjects were classified into five categories of normal , subclinical hypothyroidism , overt hypothyroidism , euthyroid sick syndrome and biochemical hyperthyroidism . subclinical hypothyroidism was seen in 1.7% of healthy controls , 10.2% of ra , 13.3% of sle and 16% of pss patients . among ra patients , the frequency of 9 subclinical hypothyroidism in females ( 11.4% ) was significantly higher than males ( 5.4% ; p < 0.01 ) . in sle and pss patients , overt hypothyroidism was seen in 1.4% of controls , 10.2% of ra , 8.3% of sle and 4% of pss patients . biochemical hyperthyroidism was seen in 0.2% of controls , 4.5% of ra , 5% of sle and none of pss patients . the euthyroid sick syndrome was seen in 0.4% of controls , 13.6% of ra , 16.7% of sle and none of pss patients . thyroid autoantibodies were present in 3.1% of controls , 12.4% of ra , 18.3% of sle and 12% of pss patients . these data showed that abnormal thyroid function tests and thyroid autoantibodies occur frequently in kuwaitis with autoimmune diseases . however , in most studies , higher prevalence of hypothyroidism , either clinical or sub - clinical , was reported in patients with sle . many of these cases were also accompanied by elevated ata suggestive of autoimmune thyroiditis ( 13 ) . it was also found that the ata levels correlated with serum levels of other sle characteristic antibodies such as anti - sm , ribonucleoprotein ( rnp ) and double stranded dna antibodies ( 6 ) . this notion can explain the results of previous studies showing that ata was often elevated in sle patients , and these patients had a higher prevalence of thyroid dysfunction , mainly clinical or subclinical hypothyroidism ( 22 , 23 ) . in most studies , thyroid function tests and ata in sle patients were not measured in a control group and were compared to existing data in the general population ( 8) . in this study , we compared the results of the study group to an age- and sex - matched control group . the prevalence of thyroid dysfunction did not differ significantly between the groups in our series . the prevalence of ata in the study group was similar to the previously reported prevalence in sle ( 7 ) . the prevalence of ata in a small control group and in an autopsy study was reported to be 27% in female patients without overt thyroid disease ( 13 , 24 , 25 ) . however , a large welsh study reported the prevalence of ata as 10% in healthy individuals aged 18 - 45 years ( 26 ) . this is similar to the data obtained in both our study and control groups and highlights the need for an appropriate control group . as autoimmune thyroiditis affects more often females than males , the absence of difference in the prevalence of ata between our study group and the control group can not be ascribed to the gender differences between the groups . it could presumably reduce the production of antibodies , which might explain the paucity of ata production in sle patients ( 27 ) . the two main autoantibodies are atg and tpo , with the latter being the most important and representative for autoimmune thyroiditis ( 14 ) . however , patients with persistently elevated ata were more likely to develop thyroid diseases ( 23 ) . the time when the tests are performed is therefore important when sle patients are evaluated for ata . showed that free t3 and free t4 concentrations were lower in lupus patients compared to control group ( 29 ) . indicated that autoimmune thyroid disease in sle is frequent and has no correlation with severity of disease ( 31 ) . suggested that autoimmune thyroid disease is more prevalent in patients with lupus ( 20 ) . in our study , we performed thyroid tests in lupus patients and compared with normal population as control group , which is representative of our population as strength point . more longitudinal prospective cohort studies are needed for more evaluation of the prevalence of thyroid dysfunction in sle patients . in conclusion , this cross - sectional study showed that thyroid dysfunction was not more prevalent in sle patients compared to healthy individuals . it has not yet been established that thyroid function tests should be performed routinely in sle patients . however , testing ata in euthyroid sle patients seems unjustified . larger controlled longitudinal studies are needed to elucidate the causal relationships between thyroid dysfunction , ata , sle and sle disease activity . larger sample size , presence of control group or a meta - analysis are needed for obtaining better results and be able to generalize them .

pesticide poisoning is a commonly underdiagnosed condition among farmers , particularly in rural areas and in many developing and developed countries . generally , pesticide - related morbidity is higher in rural farming communities because these locations suffer a scarcity of health care workers needed to diagnose and treat acute pesticide poisoning ( app ) . worldwide , studies report high proportions of farmers poisoned during pesticide application in vietnam ( 61% ) , ivory coast ( 55% ) , india ( 83.6% ) , myanmar ( 40% ) , and pakistan ( 55% ) . health care professionals ( hcps ) , who are responsible for the diagnosis and management of app , often receive limited training on pesticide hazards and management of pesticide - related illnesses and are sparsely available in rural areas , which often lack adequate health care facilities . most of the hcps often have limited experience of managing cases of app due to the fact that many poisonings , in particular nonsevere occupational poisoning , are not presented to hospitals . this limited knowledge is exacerbated due to the fact that clinical toxicology is a dynamic field of medicine in which new diagnostic and treatment methods are constantly being developed , and the effectiveness of diagnostic and treatment techniques is constantly being updated . in addition , pesticides used by farmers change over time and new products require new diagnostic , first aid , and treatment approaches . in tanzania , for example lack of experience in the management of app will therefore contribute to the inability of hcps to diagnose and manage app due to pesticide products with which they are not conversant . this means that surveillance systems that rely on diagnostic information from hcps will be severely weakened if hcps are not able to identify app with any reliability or accuracy . yet , without these data , surveillance systems will be unable to capture the full extent of the problem nor effectively inform appropriate preventive policies . as a result , communities from which the data are derived are not aware of the magnitude of pesticide poisoning as a public health problem nor are they given the opportunity to take preventive actions or develop community solutions . the health care services in tanzania are delivered and regulated by the ministry of health , community development , gender , seniors and children . the structure of the health system starts at village facilities and dispensaries followed by health centres located in both rural and urban areas . the health centres refer upwards to district and private hospitals , regional hospitals , and , finally , referral hospitals at the apex of the referral chain . health care professionals responsible for delivering health services at different levels include physicians , clinical officers or medical assistants , public health officers , and nurses , including nurse practitioners . clinical officers ( also known as medical assistants ) are hcps trained to assist physicians in clinical procedures . nurse practitioners are nurses trained to care for sick and injured patients and to assist physicians and clinical officers in providing clinical care . public health officers are responsible for protecting and improving the health of a community through preventive medicine , health education , control of communicable diseases , application of sanitary measures , and monitoring of environmental hazards . on average , the rate of medical doctors per capital in tanzania in 2005 was 138 000:1 , whereas that for clinical officers was 5000:1 . this is in contrast to most developed countries where the ratio of physicians exceeds 2 per 1000 of the population . ngowi et al reported poor competence among hcps in the recognition , diagnosis , and management of pesticide poisoning cases , thought to be due to inadequate training in toxicology and occupational health . the hcps in the same study were also poorly equipped to deliver appropriate care to pesticide poisoning victims . similar findings of low awareness among hcps of the problem of pesticide poisoning have been reported in other parts of east africa , south africa , costa rica , and cte divoire . although ngowi and colleagues addressed tanzanian health care worker practices in relation to the diagnosis and management of app , no studies in tanzania have examined hcp practices in relation to app surveillance . this study therefore addresses the gap in hcp knowledge and practices related to surveillance of app and also updates the study by ngowi which was conducted more than 10 years ago , prior to the 1997 introduction of mandatory reporting in the health service under the health information management system . this study therefore aimed to characterize the knowledge and experience of hcps in selected health facilities in tanzania in the diagnosis and management of app , common first aid measures , use of reporting systems , notification practices , and ability to interpret pesticide labels for the purpose of strengthening surveillance of app among farmers and community . the population included all physicians , clinical officers , and nurse practitioners working in kilimanjaro and arusha regions and who were directly responsible for diagnosing and treating potential app cases . the kilimanjaro and arusha regions were chosen because they have agricultural activities typical of rural tanzania and are geographically located close to tropical pesticides research institute ( tpri ) that facilitated logistics for data collection . an intended sample size of 91 participants was based on a priori estimate of 17% of hcps treating cases of app ( as reported by ngowi et al ) , a confidence level of 95% , and a margin of error of 8% . participants were interviewed using a semistructured questionnaire on their management of app cases and how they record and report the cases through the health management information system . they were also asked about their knowledge and practices that contribute to surveillance of app , their knowledge on pesticide label instructions , their experience in handling app , the type of first aid measures recommended for app , and their knowledge of adverse health effects of pesticides , precautionary measures contained on pesticide labels , and the classification of pesticides by world health organization ( who ) hazard class and by chemical groups . the data were collected by the principal investigator and 2 assistants between january and december 2005 . the assistants were laboratory technicians working at tpri for more than 15 years in pesticide - related research . for the study , the technicians received refresher training on pesticide classification , first aid measures for pesticide poisoning , pesticide labels , and how to administer the questionnaire for hcps . the data collection tool was pretested in january 2005 using a small sample of hcps ( n = 10 ) in selected facilities in arusha municipality before use in the main study . univariate descriptive statistics were estimated for frequencies and percentages of all categorical or numerical variables . cross - tabulations were conducted as follows : the variable knowledge on first aid ( low vs high ) was compared by the variable ever handled a pesticide poisoning and by years of working experience to identify associations with high knowledge of first aid measures . the variable familiarity with adverse health effects ( low vs high ) was compared by respondents educational level to identify whether education was associated with high familiarity with health effects of pesticides . the variable knowledge on pesticide classification ( low vs high ) was compared by respondents education level to identify whether education was associated with high knowledge of the who pesticide classification system . the variable knowledge on routes of exposure ( low vs high ) was compared by respondents years of working experience to identify whether years of experience was associated with high knowledge of routes of exposure . the variable ever handled a pesticide poisoning case vs never handled any case was compared with respondents years of working experience to identify whether increased years of experience was associated with treating cases of app . the variable high education vs low education was compared with years of working experience to identify whether long service was associated with education level . the variable type of health care facility ( government or private ) was compared with knowledge on first aid , knowledge on routes of exposure , familiarity with health effects , knowledge on pesticides classification , level of education , years of working experience , and status of handling of app cases . testing was used to compare distributions of dichotomous variables . to measure the strength of association between categorical independent and dependent variables , prevalence risk ratios spss statistical package version 16 and stata version 10.0 were used to analyse the data . participants gave informed consent prior to participation in the study and were free to decline participation without any fine or penalty . to ensure confidentiality , names were replaced by special codes , which were used in data analysis . the participants were assured that their responses would not affect their performance assessments by their managers . the study protocol was approved by tpri ethical committee and the national institute of medical research in tanzania ( ref nimr / hq / vol xi/371 ) as well as the university of cape town health sciences faculty research ethics committee ( 328/2004 ) . the population included all physicians , clinical officers , and nurse practitioners working in kilimanjaro and arusha regions and who were directly responsible for diagnosing and treating potential app cases . the kilimanjaro and arusha regions were chosen because they have agricultural activities typical of rural tanzania and are geographically located close to tropical pesticides research institute ( tpri ) that facilitated logistics for data collection . an intended sample size of 91 participants was based on a priori estimate of 17% of hcps treating cases of app ( as reported by ngowi et al ) , a confidence level of 95% , and a margin of error of 8% . participants were interviewed using a semistructured questionnaire on their management of app cases and how they record and report the cases through the health management information system . they were also asked about their knowledge and practices that contribute to surveillance of app , their knowledge on pesticide label instructions , their experience in handling app , the type of first aid measures recommended for app , and their knowledge of adverse health effects of pesticides , precautionary measures contained on pesticide labels , and the classification of pesticides by world health organization ( who ) hazard class and by chemical groups . the data were collected by the principal investigator and 2 assistants between january and december 2005 . the assistants were laboratory technicians working at tpri for more than 15 years in pesticide - related research . for the study , the technicians received refresher training on pesticide classification , first aid measures for pesticide poisoning , pesticide labels , and how to administer the questionnaire for hcps . the data collection tool was pretested in january 2005 using a small sample of hcps ( n = 10 ) in selected facilities in arusha municipality before use in the main study . univariate descriptive statistics were estimated for frequencies and percentages of all categorical or numerical variables . for the purpose of analysis , data were categorized as per table 1 . cross - tabulations were conducted as follows : the variable knowledge on first aid ( low vs high ) was compared by the variable ever handled a pesticide poisoning and by years of working experience to identify associations with high knowledge of first aid measures . the variable familiarity with adverse health effects ( low vs high ) was compared by respondents educational level to identify whether education was associated with high familiarity with health effects of pesticides . the variable knowledge on pesticide classification ( low vs high ) was compared by respondents education level to identify whether education was associated with high knowledge of the who pesticide classification system . the variable knowledge on routes of exposure ( low vs high ) was compared by respondents years of working experience to identify whether years of experience was associated with high knowledge of routes of exposure . the variable ever handled a pesticide poisoning case vs never handled any case was compared with respondents years of working experience to identify whether increased years of experience was associated with treating cases of app . the variable high education vs low education was compared with years of working experience to identify whether long service was associated with education level . the variable type of health care facility ( government or private ) was compared with knowledge on first aid , knowledge on routes of exposure , familiarity with health effects , knowledge on pesticides classification , level of education , years of working experience , and status of handling of app cases . testing was used to compare distributions of dichotomous variables . to measure the strength of association between categorical independent and dependent variables , prevalence risk ratios spss statistical package version 16 and stata version 10.0 were used to analyse the data . participants gave informed consent prior to participation in the study and were free to decline participation without any fine or penalty . to ensure confidentiality , the participants were assured that their responses would not affect their performance assessments by their managers . the study protocol was approved by tpri ethical committee and the national institute of medical research in tanzania ( ref nimr / hq / vol xi/371 ) as well as the university of cape town health sciences faculty research ethics committee ( 328/2004 ) . of the 91 hcps approached , 25 declined to participate , leaving a sample of 66 hcps from 32 facilities who were finally interviewed , representing a response rate of approximately 73% . in most facilities , there were 1 or 2 respondents ( table 2 ) . however , in the larger facilities , the number of respondents ranged up to 6 . health care providers interviewed by facility in northern tanzania . abbreviation : kcmc , kilimanjaro christian medical centre . most of the respondents were men ( 63.7% ) and they were from both private ( 53% ) and government ( 47% ) facilities in arusha and kilimanjaro regions . the facilities included a referral hospital , 2 regional hospitals , a district hospital , and 5 other private hospitals , health centres ( n = 6 ) , and dispensaries ( n = 16 ) . most of the respondents were clinical officers ( 57.5% ) and their experience in medical services ranged from 1 to more than 24 years . although the largest category had experience of 5 years or less ( 55% ) , the range of experience was wide and there were 3 participants with experience of more than 20 years in the field ( table 3 ) . respondents most frequent responses regarding knowledge on first aid and treatment in cases of pesticide poisoning included washing the contaminated area with water ( n = 23 ) , inducing vomiting if ingested ( n = 22 ) , and giving the poisoned victims fresh milk ( n = 19 ) . nineteen respondents ( 30% ) reported they do not know any first aid or treatment strategy used for victims poisoned by pesticides ( table 3 ) . table 3 indicates that many responses listed treatment options that were either incorrect or inappropriate , reported as first aid , such as administration of atropine or intravenous fluids , use of antihistamines , use of antibiotics , gastric lavage , or first aid measures ( 17.6% ) . for example , giving milk , antibiotics , and hydrocortisone are both ineffective and potentially dangerous ; the use of personal protective equipment ( ppe ) is only useful for prevention , and isolation of the victim is plainly mistaken ( table 3 ) . exactly , 50% of the respondents reported that they had never previously handled a pesticide poisoning case , 34.8% reported handling between 1 and 5 cases , and 15% handling 6 or more cases ( table 4 ) . the proportion of respondents who have handled an app case was marginally higher among staff with long work experience ( odds ratio = 1.32 ; 95% confidence interval [ ci ] = 0.9 - 1.5 ) compared with low experience . experience and knowledge of health care professionals on the management of pesticide poisoning in northern tanzania ( n = 66 ) . abbreviation : app , acute pesticide poisoning . when asked about the availability of medical laboratory testing , 53% of respondents indicated that their facilities had laboratories available on site , but none conducted any testing for biomarkers to diagnose pesticide poisoning . all the respondents reported having no standard diagnostic procedure for app , and all reported that they documented poisoning cases in the general health statistics abstracts reference books ( mtuha ) and patient register book . reporting poisoning cases and other disease conditions in this register is mandatory according to the ministry of health , community development , gender , seniors and children . only 5 respondents ( 8% ) reported having a high familiarity with the health effects of pesticides , whereas 50% admitted to having no awareness of pesticide toxicity ( table 4 ) . most of the respondents reported knowledge of possible pesticide exposure routes as oral ( 98.5% ) and inhalational ( 93.9% ) , whereas knowledge about absorption through the skin as a route was slightly lower ( 77% ; table 4 ) . most of the respondents ( 71% ) were unaware of the classification of pesticides by chemical group , and all respondents were unaware of the who hazard classification system for pesticides . pesticide chemical groups reported correctly by the respondents included organophosphates ( 37.8% ) , organochlorines ( 12.1% ) , carbamates ( 12.1% ) , and pyrethroids ( 1.5% ) . most respondents ( 55% ) reported that they had no knowledge of pesticide label safety instructions . of the 45% reporting some knowledge , the most common label instructions reported by respondents included instructions regarding storage out of reach of children ( n = 30 ) and use of ppe ( n = 27 ) . less common instructions reported were related to washing after handling ( n = 7 ) , refraining from eating while handling pesticides ( n = 8) , keeping pesticides away from food ( n = 4 ) , and avoiding pollution of the environment or water bodies ( n = 5 ) . eleven respondents ( 17% ) reported awareness of the signal word poisonous. products reported by the hcps as commonly associated with poisoning included both specific agents ( n = 31 ) and nonspecific agents ( n = 19 ) . organophosphates comprised 35% and pyrethroids comprised 16% of specific agents named . however , most commonly , the respondents were not able to specify a specific pesticide agent ( n = 35 ; table 5 ) . agents reported to be associated with poisoning as experienced by the health care professionals in northern tanzania . abbreviations : ca , carbamates ; in , inorganic ; oc , organochlorines ; op , organophosphates ; ot , other categories ; py , pyrethroids ; who , world health organization . there were marginally significant associations between educational levels of the respondents and high familiarity with pesticide health effects ( prevalence risk ratio [ prr ] high educated / low educated = 2.44 ; 95% ci = 1.05 - 5.65 ) and with high knowledge of pesticides classification ( prr high educated / low educated = 2.8 ; 95% ci = 1.3 - 6.2 ; table 6 ) . associations of knowledge about pesticides with work experience , education , and management of app among hcps in northern tanzania . there was a significant association between the status of health care facility with high knowledge on pesticide classification ( prr private facility / government facility = 1.5 , 95% ci = 1.1 - 2.1 ) ( table 7 ) . association of status of health facility and knowledge on first aid , knowledge on routes of exposure , familiarity with health effects , knowledge on pesticide classification , level of education , years of working experience , and handling of app cases among hcps in northern tanzania . there were marginally significant associations between educational levels of the respondents and high familiarity with pesticide health effects ( prevalence risk ratio [ prr ] high educated / low educated = 2.44 ; 95% ci = 1.05 - 5.65 ) and with high knowledge of pesticides classification ( prr high educated / low educated = 2.8 ; 95% ci = 1.3 - 6.2 ; table 6 ) . associations of knowledge about pesticides with work experience , education , and management of app among hcps in northern tanzania . there was a significant association between the status of health care facility with high knowledge on pesticide classification ( prr private facility / government facility = 1.5 , 95% ci = 1.1 - 2.1 ) ( table 7 ) . association of status of health facility and knowledge on first aid , knowledge on routes of exposure , familiarity with health effects , knowledge on pesticide classification , level of education , years of working experience , and handling of app cases among hcps in northern tanzania . this study updates and expands the scope of a previous investigation in tanzania ( ngowi et al ) into health care provider s knowledge and practices regarding poisoning arising from pesticides . the profile of pesticide agents now reflects usage shaped by tanzania s ratification of the stockholm and rotterdam conventions , domesticated into national law , as a result of which there are newer active ingredients and formulations which demand a new investigation of knowledge and practices . moreover , this study includes vegetable growing areas , with smaller production units , and including smaller health care facilities . most importantly , this study generates information for the first time on reporting of app by health workers through local health information systems , a crucial element for effective public health surveillance of app . the respondents in this substudy were hcps who had working experience ranging from 1 to 24 years . the study revealed poor knowledge on pesticide poisoning management , lack of familiarity with the adverse health effects of pesticides , low knowledge about pesticide chemical groups , and who categories but better knowledge about routes of dermal exposure . one intuitive explanation is that clinicians with longer working experience likely may have handled app cases which might have made them more knowledgeable . however , this was not supported by data in table 6 where long service was not associated with any of the knowledge measures . another explanation could be the fact that the respondents interviewed in the study period who were more recent graduates did not cover details about pesticides and their toxicity in their study curriculum , and app was not given much priority due to a perception that it is rare in hospitals . this may explain why many poisoning agents are frequently reported with nonspecific names for the causative agents , such as acaricides , bed bug insecticide , flower spray , and other unknown terms . this clearly limits the extent to which hcp reports can support effective surveillance for app . half of the respondents reported that they had never handled any pesticide poisoning cases , and among those who had , most had attended 5 or fewer cases in their careers . one reason for low experience could be the fact that pesticide poisoning cases are infrequently present to hospitals in tanzania , a finding also reported in india . alternatively , if they did attend cases but the diagnosis was missed , the provider did not know that they had treated an app case . the study also revealed that a large proportion of respondents had misconceptions about appropriate first aid . for example , 19 respondents ( 29% ) considered milk a first aid option for app and about one - third of respondents ( 33% ) reported inducing vomiting as one of the options for first aid for app . in fact , providing milk may give a false sense of security and delay proper treatment and hence may increase health risks . similarly , induction of vomiting is not appropriate for all products and may be contraindicated for certain agents . for example , pyridyls are corrosive products which can damage the oesophagus and upper airway if vomiting is induced . if the victim is unconscious , inducing vomiting could also result in potentially fatal aspiration of vomitus . a previous tanzanian study involving extension officers between 1991 and 1993 also reported the use of milk and inducing vomiting as options for first aid , along with other options such as use of lamb oil , fresh cattle dung , and salted water . this suggests that perceptions about the use of milk as an antidote for poisoning is prevalent among not only hcps but also the agriculture extension officers . misconceptions about the use of milk as an antidote to a range of workplace hazards are widespread in the region . a similar study conducted in united states involving a survey of primary care physicians revealed poor knowledge of the health risks associated with agriculture , and it recommended the training of hcps working in rural areas to address these health problems . the responses regarding the availability of laboratory testing indicate that although laboratories are available , none conduct any testing specific to diagnose pesticide poisoning . this finding agrees with data from record reviews at health care facilities in tanzania in which most of the cases were diagnosed through history and clinical signs . the lack of laboratory capacity to confirm diagnosis , a widespread phenomenon worldwide , for example , in india and also in the southern african region , may contribute to underdiagnosis of app cases reported in hospitals . if few cases are formally diagnosed , few will be reported , and surveillance data will underreport the extent of the problem and policymakers may not see the importance of building capacity for laboratory diagnosis , which in turn exacerbates the problem of underreporting . there is therefore a need to advocate for better diagnostic facilities , especially in rural health facilities to make the laboratory diagnosis of app possible . also , given that there is poor knowledge among hcps , better training in clinical diagnosis is also critically important . the role of poison control centres ( pccs ) in providing guidance to health professionals has been noted as an important strategy in some countries . however , as the who points out , many parts of the world lack access to pccs , particularly africa and parts of the eastern mediterranean and western pacific regions , and there is no fully operational pcc in tanzania able to provide this kind of support to health care providers facing a case of possible app . in a continent so lacking in key human resources for health , addressing this gap through establishing a pcc is an ambitious goal which is unlikely to be realized without sizable donor support and careful attention to sustainability . most of the hcps ( 55% ) could not report any safety instructions when interpreting pesticide labels . failure to interpret the label information may result in poor diagnosis and treatment as the label carries important information for the diagnosis and management of app . even if the label is available in an app case , it would appear that the hcp will not be able to interpret the label and handle the patient appropriately . taking into account that laboratory diagnosis is unavailable , hcps should be trained on , among other things , how to interpret label information for the identification and treatment of app cases . this is particularly important , given the lack of a pcc in tanzania able to provide timely and accurate guidance on pesticide poisoning to hcps , a problem common to much of africa and parts of the eastern mediterranean and western pacific regions . it is striking that the proportion of hcps reporting previous experience in managing a case of pesticide poisoning was much lower in this study ( 50% ) than reported by ngowi et al ( 80% ) . although the study by ngowi and colleagues visited mainly dispensaries and lower level facilities ( 65.3% ) , this study included fewer health care workers from dispensaries ( 44% ) . because dispensaries are mostly located in rural areas they are more likely to attend to poisoning cases , potentially including less severe occupational injuries . health care professionals working in these facilities may therefore have more experience in handling app cases . because facilities with higher status , for example , hospitals , receive more severe cases , which are less common , this could account for the smaller proportion of hcps in this study experienced in the management of poisoning cases . the findings in this study are consistent with that of a study conducted in east africa more than a decade ago , which found that more than 40% of the hcps interviewed could not recognize pesticide poisoning cases . the problem of management of app found in this study is also a problem in developed countries . for example , in a study conducted in washington , dc , and surrounding areas , it was reported that most of the hcps interviewed frequently did not diagnose pesticide toxicity from patient history and examination . exactly 64% of practitioners and 69% of nurses felt poorly prepared to answer patients questions . in all , 40% of practitioners and 26% of nurses felt that it was important to obtain more information on pesticides . this study found that knowledge about pesticide classification was significantly higher in private than government facilities ( 39.5% vs 12.0% ; p = .02 ) . this could arise from the fact that the proportion of health care workers who have handled app was somewhat higher in private facilities than government facilities ( 53.5% vs 39.1% ) but not statistically significantly so ( p = .27 ) . by handling app cases , hcps may come across different agents and their labels or containers , and this may , over time , make them knowledgeable . it may also be the result of private facilities being able to attract hcps with greater education because knowledge about pesticide classification and adverse health effects of pesticides was higher in respondents with high education ( table 6 ) . this association is to be expected as respondents who are graduates usually receive more intensive training , and hence would be likely to have more knowledge on pesticide health effects and classification . regarding documentation of poisoning cases , it was found that all hcps reported documenting all poisoning cases in the health statistics abstracts reference books ( mtuha ) and patient register book . however , this may be an overstatement because , in theory , documentation of poisoning cases is mandatory . incomplete registers , damaged pages , as well as failure to record cases . regarding agents , the study found that most of the specifically known agents reported to be associated with poisoning ( 87% ) were of who class i or ii . these products are , by definition , either highly or moderately hazardous , and their association with poisoning by the hcps was consistent with their toxic nature . although these products are restricted in tanzania , their handling and use are not well controlled due to weaknesses in enforcement . furthermore , among the specifically known agents , 25% of the products reported as associated with app were ops . organophosphates are cholinesterase - inhibiting agents , and although they were reported in low proportion in this substudy , their involvement in poisoning can not be underestimated . the proportion of unknown agents ( 41.3% ) was high indicating that many hcps either rarely handle app cases or the limited cases reported to them lack information . the study results may be weakened by a number of possible biases : information bias . health care professionals might have claimed greater familiarity with adverse health effects of pesticides than actually was true , implying that knowledge and familiarity might , in reality , have been worse than found in this study . selection bias . selection bias might also have affected the findings in that hcps who declined to participate might have done so because they were not conversant with app or may have been reluctant to disclose their lack of experience in managing app . again , this implies that estimates for knowledge and for experience with app reported in the study were likely to be overestimated than is the case in reality ie , a problem of over - reporting . however , the omission of the hcps from facilities in far - off remote areas may countereffect this overestimation if nonparticipants were used to seeing app cases . consequently , the direction of misclassification due to this selection bias is not obvious . the health facilities involved in the study included at least 1 respondent from referral hospitals , regional hospitals , district hospitals , health centres , dispensaries , and other hospitals . although not selected in a truly random manner , the spread of facilities and practitioners suggest that the sample includes hcps who typically staff such facilities and see cases of app . nonetheless , it is possible that the sample of facilities may differ from other facilities in the country . the findings should not be generalized without further studies to confirm the patterns in a representative sample of hcps . although interviews were conducted on an individual basis , in some situations respondents had opportunity to interact with other interviewed respondents before they underwent their own interview . in such situations , their answers were potentially influenced by their colleagues resulting in some degree of homogeneity of reporting . this would cause respondents to provide unrealistic responses which could have either underestimated or overestimated the knowledge and practices of hcps in relation to app . respondents may have poor memory of some events in particular events that took place more than 3 months earlier . the findings suggest that most hcps in the selected health care facilities in northern tanzania lacked adequate skills in the diagnosis and management of app and had very poor knowledge about what to do about app . the limited ability to diagnose app cases results in failure to recognize all poisoning cases arising from pesticide exposure , and this contributes to underreporting of app cases . a strong surveillance system requires hcps who are sufficiently skilled to make the diagnosis of app and report it effectively . to fill this gap , there is a need to include training on pesticide hazards , classification , and health effects in the training programmes for all categories of hcps in tanzania . to develop practical skills , it is recommended that hcps undergo practical training at institutions with experience in the management and study of pesticides , such as the tpri , which is the sole institution dealing specifically with pesticides in tanzania and therefore best placed to support clinicians in matters related to pesticides . currently , tpri has training programmes on pesticides in place conducted twice annually . although this study was undertaken in 2 regions of northern tanzania , and can only be generalized to these areas , the services and farming areas are fairly typical of other part of tanzania . for this reason , it is believed that the findings might well reflect a similar situation in the rest of the country . however , there is a need to conduct further studies in other parts of tanzania to see whether the findings are replicated .

background : acute pesticide poisoning ( app ) is commonly underdiagnosed in tanzania . studies in developing countries suggest that a lack of diagnostic skills among health care providers ( hcps ) undermines surveillance for app . this study aimed at characterizing experience and skills of tanzanian hcps regarding app diagnosis and management.methodology:the population included hcps responsible for managing app in kilimanjaro and arusha regions ( n = 91 ) . the resulting sample included 66 respondents ( response rate : 73% ) . the data were collected in 2005 using a standardized questionnaire.results:half of all respondents ( 50% ) reported handling at least 1 app case with 15% reporting handling more than 5 cases in the past . reported experience of handling an app case was marginally higher in respondents who reported 4 years of work experience in the health sector compared with those with < 4 years of work experience ( odds ratio = 1.32 ; 95% confidence interval = 0.9 - 1.5 ) . most of the respondents had high knowledge of exposure routes , reporting awareness of oral ( 98.5% ) , inhalational ( 93.9% ) , and dermal ( 77% ) routes . the study revealed low awareness of pesticide classification by chemical groups ( 29% ) or world health organization hazard ( 0% ) and weak knowledge on pesticide label instructions ( 55% ) . organophosphates accounted for 35% of the pesticide products reported by respondents as being responsible for poisoning . some treatment options were incorrectly reported as first aid options , and some reported first aid options were wrong or inappropriate.conclusions:the study revealed that hcps in northern tanzania lack adequate skills to diagnose and manage app . for effective surveillance of app , there is a need to include training on hazards , classification , diagnosis , and health effects in the training programmes for all hcps in tanzania .

Introduction Materials and Methods Population and sample Data collection Data analysis Ethical considerations Results Associations with knowledge among HCPs Discussion Study limitations Conclusions

pesticide poisoning is a commonly underdiagnosed condition among farmers , particularly in rural areas and in many developing and developed countries . generally , pesticide - related morbidity is higher in rural farming communities because these locations suffer a scarcity of health care workers needed to diagnose and treat acute pesticide poisoning ( app ) . worldwide , studies report high proportions of farmers poisoned during pesticide application in vietnam ( 61% ) , ivory coast ( 55% ) , india ( 83.6% ) , myanmar ( 40% ) , and pakistan ( 55% ) . health care professionals ( hcps ) , who are responsible for the diagnosis and management of app , often receive limited training on pesticide hazards and management of pesticide - related illnesses and are sparsely available in rural areas , which often lack adequate health care facilities . most of the hcps often have limited experience of managing cases of app due to the fact that many poisonings , in particular nonsevere occupational poisoning , are not presented to hospitals . in tanzania , for example lack of experience in the management of app will therefore contribute to the inability of hcps to diagnose and manage app due to pesticide products with which they are not conversant . as a result , communities from which the data are derived are not aware of the magnitude of pesticide poisoning as a public health problem nor are they given the opportunity to take preventive actions or develop community solutions . the health centres refer upwards to district and private hospitals , regional hospitals , and , finally , referral hospitals at the apex of the referral chain . health care professionals responsible for delivering health services at different levels include physicians , clinical officers or medical assistants , public health officers , and nurses , including nurse practitioners . public health officers are responsible for protecting and improving the health of a community through preventive medicine , health education , control of communicable diseases , application of sanitary measures , and monitoring of environmental hazards . ngowi et al reported poor competence among hcps in the recognition , diagnosis , and management of pesticide poisoning cases , thought to be due to inadequate training in toxicology and occupational health . similar findings of low awareness among hcps of the problem of pesticide poisoning have been reported in other parts of east africa , south africa , costa rica , and cte divoire . although ngowi and colleagues addressed tanzanian health care worker practices in relation to the diagnosis and management of app , no studies in tanzania have examined hcp practices in relation to app surveillance . this study therefore addresses the gap in hcp knowledge and practices related to surveillance of app and also updates the study by ngowi which was conducted more than 10 years ago , prior to the 1997 introduction of mandatory reporting in the health service under the health information management system . this study therefore aimed to characterize the knowledge and experience of hcps in selected health facilities in tanzania in the diagnosis and management of app , common first aid measures , use of reporting systems , notification practices , and ability to interpret pesticide labels for the purpose of strengthening surveillance of app among farmers and community . the population included all physicians , clinical officers , and nurse practitioners working in kilimanjaro and arusha regions and who were directly responsible for diagnosing and treating potential app cases . an intended sample size of 91 participants was based on a priori estimate of 17% of hcps treating cases of app ( as reported by ngowi et al ) , a confidence level of 95% , and a margin of error of 8% . participants were interviewed using a semistructured questionnaire on their management of app cases and how they record and report the cases through the health management information system . they were also asked about their knowledge and practices that contribute to surveillance of app , their knowledge on pesticide label instructions , their experience in handling app , the type of first aid measures recommended for app , and their knowledge of adverse health effects of pesticides , precautionary measures contained on pesticide labels , and the classification of pesticides by world health organization ( who ) hazard class and by chemical groups . the data were collected by the principal investigator and 2 assistants between january and december 2005 . for the study , the technicians received refresher training on pesticide classification , first aid measures for pesticide poisoning , pesticide labels , and how to administer the questionnaire for hcps . the data collection tool was pretested in january 2005 using a small sample of hcps ( n = 10 ) in selected facilities in arusha municipality before use in the main study . cross - tabulations were conducted as follows : the variable knowledge on first aid ( low vs high ) was compared by the variable ever handled a pesticide poisoning and by years of working experience to identify associations with high knowledge of first aid measures . the variable knowledge on pesticide classification ( low vs high ) was compared by respondents education level to identify whether education was associated with high knowledge of the who pesticide classification system . the variable knowledge on routes of exposure ( low vs high ) was compared by respondents years of working experience to identify whether years of experience was associated with high knowledge of routes of exposure . the variable ever handled a pesticide poisoning case vs never handled any case was compared with respondents years of working experience to identify whether increased years of experience was associated with treating cases of app . the variable type of health care facility ( government or private ) was compared with knowledge on first aid , knowledge on routes of exposure , familiarity with health effects , knowledge on pesticides classification , level of education , years of working experience , and status of handling of app cases . the population included all physicians , clinical officers , and nurse practitioners working in kilimanjaro and arusha regions and who were directly responsible for diagnosing and treating potential app cases . the kilimanjaro and arusha regions were chosen because they have agricultural activities typical of rural tanzania and are geographically located close to tropical pesticides research institute ( tpri ) that facilitated logistics for data collection . an intended sample size of 91 participants was based on a priori estimate of 17% of hcps treating cases of app ( as reported by ngowi et al ) , a confidence level of 95% , and a margin of error of 8% . they were also asked about their knowledge and practices that contribute to surveillance of app , their knowledge on pesticide label instructions , their experience in handling app , the type of first aid measures recommended for app , and their knowledge of adverse health effects of pesticides , precautionary measures contained on pesticide labels , and the classification of pesticides by world health organization ( who ) hazard class and by chemical groups . the data were collected by the principal investigator and 2 assistants between january and december 2005 . for the study , the technicians received refresher training on pesticide classification , first aid measures for pesticide poisoning , pesticide labels , and how to administer the questionnaire for hcps . the data collection tool was pretested in january 2005 using a small sample of hcps ( n = 10 ) in selected facilities in arusha municipality before use in the main study . cross - tabulations were conducted as follows : the variable knowledge on first aid ( low vs high ) was compared by the variable ever handled a pesticide poisoning and by years of working experience to identify associations with high knowledge of first aid measures . the variable knowledge on pesticide classification ( low vs high ) was compared by respondents education level to identify whether education was associated with high knowledge of the who pesticide classification system . the variable knowledge on routes of exposure ( low vs high ) was compared by respondents years of working experience to identify whether years of experience was associated with high knowledge of routes of exposure . the variable ever handled a pesticide poisoning case vs never handled any case was compared with respondents years of working experience to identify whether increased years of experience was associated with treating cases of app . the variable type of health care facility ( government or private ) was compared with knowledge on first aid , knowledge on routes of exposure , familiarity with health effects , knowledge on pesticides classification , level of education , years of working experience , and status of handling of app cases . health care providers interviewed by facility in northern tanzania . most of the respondents were men ( 63.7% ) and they were from both private ( 53% ) and government ( 47% ) facilities in arusha and kilimanjaro regions . the facilities included a referral hospital , 2 regional hospitals , a district hospital , and 5 other private hospitals , health centres ( n = 6 ) , and dispensaries ( n = 16 ) . most of the respondents were clinical officers ( 57.5% ) and their experience in medical services ranged from 1 to more than 24 years . although the largest category had experience of 5 years or less ( 55% ) , the range of experience was wide and there were 3 participants with experience of more than 20 years in the field ( table 3 ) . respondents most frequent responses regarding knowledge on first aid and treatment in cases of pesticide poisoning included washing the contaminated area with water ( n = 23 ) , inducing vomiting if ingested ( n = 22 ) , and giving the poisoned victims fresh milk ( n = 19 ) . table 3 indicates that many responses listed treatment options that were either incorrect or inappropriate , reported as first aid , such as administration of atropine or intravenous fluids , use of antihistamines , use of antibiotics , gastric lavage , or first aid measures ( 17.6% ) . for example , giving milk , antibiotics , and hydrocortisone are both ineffective and potentially dangerous ; the use of personal protective equipment ( ppe ) is only useful for prevention , and isolation of the victim is plainly mistaken ( table 3 ) . exactly , 50% of the respondents reported that they had never previously handled a pesticide poisoning case , 34.8% reported handling between 1 and 5 cases , and 15% handling 6 or more cases ( table 4 ) . the proportion of respondents who have handled an app case was marginally higher among staff with long work experience ( odds ratio = 1.32 ; 95% confidence interval [ ci ] = 0.9 - 1.5 ) compared with low experience . experience and knowledge of health care professionals on the management of pesticide poisoning in northern tanzania ( n = 66 ) . abbreviation : app , acute pesticide poisoning . all the respondents reported having no standard diagnostic procedure for app , and all reported that they documented poisoning cases in the general health statistics abstracts reference books ( mtuha ) and patient register book . only 5 respondents ( 8% ) reported having a high familiarity with the health effects of pesticides , whereas 50% admitted to having no awareness of pesticide toxicity ( table 4 ) . most of the respondents reported knowledge of possible pesticide exposure routes as oral ( 98.5% ) and inhalational ( 93.9% ) , whereas knowledge about absorption through the skin as a route was slightly lower ( 77% ; table 4 ) . most of the respondents ( 71% ) were unaware of the classification of pesticides by chemical group , and all respondents were unaware of the who hazard classification system for pesticides . pesticide chemical groups reported correctly by the respondents included organophosphates ( 37.8% ) , organochlorines ( 12.1% ) , carbamates ( 12.1% ) , and pyrethroids ( 1.5% ) . most respondents ( 55% ) reported that they had no knowledge of pesticide label safety instructions . of the 45% reporting some knowledge , the most common label instructions reported by respondents included instructions regarding storage out of reach of children ( n = 30 ) and use of ppe ( n = 27 ) . less common instructions reported were related to washing after handling ( n = 7 ) , refraining from eating while handling pesticides ( n = 8) , keeping pesticides away from food ( n = 4 ) , and avoiding pollution of the environment or water bodies ( n = 5 ) . eleven respondents ( 17% ) reported awareness of the signal word poisonous. products reported by the hcps as commonly associated with poisoning included both specific agents ( n = 31 ) and nonspecific agents ( n = 19 ) . however , most commonly , the respondents were not able to specify a specific pesticide agent ( n = 35 ; table 5 ) . agents reported to be associated with poisoning as experienced by the health care professionals in northern tanzania . there were marginally significant associations between educational levels of the respondents and high familiarity with pesticide health effects ( prevalence risk ratio [ prr ] high educated / low educated = 2.44 ; 95% ci = 1.05 - 5.65 ) and with high knowledge of pesticides classification ( prr high educated / low educated = 2.8 ; 95% ci = 1.3 - 6.2 ; table 6 ) . associations of knowledge about pesticides with work experience , education , and management of app among hcps in northern tanzania . there was a significant association between the status of health care facility with high knowledge on pesticide classification ( prr private facility / government facility = 1.5 , 95% ci = 1.1 - 2.1 ) ( table 7 ) . association of status of health facility and knowledge on first aid , knowledge on routes of exposure , familiarity with health effects , knowledge on pesticide classification , level of education , years of working experience , and handling of app cases among hcps in northern tanzania . there were marginally significant associations between educational levels of the respondents and high familiarity with pesticide health effects ( prevalence risk ratio [ prr ] high educated / low educated = 2.44 ; 95% ci = 1.05 - 5.65 ) and with high knowledge of pesticides classification ( prr high educated / low educated = 2.8 ; 95% ci = 1.3 - 6.2 ; table 6 ) . associations of knowledge about pesticides with work experience , education , and management of app among hcps in northern tanzania . there was a significant association between the status of health care facility with high knowledge on pesticide classification ( prr private facility / government facility = 1.5 , 95% ci = 1.1 - 2.1 ) ( table 7 ) . association of status of health facility and knowledge on first aid , knowledge on routes of exposure , familiarity with health effects , knowledge on pesticide classification , level of education , years of working experience , and handling of app cases among hcps in northern tanzania . this study updates and expands the scope of a previous investigation in tanzania ( ngowi et al ) into health care provider s knowledge and practices regarding poisoning arising from pesticides . moreover , this study includes vegetable growing areas , with smaller production units , and including smaller health care facilities . most importantly , this study generates information for the first time on reporting of app by health workers through local health information systems , a crucial element for effective public health surveillance of app . the study revealed poor knowledge on pesticide poisoning management , lack of familiarity with the adverse health effects of pesticides , low knowledge about pesticide chemical groups , and who categories but better knowledge about routes of dermal exposure . another explanation could be the fact that the respondents interviewed in the study period who were more recent graduates did not cover details about pesticides and their toxicity in their study curriculum , and app was not given much priority due to a perception that it is rare in hospitals . half of the respondents reported that they had never handled any pesticide poisoning cases , and among those who had , most had attended 5 or fewer cases in their careers . the study also revealed that a large proportion of respondents had misconceptions about appropriate first aid . for example , 19 respondents ( 29% ) considered milk a first aid option for app and about one - third of respondents ( 33% ) reported inducing vomiting as one of the options for first aid for app . a similar study conducted in united states involving a survey of primary care physicians revealed poor knowledge of the health risks associated with agriculture , and it recommended the training of hcps working in rural areas to address these health problems . this finding agrees with data from record reviews at health care facilities in tanzania in which most of the cases were diagnosed through history and clinical signs . the lack of laboratory capacity to confirm diagnosis , a widespread phenomenon worldwide , for example , in india and also in the southern african region , may contribute to underdiagnosis of app cases reported in hospitals . there is therefore a need to advocate for better diagnostic facilities , especially in rural health facilities to make the laboratory diagnosis of app possible . however , as the who points out , many parts of the world lack access to pccs , particularly africa and parts of the eastern mediterranean and western pacific regions , and there is no fully operational pcc in tanzania able to provide this kind of support to health care providers facing a case of possible app . most of the hcps ( 55% ) could not report any safety instructions when interpreting pesticide labels . failure to interpret the label information may result in poor diagnosis and treatment as the label carries important information for the diagnosis and management of app . this is particularly important , given the lack of a pcc in tanzania able to provide timely and accurate guidance on pesticide poisoning to hcps , a problem common to much of africa and parts of the eastern mediterranean and western pacific regions . it is striking that the proportion of hcps reporting previous experience in managing a case of pesticide poisoning was much lower in this study ( 50% ) than reported by ngowi et al ( 80% ) . although the study by ngowi and colleagues visited mainly dispensaries and lower level facilities ( 65.3% ) , this study included fewer health care workers from dispensaries ( 44% ) . the findings in this study are consistent with that of a study conducted in east africa more than a decade ago , which found that more than 40% of the hcps interviewed could not recognize pesticide poisoning cases . for example , in a study conducted in washington , dc , and surrounding areas , it was reported that most of the hcps interviewed frequently did not diagnose pesticide toxicity from patient history and examination . this study found that knowledge about pesticide classification was significantly higher in private than government facilities ( 39.5% vs 12.0% ; p = .02 ) . it may also be the result of private facilities being able to attract hcps with greater education because knowledge about pesticide classification and adverse health effects of pesticides was higher in respondents with high education ( table 6 ) . this association is to be expected as respondents who are graduates usually receive more intensive training , and hence would be likely to have more knowledge on pesticide health effects and classification . regarding documentation of poisoning cases , it was found that all hcps reported documenting all poisoning cases in the health statistics abstracts reference books ( mtuha ) and patient register book . regarding agents , the study found that most of the specifically known agents reported to be associated with poisoning ( 87% ) were of who class i or ii . furthermore , among the specifically known agents , 25% of the products reported as associated with app were ops . selection bias might also have affected the findings in that hcps who declined to participate might have done so because they were not conversant with app or may have been reluctant to disclose their lack of experience in managing app . the health facilities involved in the study included at least 1 respondent from referral hospitals , regional hospitals , district hospitals , health centres , dispensaries , and other hospitals . the findings suggest that most hcps in the selected health care facilities in northern tanzania lacked adequate skills in the diagnosis and management of app and had very poor knowledge about what to do about app . to fill this gap , there is a need to include training on pesticide hazards , classification , and health effects in the training programmes for all categories of hcps in tanzania . to develop practical skills , it is recommended that hcps undergo practical training at institutions with experience in the management and study of pesticides , such as the tpri , which is the sole institution dealing specifically with pesticides in tanzania and therefore best placed to support clinicians in matters related to pesticides . however , there is a need to conduct further studies in other parts of tanzania to see whether the findings are replicated .

a major component of the adaptive immune response to infection is the generation of protective and long - lasting humoral immunity , but factors governing selection of the particular antigens recognized are unclear . it is not uncommon for viruses encoding a small number of proteins to generate antibodies against each encoded protein . but for infectious agents containing hundreds or thousands of proteins only a subset of the proteome is recognized and little is known about the extent or the characterisitics of this subset of antigens . methods for making a complete empirical accounting of the immunoproteome have limitations , particularly when the genome of the organism is large . here we describe a b. melitensis proteome microarray that enables this problem to be directly addressed by applying an unbiased systems biology approach to identify immunodominant and serodiagnostic antigens and to classify the reactive antigens based on functional and physical properties . protein microarrays can be used with relative ease to probe the entire proteome of different infectious microorganisms including bacteria , viruses and parasites . this approach permits assessing the repertoire of antibodies produced in response to infections or vaccinations from large collections of individual patient sera , and can be used to perform large - scale sero - epidemiological and sero - surveillance analyses not possible with other technologies , while consuming small quantities ( < 2 l ) of each serum sample . probing large numbers of patient specimens empowers the statistical tests resulting in more reliable conclusions while correcting for false discovery required for multiple comparison testing inherent in microarray analysis . moreover , microarrays can display all proteins of an infectious agent and may allow for identification of novel antigens , otherwise undetectable by methods like 2-d gels that are highly biased by microbial protein expression patterns . brucella melitensis , the most virulent species infecting humans , also infects goats , sheep and cattle , in central and south america , the middle east , east asia , and some southern european countries . consumption of animal products , direct animal contact and inhalation of aerosolized bacteria in the laboratory setting ( indicating the potential for air - borne spread via a bioterrorism - type attack ) are well - recognized routes of infection . the current knowledge of protein antigens recognized by humans and reservoir animals is based on limited numbers of studies on brucella melitensis and brucella abortus . we recently constructed a pilot proteome array consisting of 1406 b. melitensis proteins and observed marked differences in immune responses between humans and goats.(5 ) here we have expanded our previous study to the full proteome microarray consisting of 3046 b. melitensis proteins , by including expression of newly cloned 1640 bm genes . in addition to developing a better classifier capable of predicting disease based on serological response , more importantly , this study allows an estimate of the extent of immune reactivity against the whole proteome , and prediction of proteomic features that may dictate immune recognition for other yet uncharacterized gram - negative bacteria . human sera were obtained from patients enrolled in a prospective clinical study of brucellosis in lima , peru . the human subjects part of the study was approved by the humans research protections committee of the university of california san diego , the comite de tica of universidad peruana cayetano heredia , lima , peru and the comite de tica of asociacin benfica prisma , lima , peru , all of whom have maintained federal wide assurances with the united states department of health and human services . all patients provided written informed consent prior to enrollment in the study , and signed consent forms have been stored in locked files in study offices at upch and ab prisma , lima , peru . forty- two patients were confirmed to have acute brucellosis by positive culture , positive rose bengal test and by tube agglutination tests titers 1/160 . eighteen patients presenting with brucellosis - compatible syndromes were culture negative and rose bengal positive , and treated according to standard antibiotic therapy within 2 days of serum sampling . additional control patient samples included 13 sera from rose bengal - negative patients , 44 samples from ambulatory healthy controls from north lima where brucellosis occasionally affects patients , and sera from humans in the u.s . where brucellosis is not found . approximately 10 g of autoclaved pellet of b. melitensis 16 m were used to isolate lps using the hot phenol - water method.(32 ) the purified lps was treated with rnase , dnase and proteinase - k , and the hot phenol - water treament was repeated . b. melitensis lps obtained from both upper phenol saturated aqueous layer ( aqueous phase ) and lower water saturated phenol layer ( phenol phase ) were pooled . the lps from e. coli 055:b5 and b. melitensis 16 m were analyzed on a gradient 412% tris - glycine sodium dodecyl sulfate ( sds ) polyacrylamide gel ( invitrogen corp . , the presence of lps in the gels was detected with a periodic acid silver stain(33 ) and protein using coomassie blue stain . b. melitensis lps was quantified using a colorimetric assay to measure 2-keto-3-deoxyoctonate ( kdo ) concentration.(34)e . all orfs from brucella melitensis 16 m genomic dna were identified using genbank nc_003317 and nc_003318 , and 1640 orfs that were absent from pilot chip were amplified and cloned using a high - throughput pcr and recombination cloning method described previously.(5 ) microarrays and immunostrips were fabricated and probed as described before.(5 ) plasmids were expressed at 24 c for 16 h in in vitro transcription / translation e. coli reactions ( expressway maxi kits from invitrogen ) , according to the manufacturer s instructions . for microarrays , 10 l of reaction was mixed with 3.3 l 0.2% tween 20 to give a final concentration of 0.05% tween 20 , and printed onto nitrocellulose coated glass fast slides ( whatman ) using an omni grid 100 microarray printer ( genomic solutions ) . for immunostrips , b. melitensis lps was printed at 0.01 mg / ml . all antigens were printed on optitran ba - s 85 0.45 m nitrocellulose membrane ( whatman ) using biojet dispenser ( biodot ) at 1 l / cm , and cut into 3 mm strips . human sera samples were diluted to 1:200 with 10 mg / ml e. coli lysate ( mclab ) . microarray slides were incubated in biotin - conjugated secondary antibody ( jackson immunoresearch ) diluted 1/200 in blocking buffer , and detected by incubation with streptavidin - conjugated surelight p-3 ( columbia biosciences ) . microarray slides were scanned and analyzed using a perkin - elmer scanarray express ht microarray scanner . human sera were diluted to 1/200 in 5% bsa solution containing 20% e. coli lysate ( mclab ) . strips were then incubated in alkaline phosphatase conjugated donkey antihuman immunoglobulin ( anti - igg , fc fragment - specific , jackson immunoresearch ) secondary antibody , diluted to 1/2000 , and reactive bands were visualized by incubating with 1-step nitro - blue tetrazolium chloride/5-bromo-4-chloro-3-indolyphosphate p - toluidine salt ( nbt / bcip ) developing buffer ( thermo fisher scientific ) . immunostrips were scanned with hewlett - packard document scanner , and were quantified using image j software . the mass spectrometry data set characterization of host and pathogen proteins affected by brucella abortus infection was from caprion proteomics studies of b abortus , downloadable from pathogen portal web site hosted by proteomics resource center at http://www.pathogenportal.org/portal/portal/pathport/home . samples were processed for intact bacteria or cell envelope preparations from exponentially growing bacteria according to the caprion protocol 24401 and 24302 , as described before . briefly , to process intact bacteria for ms analysis , 50 l aliquots of 20 g protein samples were denatured in 50 mm ammonium bicarbonate ( sigma - aldrich , st . after sonication , samples were centrifuged at 2000 g for 1 min and a 950 l cold ( 20 c ) chloroform / methanol solution ( 2:1 v / v ) were added . samples were vortex mixed and incubated at 20 c for 2 h. subsequently , 100 l cold ( 20 c ) methanol was added and samples were clarified by centrifugation at 21 000 g for 10 min at 4 c . the supernatants were dried under vacuum , resuspended in 100 mm ammonium bicarbonate containing 8 m urea and 1% ( w / v ) of acid - labile surfactant ( als ; waters , milford , ma ) and sonicated for 10 min . after centrifugation for 20 s at 2000 g , samples were incubated for 1 h at ambient temperature . a volume of 450 l of 100 mm ammonium bicarbonate containing 5% ( v / v ) of acetonitrile was added . lys - c ( wako , richmond , va ) was added to yield a 1:50 enzyme to protein ratio . trypsin ( promega , madison , wi ) was then added at a 1:50 enzyme to protein ratio and samples were incubated for an additional 16 h. following proteolysis , tcep ( pierce , rockford , il ) was added to a final concentration of 10 mm and samples were incubated for 30 min at ambient temperature and then lyophilized . to cleave the acid labile surfactant n hcl and 100 l of water were added into each sample with subsequent incubation for another half hour at ambient temperature . to process cell envelope for ms analysis , supernatants of exponentially growing b. abortus strains were clarified , centrifuged at 100 000 g for 6 h at 4 c , pellets resuspended in double distilled water . the sample was sonicated , and 50 mm ammonium bicarbonate containing 2% acid - labile surfactant and 8 m urea were added . the sample was vortex mixed for 1 h. a sample of 50 l of the om suspension was added to a final volume of 1 ml of a chloroform / methanol solution ( 2:1 v / v ) . the sample was vortex mixed and incubated at 20 c for 2 h. subsequently , 100 l of cold methanol ( 20 c ) was added , and the sample was cleared by centrifugation for 10 min at 21 000 g. the supernatant was dried under vacuum and resuspended in 4 m urea ; 50 mm ammonium bicarbonate ph 8.0 . the samples were then diluted 4:1 with 50 mm ammonium bicarbonate buffer ph 8.0 , and trypsin was added at a 1:25 protein ratio for an additional 16 h. following proteolysis , the samples were distributed into 96-well plates for mass spectrometry analysis . peptide digests were analyzed by liquid chromatography coupled to mass spectrometry ( lc - ms ) as described . ms system consisted of a caplc ( waters , milford , ma ) with a cooled autosampler and a qtof ultima ( waters , milford , ma ) controlled by masslynx version 4.0 software . the peptide concentrations were normalized and the samples were injected into a reversed - phase column ( jupiter c18 , phenomenex , torrance , ca ) for hplc separation . ms spectra to mascot software ( matrixscience , boston , ma ) for searching against the national center for biotechnology information protein database ( ncbi ) . numbers of different peptides for each identified protein were counted and listed in table s4 ( supporting information ) . data analysis was performed using the r ( http://www.r-project.org ) and sas ( http://www.sas.com/ ) statistical software . it has been noted in literature that data derived from microarray platforms is heteroskedatic . to stabilize the variance , the vsn normalization method implemented as part of the bioconductor suite ( www.bioconductor.org ) in addition to removing heteroskedacity , this procedure corrects for nonspecific noise effects by finding maximum likelihood shifting and scaling parameters for each array such that control probe variance is minimized . this calibration has been shown to be effective on a number of platforms.(42 ) differentially reactive proteins between groups were determined using a bayes regularized t test adapted from cyber - t for protein arrays , which has been shown to be more effective than other differential expression techniques.(44 ) to account for multiple comparison conditions , the benjamini and hochberg ( bh ) method was used to control the false discovery rate.(45 ) after benjamini and hochberg correction , p - value smaller than 0.05 was considered significant , and the corresponding protein was considered differentially reactive and serodiagnostic . multiplex classifiers were constructed using linear and nonlinear support vector machines ( svms ) using the e1071 r package . plots of receiver operating characteristic ( roc ) curves were made with the rocr r package . sensitivity specificity and area under the curve ( auc ) were determined from the resulting roc curves . principle component analysis ( pca ) to visually reduce and summarize signal intensity variance among the subjects was conducted in jmp software ( www.jmp.com ) . hierarchical clustering was used to group sera samples into subsets , such that those within each cluster ( subset ) are more closely related to one another than samples assigned to other clusters . clustering is based on the degree of similarity between the protein intensity for each individual . mev v4.6 ( tm4microarray software suite , www.tm4.org ) was used to perform the clustering analysis . tmhmm v2.0 was utilized for transmembrane domains prediction(46 ) ( http://www.cbs.dtu.dk/services/tmhmm/ ) , signalp v3.0 for signal peptide prediction(47 ) ( http://www.cbs.dtu.dk/services/signalp/ ) , psortb v3.0 . pi / mw tool from swiss institute of bioinformatics was used to determine isolectric point ( http://ca.expasy.org/tools/pi_tool.html ) . p value for enrichment statistical analysis was calculated using fisher s exact test in the r environment . the 3198 predicted orfs of b. melitensis ( bm ) 16 m were subjected to amplification from genomic dna , and 1406 orfs were cloned and printed on pilot chip.(5 ) in this study , another 1640 orfs were successfully cloned using our high throughput recombination method . about one - fourth of the cloned genes were sequenced and > 99% of sequenced clones had the correct sequence . all 3046 bm orfs cloned in pxt7 vector ( > 95% of proteome ) were expressed under t7 promoter in the e. coli in vitro transcription / translation system , and printed on microarrays . over 98% of protein spots bm protein arrays were probed with sera from acute brucellosis patients in lima , peru obtained within 13 weeks of the onset of symptoms , and sera from bm culture - positive humans ( figure s1b , supporting information ) showed robust reactivity against a collection of antigens compared to unexposed individuals . among the 3046 antigens tested , 1464 antigens reacted with at least one culture positive individual , accounting for 48% of the proteome ( figure 1 ) . within this immunoproteome , , with mean reactivity greater than the mean of the no dna controls plus 2.5 standard deviations among culture positive individuals ( figure 2 , figure s2 and table s1 , supporting information ) . of these , 33 protein antigens were serodiagnostic , and were significantly differentially reactive between nave and culture positive patients from peru ( benjamini and hochberg adjusted cyber - t p - value < 0.05 ) . most of these antigens also reacted strongly with sera from brucellosis cases that were culture negative but positive for the rose bengal diagnostic test . an unbiased hierarchical clustering analysis segregated most of the individual specimens into two groups , with a few infected cases with weak reactivity sporadically clustered among samples diagnosed as uninfected ( figure s2 , supporting information ) . we also identified 90 cross - reactive antigens that reacted similarly among all human samples , whether from nave individuals or individuals diagnosed to be infected . the raised bars of any color represent 1464 reactive proteins , which define the immunoproteome ( approximately 48% of the proteome ) . there are 538 antigens reactive in 1 sample , 648 antigens reactive in 25 samples , 143 antigens reactive in 69 samples and 135 antigens reactive in 1042 samples . bmei0536 and bmei1079 reacted in all 42 bm culture positive samples . all serodiagnostic antigens except bmei0503 reacted in 1042 samples ; bmei0503 reacted in 9 samples . discovery of new human serodiagnostic antigens by probing the full b. melitensis proteome arrays with a collection of b. melitensis peruvian nave and culture positive human sera . the mean sera reactivity of the 3046 antigens was compared between the culture positive and peruvian naive groups . antigens with benjamini hochberg corrected p - value less than 0.05 ( serodiagnostic ) are organized to the left and cross - reactive ( bh_p > 0.05 ) antigens to the right . shown are the 33 serodiagnostic protein antigens , bm lps , and a subset of cross - reactive antigens . we performed unsupervised principal component analysis and showed that the 33 serodiagnostic antigens clearly separated the peruvian naive from culture positive groups ( figure 3a ) . to further determine the accuracy of distinguishing brucellosis cases from controls , cross - validation receiver operating characteristic ( roc ) curves and area under the curve ( auc ) the top four orfs , bmei0536 ( bp26 ) , bmei0805 ( hypothetical protein ) , bmei1330 ( protease do ) , and bmei 1890 ( transporter ) , as wells as bm lps , all have an individual antigen auc greater than 0.90 ( table s1 , supporting information ) . three of the antigens were identified previously on microarrays.(5 ) antigen bp26 ( bmei0536 ; auc 0.991 ; bh p - value < 1 10 ) gave the best single antigen discrimination with 95% sensitivity and specificity rate . variability and mean value of accuracy from this model has been shown in the auc boxplot . the top 5 antigens all produced sensitivity and specificity over 96% ( figure 3c ) . with the top 3 antigens , this classifier yielded a highest sensitivity and specificity rate of 98.6 and 98.6% , respectively . altogether 33 protein antigens plus lps produced sensitivity and specificity over 93% ( a ) unsupervised principal component analysis of the signal intensity for samples from peruvian naives and culture positive groups revealed that these two groups could be segregated on the basis of 33 selected serodianosis protein antigens and 2 bm lps ( at 0.1 mg / ml or 0.01 mg / ml ) . the serodiagnostic antigens were ranked by single antigen auc . ( b ) cross - validation auc boxplot with variance and mean value and ( c ) cross - validation roc graphs show classifiers with increasing number of human serodiagnostic antigens . with the top 3 antigens , this classifier yielded a highest sensitivity and specificity rate of 98.6% and 98.6% , respectively . all 33 antigens plus bm lps also produced sensitivity and specificity over 93% . eighteen serodiagnostic proteins and bm lps the individual strips were probed with 31 different randomly selected culture positive sera and 31 peruvian naive sera . brucellosis patients reacted strongly against the serodiagnostic antigens with variable signal intensities among the patients . the roc curve ( figure 4b ) shows that this immunostrip test yielded a high auc of 0.97 , with sensitivity rate of 96.7% and specificity rate of 96.7% . ( a ) eineteen serodiagnostic antigens and bm lps at 0.01 mg / ml were printed onto nitrocellulose membrane in adjacent stripes using a biodot jet dispenser as described in materials and methods . strips were probed with culture positive or peruvian naive sera diluted 1/200 followed by alkaline phosphatase conjugated secondary antibody and enzyme substrate . weak reactivity in the nave healthy controls ( b ) cross - validation roc curve was generated and sensitivity and specificity of immunostrips test is 96% and 96% , respectively . serological tests are routinely used to diagnose brucellosis . while the qualitative agglutination test ( rose bengal ) is 90% sensitive for screening for acute / subacute brucellosis , quantitative agglutination testing ( e.g. , tube agglutination test ( tat ) ) with titer typically greater than 1/160 used as a more precise diagnostic criterion , can also be used to diagnose relapse . often blood cultures are negative at the time of initial presentation or relapse , possibly because of low levels of bacteremia or antecedent use of antibiotics . without being able to ascertain whether the sera used in this study were obtained from patients who might have taken antibiotics prior to blood cultures , we queried our data set to determine whether sera from blood culture - positive , rose bengal positive brucellosis patients ( gold standard diagnosis ) vs blood culture - negative , rose bengal positive ( tat not done ) subjects ( presumptive positive , unconfirmed ) recognized different proteins on the protein microarrays . sera from blood culture - negative , rose bengal positive subjects reacted with 10 b. melitensis antigens much more strongly than blood culture - positive , rose bengal positive individuals ( figure s3 , supporting information ) . this unique set of 10 antigens was distinct from the 33 antigens that distinguish culture positive cases from peruvian naves . these results indicate that protein microarray analysis was able to delineate distinct antibody profiles in blood culture positive and negative patients , which has the potential to identify antigens capable of differentiating active acute / subacute brucellosis from previously exposed patients . these findings may suggest novel biological interactions between bacteria and host that need to be further explored because culture negativity / rose bengal seropositivity might be associated with some biological difference between human hosts , perhaps an immune response that might lower bacterial loads to levels difficult to culture from blood . alternatively , it is possible that antibiotic use might lead to an altered host immune response to killed bacteria that no longer would subvert the host immune responses ( typical of intracellular pathogens such as brucella ) . to better understand the determinants of antigenicity in bacteria , we performed a functional enrichment analysis of the serodominant and serodiagnostic antigens identified in this study . a total of 543 proteins are unassociated with cogs , and 39 proteins are associated with cogs but have not been assigned to a category , shown as some proteins have multiple cog assignments so there are 3,291 cogs in total for 3046 proteins on chip . proteins with predicted cog- u function , involved in intracellular trafficking and secretion , were 6.2- fold enriched in serodiagnostic antigens and 4.3- fold enriched in serodominant antigens . proteins in the cog - m category , cell envelope biogenesis and outer membrane , were 2-fold enriched in serodominant antigens ( p - value 0.01 ) . proteins in cog - n category , cell motility and secretion , were 3.5-fold enriched in serodominant antigens ( p = 0.027 ) . proteins with predicted cog - o function , posttranslational modification , protein turnover , chaperones , were also significantly enriched at 3.0-fold in serodominant antigens . there was only 1 serodominant antigen out of 176 cog - k category proteins , predicted to be involved in transcription and identified as serodominant , which was 0.1- fold enrichment and significantly underrepresented with p value of 0.014 ( figure 5a , table s3a , supporting information ) . overview of an enrichment analysis of bm proteome classified with cogs and computational predictions categories . the size of each slice represents the number of proteins assigned to each cog category . proteins classified with cogs - u , m , n , o categories are significantly overrepresented in serodominant antigens and shown in extracted red . cog - k proteins are significantly underrepresented in serodominant antigens are shown in extracted black . cog categories with < 5 proteins were not included in the figure ( cogs - b , w ) . significant over- and under - representations are shown in extracted red or black , respectively . numbers of serodominant antigens within these categories / number of total proteins within these categories are in parentheses in the legend . results of the complete cog analysis and computational predictions are in table s3 ( supporting information ) . ( c ) correlation between enrichment fold and peptide numbers detected by mass spectrometry . as peptide numbers for serdiagnostic antigens in contrast , enrichment fold for cross - reactive antigens decreases as peptide numbers for cross - reactive antigens increase . as shown in figure 5b and table s3b ( supporting information ) , proteins with 1 transmembrane domain were significantly enriched in serodominant , serodiagnostic and cross - reactive antigens groups , with enrichment fold at 4.3 , 5.0 , and 4.0 , respectively . interestingly , proteins with more than 1 transmembrane domain were also significantly underrepresented among the serodominant and cross - reactive antigens . proteins with predicted signal peptides ( signalp score > 0.7 ) were significantly enriched in all three groups at a range of 2.8- to 3.6-fold . conversely , proteins without signal peptides ( signalp score < 0.7 ) were significantly underrepresented . psortb predicted 4 serodiagnostic and 5 serodominant outermembrane antigens , resulting in 11.5- and 3.9-fold enrichment , respectively . however , psortb cytoplasmic proteins were 0.5- to 0.7-fold underrepresented in serodominant and serodiagnostic antigens groups . we also found that proteins with isoelectric points lower than 5 were 1.7- to 2.0-fold enriched in serodominant and cross - reactive antigen groups , whereas , proteins with pi = 9 to 14 were 0.6- and 0.5-fold underrepresented in these two groups . another potential feature that could account for protein antigenicity is the level of in vivo expression ; proteins expressed at a high level in vivo would be expected more likely to be seen by the immune system than proteins expressed at a low level . to test this hypothesis , we compared the group of antigenic b. melitensis proteins with an existing data set of expressed protein peptide data from a very closely related species , b. abortus . in this study , cytosolic proteins and cell envelope proteins from b. abortus in the log phase of in vitro growth , were extracted and subjected to lc mass spectrometry ( ms ) analysis as described in materials and methods . the number of different peptides identified for each protein was listed in table s4 ( supporting information ) . two - hundred forty proteins were exclusively expressed in the cytosol of b. abortus with 1 to12 detected peptides , and 53 proteins exclusively expressed in the cell envelope of b. abortus with 1 to 15 detected peptides , and 80 overlapping proteins expressed in both conditions . of these , 356 proteins were printed on our proteome chip . not surprisingly , expression was a significant enriching feature with 5.2-fold enrichment among serodiagnostic antigens ( p - value 1.87 10 ) , and 2.9-fold in serodominant antigens ( p - value 5.28 10 ) ( table 1 ) . interestingly , the fold enrichment was directly proportional to the number of peptides detected ( figure 5c ) . there was 8.4- fold enrichment of serodiagnostic antigens in proteins detected with more than 1 peptide ( p - value 3.56 10 ) , 16.2- fold in proteins detected with 5 or more peptides ( p - value 1.30 10 ) , 23.1- fold enrichment in proteins detected with 7 or more peptides ( p - value 1.90 10 ) , and 20.5-fold enrichment in proteins detected with 9 or more peptides ( p - value 3.91 10 ) . conversely , proteins that were not identified by ms were significantly underrepresented at 0.4-fold among the serodiagnostic and 0.8-fold among serodominant antigens . the data suggests that protein expression level is an important factor contributing to antigenicity of protein antigens . this was also supported by data in other experimental systems from different research groups.(55 ) significant enrichment or underrepresentation for certain features are underlined and bold . there are a total of 10 enriching features that fall into 3 categories : ( i ) functionally annotated cogs u , m , n and o , ( ii ) computationally predicted features ( tmhmm = 1 , signalp > 0.7 , psort outermembrane , psort periplasmic , and pi < 5 ) , and ( iii ) ms evidence of expression . the venn diagrams summarize the number of enriched proteins found in each of these 3 categories , showing overlap among the proteins found in each category ( figure 6 ) . there are 338 proteins in the enriched cog categories , 696 computationally predicted proteins , and 356 proteins that were positive by ms ( table 2 ) . accounting for the overlap between categories , 1128 proteins in all enriched categories represented 37% of the proteome . cogs accounted for 30% of the serodiagnostic hits , the computationally predicted features accounted for 61% of the hits , and ms positive proteins contained 61% of the hits . all together the three categories account for 37% of the proteome and include 91% of the serodiagnostic antigens . but there is only 1 unique serodiagnostic cog antigen that is not represented in either the computationally predicted or ms+ categories ( bmei1060 , table s1 , supporting information ) . so by combining the pool of computationally predicted and ms+ proteins representing 30% of the proteome , 88% of the serodiagnostic antigens can be predicted . enriching features are classified into three categories : ( 1 ) cogs - umno , for proteins assigned to one or more of these four cogs ; ( 2 ) computational predictions with enriching features , for proteins predicted to have one or more of the following features , that is , tmhmm = 1 , signalp > 0.7 , psort outermembrane , psort periplasmic , or pi < 5 , and ( 3 ) protein expression as detected by peptide presence in mass spectrometry . separately shown are numbers of antigens with enriching features , including all such 1128 antigens on chip , 99 serodominant , and 30 serodiagnostic antigens . numbers of antigens having more than one category of enriching features are also presented in the overlapping region of such categories . enriching features are classified into three categories : ( 1 ) cogs - umno , for proteins assigned to one or more of these four cogs ; ( 2 ) computational predictions with enriching features , for proteins predicted to have one or more of the following features , i.e. , tmhmm = 1 , signalp > 0.7 , psort outermembrane , psort periplasmic , or pi < 5 ; and ( 3 ) protein expression as detected by mass spectrometry . today with increasing efficiency , accuracy and speed we access completed genome sequences from thousands of infectious microorganisms . however , systems- level studies to understand the complete network of antibody responses have not been widely performed . this work was motivated by the disturbing lack of a detailed scientific understanding of the antibody responses to infectious diseases . we have been taking a systems biology approach to account for all of the antibodies that develop after exposure to infectious microorganisms and to identify specific antibody signatures associated with each disease , in order to understand the molecular basis for antigen selection by the immune system and to predict serodiagnostic and vaccine antigens targets . our previous study identified a set of serodiagnostic antigens from a randomly selected half of the b. melitensis proteome . however , an important difference between the present study and this previous report is that analysis of the complete proteome not only has allowed us to delineate additional serodiagnostic antigens but , more fundamentally , also provided the basis for a rigorous , comprehensive and quantitative determination of basic biological characteristics of the entire set of the serodominant antigens on a genomic level . the current standard serological screening assay , an agglutination test that uses tinted , killed bacteria as antigen ( rose bengal ) is based primarily on identification of antibodies to lps in patient serum . in the present microarray study , we confirmed lps reactivity ( with purified lps spotted onto the microarray ) , and identified novel protein antigens . although antibody responses against all 33 protein antigens used together can predict disease with 92% accuracy , using the serological responses against the top 3 individual antigens together improves accuracy to > 98% for diagnosis of acute human brucellosis . one limitation is that the humans studied here only had acute or subacute brucellosis - fever but not focal or chronic disease . future studies are needed to assess suspected cases that are agglutination test negative , for example chronic neurobrucellosis or focal disease such as orchitis or vertebral osteomyelitis . eleven of the 33 serodiagnostic antigens identified in this study were also identified on the pilot array(5 ) ( table s1 , supporting information ) . our findings are in good agreement with published studies that identified well characterized antigens from other brucella spp , including bp26 ( bmei0536 ) , htra / degp ( bmei1330),(28 ) omp16 ( bmei0340),(29 ) the chaperonin groel protein ( bmeii1048 ) and omp 10 ( bmeii0017 ) . in addition to the well characterized antigens , we also identified 21 novel serodiagnostic antigens on the current array , including top antigen hypothetical protein bmei0805 . two pyruvate dehydrogenase complex molecules and the associated acetyl coa hydrolase , which together form a large multimeric structure consisting of 60 subunits , are also differentially recognized ; this large enzyme complex is found in most bacteria and is frequently a target of immune recognition for other infections . protein microarrays enable enrichment analyses to identify proteomic features that are enriched in the immunodominant antigen set , and development of protein antigenicity prediction tools based on enriched features . the prediction tools then can be applied on a high - throughput scale to existing or new proteomes to identify key antigenic proteins that may have serodiagnostic or protective characteristics . the proteomic features fall into 3 categories : ( i ) cog annotations , ( ii ) computationally predicted proteomic features , and ( iii ) proteins with ms evidence of expression in viable organisms . no single proteomic feature or category of features is sufficient to identify all these signature antigens . our data suggests that cloning of 37% of genome with these enriching features would reveal more than 90% of serodiagnostic antigens . we have classified the reactive immunodominant antigens for numerous agents and have consistently found these features predict antigenicity . these results describe the relationship between antigenicity and in vivo expression of individual proteins . in our study , we utilized the expression of proteins quantified by ms of a closely related strain , b. abortus during in vitro growth . we found that mass spec positive antigens were significantly enriched among serodiagnostic antigens but not among the cross- reactive antigens . this validates the classification of the serodiagnostic antigens being derived from actively replicating organisms , and validates the classification of cross - reactive antigens being derived from previous exposure to nonbrucellosis infections . although the number of peptides observed per protein has been applied to estimating protein abundance , we are aware that mass spectra of cultured organisms may not reflect the expression during infection in vivo . first , nutrients are not limiting in log phase growth in vitro , whereas in vivo , brucella is likely to be within a nutrient - limited intracellular environment.(63 ) second , the ms method may not be particularly quantitative , especially for membrane proteins . our protein array technology is also able to provide strong evidence of the comprehensive set of proteins expressed in vivo within a mammalian host by b. melitensis , by virtue of their exposure to the host immune system . the expression and abundance of proteins during in vivo growth merits further examination by other novel technologies , such as measurement of transcript abundance from rna - seq ( deep sequencing ) technique.(64 ) the b. melitensis immunoproteome comprises 1464 antigens that are significantly reactive in at least one of the individuals in this study . answers to questions of why and how the immune system focuses on 4% of the potential target antigens are not yet apparent from this work . one might expect that an immune response against a larger collection of antigens could result in a more effective immune response attack against the infectious agent . but antibody responses against thousands of antigens from hundreds of clinical infections could accumulate during a lifetime , leading to cross reactivity against autologous antigens and autoimmune chaos . the observation that dozens of organism- specific antibody responses develop after b. melitensis infection is consistent with similar observations from other infectious agents , and has implications for subunit vaccine discovery and development . mimicking the natural response to infection could be considered a viable strategy for vaccine development but most subunit vaccines aim to derive protection from immunization with only a single antigen . attenuated or killed whole organism vaccines produce an antibody reactivity profile against dozens of antigens more similar to natural infection.(6 ) this systematic genome scale analysis of human antibody responses against b. melitensis proteins provides a top hit list of antigens worthy of assessing for improved diagnostics , and furthermore , enables development of a predictive model of proteomic features that determine whether a protein is antigenic and produces antibodies that confer protection . this systems biology approach provides an empirical basis for understanding the breadth and specificity of the immune response to b. melitensis and a new framework for comparing the humoral responses against other organisms .

a complete understanding of the factors that determine selection of antigens recognized by the humoral immune response following infectious agent challenge is lacking . here we illustrate a systems biology approach to identify the antibody signature associated with brucella melitensis ( bm ) infection in humans and predict proteomic features of serodiagnostic antigens . by taking advantage of a full proteome microarray expressing previously cloned 1406 and newly cloned 1640 bm genes , we were able to identify 122 immunodominant antigens and 33 serodiagnostic antigens . the reactive antigens were then classified according to annotated functional features ( cogs ) , computationally predicted features ( e.g. , subcellular localization , physical properties ) , and protein expression estimated by mass spectrometry ( ms ) . enrichment analyses indicated that membrane association and secretion were significant enriching features of the reactive antigens , as were proteins predicted to have a signal peptide , a single transmembrane domain , and outer membrane or periplasmic location . these features accounted for 67% of the serodiagnostic antigens . an overlay of the seroreactive antigen set with proteomic data sets generated by ms identified an additional 24% , suggesting that protein expression in bacteria is an additional determinant in the induction of brucella - specific antibodies . this analysis indicates that one - third of the proteome contains enriching features that account for 91% of the antigens recognized , and after b. melitensis infection the immune system develops significant antibody titers against 10% of the proteins with these enriching features . this systems biology approach provides an empirical basis for understanding the breadth and specificity of the immune response to b. melitensis and a new framework for comparing the humoral responses against other microorganisms .

Introduction Materials and Methods Results Discussion

a major component of the adaptive immune response to infection is the generation of protective and long - lasting humoral immunity , but factors governing selection of the particular antigens recognized are unclear . but for infectious agents containing hundreds or thousands of proteins only a subset of the proteome is recognized and little is known about the extent or the characterisitics of this subset of antigens . methods for making a complete empirical accounting of the immunoproteome have limitations , particularly when the genome of the organism is large . here we describe a b. melitensis proteome microarray that enables this problem to be directly addressed by applying an unbiased systems biology approach to identify immunodominant and serodiagnostic antigens and to classify the reactive antigens based on functional and physical properties . moreover , microarrays can display all proteins of an infectious agent and may allow for identification of novel antigens , otherwise undetectable by methods like 2-d gels that are highly biased by microbial protein expression patterns . brucella melitensis , the most virulent species infecting humans , also infects goats , sheep and cattle , in central and south america , the middle east , east asia , and some southern european countries . the current knowledge of protein antigens recognized by humans and reservoir animals is based on limited numbers of studies on brucella melitensis and brucella abortus . we recently constructed a pilot proteome array consisting of 1406 b. melitensis proteins and observed marked differences in immune responses between humans and goats. (5 ) here we have expanded our previous study to the full proteome microarray consisting of 3046 b. melitensis proteins , by including expression of newly cloned 1640 bm genes . in addition to developing a better classifier capable of predicting disease based on serological response , more importantly , this study allows an estimate of the extent of immune reactivity against the whole proteome , and prediction of proteomic features that may dictate immune recognition for other yet uncharacterized gram - negative bacteria . the human subjects part of the study was approved by the humans research protections committee of the university of california san diego , the comite de tica of universidad peruana cayetano heredia , lima , peru and the comite de tica of asociacin benfica prisma , lima , peru , all of whom have maintained federal wide assurances with the united states department of health and human services . eighteen patients presenting with brucellosis - compatible syndromes were culture negative and rose bengal positive , and treated according to standard antibiotic therapy within 2 days of serum sampling . additional control patient samples included 13 sera from rose bengal - negative patients , 44 samples from ambulatory healthy controls from north lima where brucellosis occasionally affects patients , and sera from humans in the u.s . , the presence of lps in the gels was detected with a periodic acid silver stain(33 ) and protein using coomassie blue stain . all orfs from brucella melitensis 16 m genomic dna were identified using genbank nc_003317 and nc_003318 , and 1640 orfs that were absent from pilot chip were amplified and cloned using a high - throughput pcr and recombination cloning method described previously. (5 ) plasmids were expressed at 24 c for 16 h in in vitro transcription / translation e. coli reactions ( expressway maxi kits from invitrogen ) , according to the manufacturer s instructions . strips were then incubated in alkaline phosphatase conjugated donkey antihuman immunoglobulin ( anti - igg , fc fragment - specific , jackson immunoresearch ) secondary antibody , diluted to 1/2000 , and reactive bands were visualized by incubating with 1-step nitro - blue tetrazolium chloride/5-bromo-4-chloro-3-indolyphosphate p - toluidine salt ( nbt / bcip ) developing buffer ( thermo fisher scientific ) . the samples were then diluted 4:1 with 50 mm ammonium bicarbonate buffer ph 8.0 , and trypsin was added at a 1:25 protein ratio for an additional 16 h. following proteolysis , the samples were distributed into 96-well plates for mass spectrometry analysis . peptide digests were analyzed by liquid chromatography coupled to mass spectrometry ( lc - ms ) as described . ms system consisted of a caplc ( waters , milford , ma ) with a cooled autosampler and a qtof ultima ( waters , milford , ma ) controlled by masslynx version 4.0 software . tmhmm v2.0 was utilized for transmembrane domains prediction(46 ) ( http://www.cbs.dtu.dk/services/tmhmm/ ) , signalp v3.0 for signal peptide prediction(47 ) ( http://www.cbs.dtu.dk/services/signalp/ ) , psortb v3.0 . the 3198 predicted orfs of b. melitensis ( bm ) 16 m were subjected to amplification from genomic dna , and 1406 orfs were cloned and printed on pilot chip. about one - fourth of the cloned genes were sequenced and > 99% of sequenced clones had the correct sequence . all 3046 bm orfs cloned in pxt7 vector ( > 95% of proteome ) were expressed under t7 promoter in the e. coli in vitro transcription / translation system , and printed on microarrays . over 98% of protein spots bm protein arrays were probed with sera from acute brucellosis patients in lima , peru obtained within 13 weeks of the onset of symptoms , and sera from bm culture - positive humans ( figure s1b , supporting information ) showed robust reactivity against a collection of antigens compared to unexposed individuals . among the 3046 antigens tested , 1464 antigens reacted with at least one culture positive individual , accounting for 48% of the proteome ( figure 1 ) . of these , 33 protein antigens were serodiagnostic , and were significantly differentially reactive between nave and culture positive patients from peru ( benjamini and hochberg adjusted cyber - t p - value < 0.05 ) . the raised bars of any color represent 1464 reactive proteins , which define the immunoproteome ( approximately 48% of the proteome ) . discovery of new human serodiagnostic antigens by probing the full b. melitensis proteome arrays with a collection of b. melitensis peruvian nave and culture positive human sera . shown are the 33 serodiagnostic protein antigens , bm lps , and a subset of cross - reactive antigens . we performed unsupervised principal component analysis and showed that the 33 serodiagnostic antigens clearly separated the peruvian naive from culture positive groups ( figure 3a ) . to further determine the accuracy of distinguishing brucellosis cases from controls , cross - validation receiver operating characteristic ( roc ) curves and area under the curve ( auc ) the top four orfs , bmei0536 ( bp26 ) , bmei0805 ( hypothetical protein ) , bmei1330 ( protease do ) , and bmei 1890 ( transporter ) , as wells as bm lps , all have an individual antigen auc greater than 0.90 ( table s1 , supporting information ) . three of the antigens were identified previously on microarrays. altogether 33 protein antigens plus lps produced sensitivity and specificity over 93% ( a ) unsupervised principal component analysis of the signal intensity for samples from peruvian naives and culture positive groups revealed that these two groups could be segregated on the basis of 33 selected serodianosis protein antigens and 2 bm lps ( at 0.1 mg / ml or 0.01 mg / ml ) . the serodiagnostic antigens were ranked by single antigen auc . with the top 3 antigens , this classifier yielded a highest sensitivity and specificity rate of 98.6% and 98.6% , respectively . weak reactivity in the nave healthy controls ( b ) cross - validation roc curve was generated and sensitivity and specificity of immunostrips test is 96% and 96% , respectively . while the qualitative agglutination test ( rose bengal ) is 90% sensitive for screening for acute / subacute brucellosis , quantitative agglutination testing ( e.g. without being able to ascertain whether the sera used in this study were obtained from patients who might have taken antibiotics prior to blood cultures , we queried our data set to determine whether sera from blood culture - positive , rose bengal positive brucellosis patients ( gold standard diagnosis ) vs blood culture - negative , rose bengal positive ( tat not done ) subjects ( presumptive positive , unconfirmed ) recognized different proteins on the protein microarrays . these results indicate that protein microarray analysis was able to delineate distinct antibody profiles in blood culture positive and negative patients , which has the potential to identify antigens capable of differentiating active acute / subacute brucellosis from previously exposed patients . to better understand the determinants of antigenicity in bacteria , we performed a functional enrichment analysis of the serodominant and serodiagnostic antigens identified in this study . a total of 543 proteins are unassociated with cogs , and 39 proteins are associated with cogs but have not been assigned to a category , shown as some proteins have multiple cog assignments so there are 3,291 cogs in total for 3046 proteins on chip . proteins with predicted cog- u function , involved in intracellular trafficking and secretion , were 6.2- fold enriched in serodiagnostic antigens and 4.3- fold enriched in serodominant antigens . proteins in the cog - m category , cell envelope biogenesis and outer membrane , were 2-fold enriched in serodominant antigens ( p - value 0.01 ) . cog categories with < 5 proteins were not included in the figure ( cogs - b , w ) . as shown in figure 5b and table s3b ( supporting information ) , proteins with 1 transmembrane domain were significantly enriched in serodominant , serodiagnostic and cross - reactive antigens groups , with enrichment fold at 4.3 , 5.0 , and 4.0 , respectively . interestingly , proteins with more than 1 transmembrane domain were also significantly underrepresented among the serodominant and cross - reactive antigens . another potential feature that could account for protein antigenicity is the level of in vivo expression ; proteins expressed at a high level in vivo would be expected more likely to be seen by the immune system than proteins expressed at a low level . to test this hypothesis , we compared the group of antigenic b. melitensis proteins with an existing data set of expressed protein peptide data from a very closely related species , b. abortus . in this study , cytosolic proteins and cell envelope proteins from b. abortus in the log phase of in vitro growth , were extracted and subjected to lc mass spectrometry ( ms ) analysis as described in materials and methods . two - hundred forty proteins were exclusively expressed in the cytosol of b. abortus with 1 to12 detected peptides , and 53 proteins exclusively expressed in the cell envelope of b. abortus with 1 to 15 detected peptides , and 80 overlapping proteins expressed in both conditions . not surprisingly , expression was a significant enriching feature with 5.2-fold enrichment among serodiagnostic antigens ( p - value 1.87 10 ) , and 2.9-fold in serodominant antigens ( p - value 5.28 10 ) ( table 1 ) . there was 8.4- fold enrichment of serodiagnostic antigens in proteins detected with more than 1 peptide ( p - value 3.56 10 ) , 16.2- fold in proteins detected with 5 or more peptides ( p - value 1.30 10 ) , 23.1- fold enrichment in proteins detected with 7 or more peptides ( p - value 1.90 10 ) , and 20.5-fold enrichment in proteins detected with 9 or more peptides ( p - value 3.91 10 ) . conversely , proteins that were not identified by ms were significantly underrepresented at 0.4-fold among the serodiagnostic and 0.8-fold among serodominant antigens . the data suggests that protein expression level is an important factor contributing to antigenicity of protein antigens . there are a total of 10 enriching features that fall into 3 categories : ( i ) functionally annotated cogs u , m , n and o , ( ii ) computationally predicted features ( tmhmm = 1 , signalp > 0.7 , psort outermembrane , psort periplasmic , and pi < 5 ) , and ( iii ) ms evidence of expression . there are 338 proteins in the enriched cog categories , 696 computationally predicted proteins , and 356 proteins that were positive by ms ( table 2 ) . cogs accounted for 30% of the serodiagnostic hits , the computationally predicted features accounted for 61% of the hits , and ms positive proteins contained 61% of the hits . all together the three categories account for 37% of the proteome and include 91% of the serodiagnostic antigens . so by combining the pool of computationally predicted and ms+ proteins representing 30% of the proteome , 88% of the serodiagnostic antigens can be predicted . enriching features are classified into three categories : ( 1 ) cogs - umno , for proteins assigned to one or more of these four cogs ; ( 2 ) computational predictions with enriching features , for proteins predicted to have one or more of the following features , that is , tmhmm = 1 , signalp > 0.7 , psort outermembrane , psort periplasmic , or pi < 5 , and ( 3 ) protein expression as detected by peptide presence in mass spectrometry . separately shown are numbers of antigens with enriching features , including all such 1128 antigens on chip , 99 serodominant , and 30 serodiagnostic antigens . numbers of antigens having more than one category of enriching features are also presented in the overlapping region of such categories . enriching features are classified into three categories : ( 1 ) cogs - umno , for proteins assigned to one or more of these four cogs ; ( 2 ) computational predictions with enriching features , for proteins predicted to have one or more of the following features , i.e. , tmhmm = 1 , signalp > 0.7 , psort outermembrane , psort periplasmic , or pi < 5 ; and ( 3 ) protein expression as detected by mass spectrometry . this work was motivated by the disturbing lack of a detailed scientific understanding of the antibody responses to infectious diseases . we have been taking a systems biology approach to account for all of the antibodies that develop after exposure to infectious microorganisms and to identify specific antibody signatures associated with each disease , in order to understand the molecular basis for antigen selection by the immune system and to predict serodiagnostic and vaccine antigens targets . our previous study identified a set of serodiagnostic antigens from a randomly selected half of the b. melitensis proteome . however , an important difference between the present study and this previous report is that analysis of the complete proteome not only has allowed us to delineate additional serodiagnostic antigens but , more fundamentally , also provided the basis for a rigorous , comprehensive and quantitative determination of basic biological characteristics of the entire set of the serodominant antigens on a genomic level . in the present microarray study , we confirmed lps reactivity ( with purified lps spotted onto the microarray ) , and identified novel protein antigens . eleven of the 33 serodiagnostic antigens identified in this study were also identified on the pilot array(5 ) ( table s1 , supporting information ) . in addition to the well characterized antigens , we also identified 21 novel serodiagnostic antigens on the current array , including top antigen hypothetical protein bmei0805 . protein microarrays enable enrichment analyses to identify proteomic features that are enriched in the immunodominant antigen set , and development of protein antigenicity prediction tools based on enriched features . the proteomic features fall into 3 categories : ( i ) cog annotations , ( ii ) computationally predicted proteomic features , and ( iii ) proteins with ms evidence of expression in viable organisms . our data suggests that cloning of 37% of genome with these enriching features would reveal more than 90% of serodiagnostic antigens . we have classified the reactive immunodominant antigens for numerous agents and have consistently found these features predict antigenicity . in our study , we utilized the expression of proteins quantified by ms of a closely related strain , b. abortus during in vitro growth . we found that mass spec positive antigens were significantly enriched among serodiagnostic antigens but not among the cross- reactive antigens . this validates the classification of the serodiagnostic antigens being derived from actively replicating organisms , and validates the classification of cross - reactive antigens being derived from previous exposure to nonbrucellosis infections . although the number of peptides observed per protein has been applied to estimating protein abundance , we are aware that mass spectra of cultured organisms may not reflect the expression during infection in vivo . our protein array technology is also able to provide strong evidence of the comprehensive set of proteins expressed in vivo within a mammalian host by b. melitensis , by virtue of their exposure to the host immune system . (64 ) the b. melitensis immunoproteome comprises 1464 antigens that are significantly reactive in at least one of the individuals in this study . answers to questions of why and how the immune system focuses on 4% of the potential target antigens are not yet apparent from this work . one might expect that an immune response against a larger collection of antigens could result in a more effective immune response attack against the infectious agent . but antibody responses against thousands of antigens from hundreds of clinical infections could accumulate during a lifetime , leading to cross reactivity against autologous antigens and autoimmune chaos . the observation that dozens of organism- specific antibody responses develop after b. melitensis infection is consistent with similar observations from other infectious agents , and has implications for subunit vaccine discovery and development . (6 ) this systematic genome scale analysis of human antibody responses against b. melitensis proteins provides a top hit list of antigens worthy of assessing for improved diagnostics , and furthermore , enables development of a predictive model of proteomic features that determine whether a protein is antigenic and produces antibodies that confer protection . this systems biology approach provides an empirical basis for understanding the breadth and specificity of the immune response to b. melitensis and a new framework for comparing the humoral responses against other organisms .

the final product of epidermal differentiation , the stratum corneum ( sc ) , constitutes a barrier that efficiently separates the body s internal milieu from the terrestrial environment . it is composed of 1020 layers of dead cornified cells embedded in a highly hydrophobic extracellular matrix . quasi equimolar proportions of three lipid families , i.e. , ceramides , cholesterol and free fatty acids , are necessary for the adequate molecular organization of extracellular spaces and the resulting relative impermeability to water and other substances ( bouwstra et al . 2003 ; feingold and elias 2014 ) . the physical barrier of sc is highly interactive in terms of its constant response to changing environmental conditions and insults . such a rapid adaptation is possible because of perpetual epidermal renewal accompanied by relatively rapid sc recycling , with a turnover time of approximately two weeks ( hoath and leahy 2003 ; elias and choi 2005 ; haftek 2014 ) . a secondary barrier composed of epidermal tight junctions is located in the stratum granulosum and appears to play an important role in sc formation , notably in the case of the acute abrogation of the principal sc fence ( abdayem et al . 2014 ; for a review , see the following paper by j.m . although sc barrier function depends greatly upon its biochemical composition , no effective barrier would exist without the appropriate tissue structure . the flattened cornified keratinocytes , namely the corneocytes , are delineated by highly insoluble cornified envelopes together with equally cross - linked lipid envelopes . the latter are constituted by a monolayer of ceramides that replace plasma membranes of the living cells . lipid envelopes constitute the scaffold for the molecular arrangement of extracellular lipids to form stacked bilayer sheets in inter - corneocyte spaces . this layered lipid structure is essential for providing an adequate degree of waterproofing and the sc permeability barrier ( van smeden et al . corneocytes remain connected via cell - cell junctions persisting in the sc and their desquamation at the top of the skin depends on the gradual degradation of these cell attachments ( haftek et al . 2011 ; haftek 2014 ; ishida - yamamoto and igawa 2014 ; for a review , see the previous paper by a. ishida - yamamoto ) . the principal mechanical junctions of the sc , namely the corneodesmosomes , are modified desmosomes from the uppermost nucleated epidermal layers . they retain the molecular composition of the stratum granulosum junctions , notably desmosomal cadherins characteristic of differentiated keratinocytes , i.e. , desmoglein 1 and desmocollin 1 but are immobilized at the cell periphery through an extensive enzymatic cross - linking mediated by transglutaminases 1 , 3 and 5 ( haftek et al . the keratinocytes of the granular layer synthesize and excrete into the extracellular spaces a new glycoprotein , namely corneodesmosin , which spontaneously embeds within the intercellular portions of the stratum granulosum desmosomes occupied by cadherins ( serre et al . corneodesmosin reinforces the junctions and must be degraded by proteases , together with the desmosomal cadherins , to permit desquamation ( simon et al . 2002 ) . a complex interplay of serine proteases ( kallikreins ) and cysteine proteases ( cathepsins ) with their respective inhibitors ( all excreted through the same vesiculo - tubular system of lamellar granules as the intercellular lipids ) is orchestrated by the modifications of sc ph and hydration to result in the progressive digestion of the corneodesmosomes ( haftek et al . 1998 ; hachem et al . 2003 ; caubet et al . 2004 ; rawlings and voegeli 2013 ; fig . 1 ) . during this degradation process , first the junctions from between the consecutive layers of corneocytes disappear , leaving intact the lateral cell - cell attachments . this results in the subdivision of the sc into a highly cohesive part , the sc compactum , with corneodesmosomes all around the cells and the sc disjunctum , with side - to - side cell connexions only . once again , the peculiar spatial regulation of this desquamation process might be dependent on structural features : in this case , the persistence of strategically located fusions between the adjacent cornified cell envelopes , i.e. , cross - linked remnants of tight junctions ( haftek et al . functional consequences of this situation can be measured based on the energy necessary for intercellular sc delamination . indeed , such energy values diminish together with the lowering of corneodesmosome density from the deeper parts of the sc towards the surface ( wu et al . the balance between the sc formation and desquamation impacts in an evident way on sc thickness and its barrier function . in many cases , intercellular proteins of corneodesmosome are substrates for serine proteases kallikreins ( klk ) , elastase 2 ( ela2 ) and cysteine proteases cathepsins . plasma - membrane - attached serine proteases of matriptase / mt - sp1/cap3prostasin / cap1/prss8 cascade can cross - activate and act through the protease activated receptor 2 on filaggrin and occludin processing , the thymic stromal lymphopoietin ( tslp)mediated adaptive inflammatory response and epithelial sodium channel ( enac ) activation . all these interactions are crucial for the maintenance of epidermal homeostasis and stratum corneum barrier function interplay of proteolytic enzymes with their inhibitors taking place in human epidermis . intercellular proteins of corneodesmosome are substrates for serine proteases kallikreins ( klk ) , elastase 2 ( ela2 ) and cysteine proteases cathepsins . plasma - membrane - attached serine proteases of matriptase / mt - sp1/cap3prostasin / cap1/prss8 cascade can cross - activate and act through the protease activated receptor 2 on filaggrin and occludin processing , the thymic stromal lymphopoietin ( tslp)mediated adaptive inflammatory response and epithelial sodium channel ( enac ) activation . all these interactions are crucial for the maintenance of epidermal homeostasis and stratum corneum barrier function homozygous nonsense mutations in the corneodesmosin ( cdsn ) gene leading to the complete absence of the encoded protein or to residual expression of small non - functional fragments result in peeling skin disease ( psd ) , a generalized form of peeling skin syndrome , classified as inflammatory form b ( mim270300 ; oji et al . 2010 ; israeli et al . 2011 ; mazereeuw - hautier et al . the extracellular portions of the corneodesmosomes prove less resistant to mechanical stress and are easily cleaved , especially at the bottom of the sc , at the interface with the granular layer . this mechanical separation of the entire sheet of full thickness sc leaves largely denuded areas with practically no barrier at all . rupture of the permeability barrier results , in turn , in cytokine production by keratinocytes ( wood et al . a rescue response of the uppermost viable epidermal layers aimed at the re - establishment of the sc barrier is also induced . most probably , it comprises lamellar granule / lipid release , as suggested by the mouse experiments of the elias group ( menon et al . 1992 ) and the up - regulation of the tight junction structures , as documented by a significant increase in the tight junction remnants persisting in the sc of psd ( haftek et al . in contrast , specific dominant cdsn mutations are associated with autosomal dominant hypotrichosis simplex ( mim146520 ; levy - nissenbaum et al . the truncated mutant corneodesmosin has been found to exert a toxic effect on hair follicles through the formation of amyloid deposits ( caubet et al . psd must be differentiated from another generalized but non - inflammatory form a , the etiology of which has been recently linked to a mutation in chst8 gene encoding a golgi sulfotransferase ( cabral et al . 2013 ) with its major variant being caused by mutations in tgm5 , encoding transglutaminase 5 ( mim609796 ; cassidy et al . 2005 ; szczecinska et al . homozygous bi - allelic mutations of the desmoglein 1 gene ( dsg1 ) observed in rare consanguineous families also impact corneodesmosome function . they result in severe dermatitis , multiple allergies and metabolic wasting syndrome ( mim 615508 ; samuelov et al . 2013 ) , although cases without metabolic wasting have also been observed ( has et al . although the main structural changes , such as irregular desmosome distribution , hypergranulosis with focal absence of the granular layer and widespread acantholysis within the stratum spinosum and granulosum , result in subcorneal and intragranular separation , a modified sc is also observed showing mixed ortho- and parakeratosis . corneodesmosome distribution also remains uneven and the disorder is characterized by compromised barrier function , which may expose the immune system to abnormal stimulation and lead to multiple allergies . as can be logically predicted , analogical sc defects involving the other desmosomal cadherin engaged in the process of sc cohesion , desmocollin 1 , the protease - antiprotease system efficiently regulates the normal process of sc formation and desquamation ( egelrud 2000 ; rawlings and voegeli 2013 ) . observation of several pathological states in man and in rodents has helped partially to unravel these complex interactions . however , whether a common pathway and a coordinated regulation of their activity are involved in the terminal differentiation of epidermal keratinocytes remains unclear . netherton syndrome is an autosomal recessive genodermatosis ( mim 256500 ) characterized by congenital ichthyosiform erythroderma , invaginated distal hair shafts and atopic disease . it is caused by mutations in the spink5 gene encoding lekti 1 , a serine protease inhibitor ( hovnanian 2013 ) . specific and measured neutralization of kallikreins 5 , 7 and 14 by lekti is necessary for the limitation of corneodesmosome degradation and hence , in netherton syndrome , premature desquamation occurs associated with inflammatory reaction and severe barrier impairment leading to multiple allergies ( deraison et al . an inverse pathomechanism takes place in recessive x - linked ichthyosis ( xli , mim 308100 ) in which deletions in the steroid sulfatase ( sts ) gene result in insufficiency of the enzyme and sc retention ( elias et al . steroid sulfatase is necessary for the conversion of cholesterol sulfate to cholesterol , a fundamental building brick of the intercellular lipid lamellae of sc . in xli , reduction in the cholesterol molecules within the horny layer and the accumulation of cholesterol sulfate provoke disequilibrium in the extracellular lipid species leading to phase separation and suboptimal barrier function . moreover , the accumulation of cholesterol sulfate substrate ( up to 20-fold that of normal values ) has been revealed to be a potent inhibitor of sc kallikreins in vitro ( sato et al . 1998 ) and is thus able significantly to slow down corneodesmosome degradation . other mechanisms leading to corneodesmosome retention in xli include ( 1 ) low sc ph , out of the neutral to basic operating optima of kallikreins ( ohman and vahlquist 1998 ) and ( 2 ) the increased presence of ca in the intercellular domains of the lower part of ichthyotic sc possibly contributing to the stabilization of corneodesmosome attachments ( elias et al . autosomal recessive ichthyosis with hypotrichosis ( arih , omim 610765 ) , an inherited disorder linked to mutations in the st14 gene coding for a serine protease matriptase , is characterized by the absence of the proteolytic activity of this type ii transmembrane enzyme ( chen et al . histologically , impaired corneodesmosome degradation , acanthosis and sc accumulation can be observed ( basel - vanagaite et al . another inherited disease linked to various kinds of mutation of the matriptase gene and resulting in the total loss of the expression of the protein is ifah ( ichthyosis , follicular atrophoderma , hypotrichosis and hypohidrosis ; omim 602400 ; chen et al . the sc barrier defect observed in this latter affliction has been associated with the impaired processing of profilaggrin ( alef et al . clinical differences in phenotype between arih and ifah can possibly be explained by the presence of modified matriptase fragments in the former , in the light of evidence that reciprocal cross - activation of zymogen forms of matriptase and its downstream partner prostasin / prss8/cap-1 occurs and is independent of the activation state of the enzymes ( friis et al . although the epidermal distribution of human and rodent matriptase diverges significantly ( chen et al . 2014 ) , we should note that reduced filaggrin formation from its profilaggrin precursor has also been reported in matriptase knockout ( ko ) mice , additionally impacting sc lipid matrix formation and cornified envelope morphogenesis ( list et al . together , these observations point to a role of the matriptase - activated cascade spanning from keratinocyte proliferation , through their terminal differentiation , to desquamation . interestingly , impaired filaggrin processing has been reported in mice models mimicking the human autosomal recessive congenital ichthyosis group of diseases ( arci ; jobard et al . 2002 ) through an altered function of arachidonic acid converting enzymes such as 12r - lipoxygenase ( epp et al . because such enzymes have no obvious roles in the processing of profilaggrin , defects in the epidermal differentiation processes , even at early stages of differentiation , could affect downstream filaggrin processing . ichthyosis vulgaris ( mim 146700 ) and atopic dermatitis ( atod2 ; mim 605803 ) can be associated with loss - of - function mutations in the filaggrin gene ( flg ; smith et al . as discussed previously , no straightforward explanation has been proposed for the pathological mechanism involved in which low filaggrin results in impaired barrier function . nevertheless , recent experimental data suggest the existence of a feedback mechanism involving the n - terminal fragment of filaggrin , which would thus be responsible for controlling epidermal homeostasis ( aho et al . free amino acids originating from filaggrin degradation are the main contributors to the nmf ( natural moisturizing factor ) of the sc . therefore , the absence or largely reduced presence of filaggrin should impact nmf quantities and , consequently , sc hydration ( dapic et al . 2013 ) . because filaggrin is also incorporated into cornified envelopes ( simon et al . 1996 ) , one can hypothesize that low filaggrin levels influence the quality of these structures and , in this way , modify the fate of the intercellular lipid lamellae and corneodesmosomes . indeed , an abnormal distribution of corneodesmosome proteins persisting on the flat lower / ventral sides of superficial corneocytes has been reported in atopic dermatitis , mostly in skin lesions and to a lower extent in non - involved skin ( igawa et al . this is in stark contrast to the strips from normal subjects in which staining is present only at the lateral rims of corneocytes ( oyama et al . 2013 ; singh et al . 2014 ; fig . 2 ) and with psd in which corneodesmosin is not detected . patterns of corneodesmosome distribution assessed with immunofluorescence by using antibodies to corneodesmosin , desmoglein 1 and desmocollin 1 are similar in atopic dermatitis and in ichthyosis vulgaris , although no information has been provided concerning the eventual existence of flg mutations in the former . this has confirmed the presence of wide - ranging defects in the cornification occurring in atopic dermatitis ( guttman - yassky et al . 2009 ) but , unfortunately , does not permit any connection to be made between them and the eventual occurrence of a filaggrin defect . by the way , in netherton syndrome , which also presents a defective sc barrier function and atopy , the staining pattern is similar , although the corneocytes were stripped in an irregular way . the method of immunofluorescent labeling of corneodesmosome proteins on tape - stripped corneocytes was originally used by oyama et al . ( 2010 ) who studied the lesional skin of two other inflammatory dermatoses with altered terminal differentiation , namely psoriasis and lichen planus . these authors found a diffuse pattern of desmoglein 1 distribution all over the surface of corneocytes from the psoriatic scale , reminiscent of the above - described findings in ichthyosis vulgaris , suggesting that corneodesmosome retention at the ventral / dorsal surfaces of cells might not be pathognomonic but rather related to the relative this point of view seems to be strengthened by the biochemical analysis of the pattern of the cleavage of corneodesmosome proteins in psoriasis , as presented by simon et al . the authors detected a near full - length form of corneodesmosin that has not been previously observed in normal sc and altered proteolysis of desmoglein 1 , desmocollin 1 and plakoglobin , indicating a reduced degradation of all corneodesmosomal proteins in psoriatic lesions . studies of dandruff have shown that the persistence of non - peripheral corneodesmosomes is a characteristic feature of the perturbed desquamation seen in this scalp affliction ( singh et al . the reported observations of the concomitant increased expression of lekti-1 and scca1 serine protease inhibitors are consistent with the view that the dandruff condition is characterized by an imbalance in protease / protease inhibitor interaction in the sc.fig . a , b labeling of native superficial tape - stripped corneocytes revealed with 1-nm immunogold enhanced with silver coating and observed by scanning electron microscopy . c post - embedding labeling with 10-nm gold granules on a vertical section of normal stratum corneum compactum as visualized by transmission electron microscopy ( arrows corneodesmosomes present both at the lateral and at the ventral / dorsal faces of the cells , sg stratum granulosum , sc1 , sc2 successive horny layers ) . bars 50 m ( a ) , 20 m ( b ) , 200 nm ( c ) corneodesmosome distribution as highlighted by immunogold labeling with an anti - corneodesmosin monoclonal antibody . a , b labeling of native superficial tape - stripped corneocytes revealed with 1-nm immunogold enhanced with silver coating and observed by scanning electron microscopy . c post - embedding labeling with 10-nm gold granules on a vertical section of normal stratum corneum compactum as visualized by transmission electron microscopy ( arrows corneodesmosomes present both at the lateral and at the ventral / dorsal faces of the cells , sg stratum granulosum , sc1 , sc2 successive horny layers ) . bars 50 m ( a ) , 20 m ( b ) , 200 nm ( c ) homozygous nonsense mutations in the corneodesmosin ( cdsn ) gene leading to the complete absence of the encoded protein or to residual expression of small non - functional fragments result in peeling skin disease ( psd ) , a generalized form of peeling skin syndrome , classified as inflammatory form b ( mim270300 ; oji et al . 2010 ; israeli et al . 2011 ; mazereeuw - hautier et al . the extracellular portions of the corneodesmosomes prove less resistant to mechanical stress and are easily cleaved , especially at the bottom of the sc , at the interface with the granular layer . this mechanical separation of the entire sheet of full thickness sc leaves largely denuded areas with practically no barrier at all . rupture of the permeability barrier results , in turn , in cytokine production by keratinocytes ( wood et al . a rescue response of the uppermost viable epidermal layers aimed at the re - establishment of the sc barrier is also induced . most probably , it comprises lamellar granule / lipid release , as suggested by the mouse experiments of the elias group ( menon et al . 1992 ) and the up - regulation of the tight junction structures , as documented by a significant increase in the tight junction remnants persisting in the sc of psd ( haftek et al . in contrast , specific dominant cdsn mutations are associated with autosomal dominant hypotrichosis simplex ( mim146520 ; levy - nissenbaum et al . the truncated mutant corneodesmosin has been found to exert a toxic effect on hair follicles through the formation of amyloid deposits ( caubet et al . psd must be differentiated from another generalized but non - inflammatory form a , the etiology of which has been recently linked to a mutation in chst8 gene encoding a golgi sulfotransferase ( cabral et al . 2013 ) with its major variant being caused by mutations in tgm5 , encoding transglutaminase 5 ( mim609796 ; cassidy et al . 2005 ; szczecinska et al . homozygous bi - allelic mutations of the desmoglein 1 gene ( dsg1 ) observed in rare consanguineous families also impact corneodesmosome function . they result in severe dermatitis , multiple allergies and metabolic wasting syndrome ( mim 615508 ; samuelov et al . 2013 ) , although cases without metabolic wasting have also been observed ( has et al . although the main structural changes , such as irregular desmosome distribution , hypergranulosis with focal absence of the granular layer and widespread acantholysis within the stratum spinosum and granulosum , result in subcorneal and intragranular separation , a modified sc is also observed showing mixed ortho- and parakeratosis . corneodesmosome distribution also remains uneven and the disorder is characterized by compromised barrier function , which may expose the immune system to abnormal stimulation and lead to multiple allergies . as can be logically predicted , analogical sc defects involving the other desmosomal cadherin engaged in the process of sc cohesion , desmocollin 1 , the protease - antiprotease system efficiently regulates the normal process of sc formation and desquamation ( egelrud 2000 ; rawlings and voegeli 2013 ) . observation of several pathological states in man and in rodents has helped partially to unravel these complex interactions . however , whether a common pathway and a coordinated regulation of their activity are involved in the terminal differentiation of epidermal keratinocytes remains unclear . netherton syndrome is an autosomal recessive genodermatosis ( mim 256500 ) characterized by congenital ichthyosiform erythroderma , invaginated distal hair shafts and atopic disease . it is caused by mutations in the spink5 gene encoding lekti 1 , a serine protease inhibitor ( hovnanian 2013 ) . specific and measured neutralization of kallikreins 5 , 7 and 14 by lekti is necessary for the limitation of corneodesmosome degradation and hence , in netherton syndrome , premature desquamation occurs associated with inflammatory reaction and severe barrier impairment leading to multiple allergies ( deraison et al . an inverse pathomechanism takes place in recessive x - linked ichthyosis ( xli , mim 308100 ) in which deletions in the steroid sulfatase ( sts ) gene result in insufficiency of the enzyme and sc retention ( elias et al . steroid sulfatase is necessary for the conversion of cholesterol sulfate to cholesterol , a fundamental building brick of the intercellular lipid lamellae of sc . in xli , reduction in the cholesterol molecules within the horny layer and the accumulation of cholesterol sulfate provoke disequilibrium in the extracellular lipid species leading to phase separation and suboptimal barrier function . moreover , the accumulation of cholesterol sulfate substrate ( up to 20-fold that of normal values ) has been revealed to be a potent inhibitor of sc kallikreins in vitro ( sato et al . 1998 ) and is thus able significantly to slow down corneodesmosome degradation . other mechanisms leading to corneodesmosome retention in xli include ( 1 ) low sc ph , out of the neutral to basic operating optima of kallikreins ( ohman and vahlquist 1998 ) and ( 2 ) the increased presence of ca in the intercellular domains of the lower part of ichthyotic sc possibly contributing to the stabilization of corneodesmosome attachments ( elias et al . autosomal recessive ichthyosis with hypotrichosis ( arih , omim 610765 ) , an inherited disorder linked to mutations in the st14 gene coding for a serine protease matriptase , is characterized by the absence of the proteolytic activity of this type ii transmembrane enzyme ( chen et al . histologically , impaired corneodesmosome degradation , acanthosis and sc accumulation can be observed ( basel - vanagaite et al . another inherited disease linked to various kinds of mutation of the matriptase gene and resulting in the total loss of the expression of the protein is ifah ( ichthyosis , follicular atrophoderma , hypotrichosis and hypohidrosis ; omim 602400 ; chen et al . the sc barrier defect observed in this latter affliction has been associated with the impaired processing of profilaggrin ( alef et al . clinical differences in phenotype between arih and ifah can possibly be explained by the presence of modified matriptase fragments in the former , in the light of evidence that reciprocal cross - activation of zymogen forms of matriptase and its downstream partner prostasin / prss8/cap-1 occurs and is independent of the activation state of the enzymes ( friis et al . although the epidermal distribution of human and rodent matriptase diverges significantly ( chen et al . 2014 ) , we should note that reduced filaggrin formation from its profilaggrin precursor has also been reported in matriptase knockout ( ko ) mice , additionally impacting sc lipid matrix formation and cornified envelope morphogenesis ( list et al . , these observations point to a role of the matriptase - activated cascade spanning from keratinocyte proliferation , through their terminal differentiation , to desquamation . interestingly , impaired filaggrin processing has been reported in mice models mimicking the human autosomal recessive congenital ichthyosis group of diseases ( arci ; jobard et al . 2002 ) through an altered function of arachidonic acid converting enzymes such as 12r - lipoxygenase ( epp et al . because such enzymes have no obvious roles in the processing of profilaggrin , defects in the epidermal differentiation processes , even at early stages of differentiation , could affect downstream filaggrin processing . ichthyosis vulgaris ( mim 146700 ) and atopic dermatitis ( atod2 ; mim 605803 ) can be associated with loss - of - function mutations in the filaggrin gene ( flg ; smith et al . as discussed previously , no straightforward explanation has been proposed for the pathological mechanism involved in which low filaggrin results in impaired barrier function . nevertheless , recent experimental data suggest the existence of a feedback mechanism involving the n - terminal fragment of filaggrin , which would thus be responsible for controlling epidermal homeostasis ( aho et al . free amino acids originating from filaggrin degradation are the main contributors to the nmf ( natural moisturizing factor ) of the sc . therefore , the absence or largely reduced presence of filaggrin should impact nmf quantities and , consequently , sc hydration ( dapic et al . 2013 ) . because filaggrin is also incorporated into cornified envelopes ( simon et al . 1996 ) , one can hypothesize that low filaggrin levels influence the quality of these structures and , in this way , modify the fate of the intercellular lipid lamellae and corneodesmosomes . indeed , an abnormal distribution of corneodesmosome proteins persisting on the flat lower / ventral sides of superficial corneocytes has been reported in atopic dermatitis , mostly in skin lesions and to a lower extent in non - involved skin ( igawa et al . this is in stark contrast to the strips from normal subjects in which staining is present only at the lateral rims of corneocytes ( oyama et al . 2013 ; singh et al . 2014 ; fig . 2 ) and with psd in which corneodesmosin is not detected . patterns of corneodesmosome distribution assessed with immunofluorescence by using antibodies to corneodesmosin , desmoglein 1 and desmocollin 1 are similar in atopic dermatitis and in ichthyosis vulgaris , although no information has been provided concerning the eventual existence of flg mutations in the former . this has confirmed the presence of wide - ranging defects in the cornification occurring in atopic dermatitis ( guttman - yassky et al . 2009 ) but , unfortunately , does not permit any connection to be made between them and the eventual occurrence of a filaggrin defect . by the way , in netherton syndrome , which also presents a defective sc barrier function and atopy , the staining pattern is similar , although the corneocytes were stripped in an irregular way . the method of immunofluorescent labeling of corneodesmosome proteins on tape - stripped corneocytes was originally used by oyama et al . ( 2010 ) who studied the lesional skin of two other inflammatory dermatoses with altered terminal differentiation , namely psoriasis and lichen planus . these authors found a diffuse pattern of desmoglein 1 distribution all over the surface of corneocytes from the psoriatic scale , reminiscent of the above - described findings in ichthyosis vulgaris , suggesting that corneodesmosome retention at the ventral / dorsal surfaces of cells might not be pathognomonic but rather related to the relative this point of view seems to be strengthened by the biochemical analysis of the pattern of the cleavage of corneodesmosome proteins in psoriasis , as presented by simon et al . the authors detected a near full - length form of corneodesmosin that has not been previously observed in normal sc and altered proteolysis of desmoglein 1 , desmocollin 1 and plakoglobin , indicating a reduced degradation of all corneodesmosomal proteins in psoriatic lesions . studies of dandruff have shown that the persistence of non - peripheral corneodesmosomes is a characteristic feature of the perturbed desquamation seen in this scalp affliction ( singh et al . the reported observations of the concomitant increased expression of lekti-1 and scca1 serine protease inhibitors are consistent with the view that the dandruff condition is characterized by an imbalance in protease / protease inhibitor interaction in the sc.fig . a , b labeling of native superficial tape - stripped corneocytes revealed with 1-nm immunogold enhanced with silver coating and observed by scanning electron microscopy . c post - embedding labeling with 10-nm gold granules on a vertical section of normal stratum corneum compactum as visualized by transmission electron microscopy ( arrows corneodesmosomes present both at the lateral and at the ventral / dorsal faces of the cells , sg stratum granulosum , sc1 , sc2 successive horny layers ) . bars 50 m ( a ) , 20 m ( b ) , 200 nm ( c ) corneodesmosome distribution as highlighted by immunogold labeling with an anti - corneodesmosin monoclonal antibody . a , b labeling of native superficial tape - stripped corneocytes revealed with 1-nm immunogold enhanced with silver coating and observed by scanning electron microscopy . c post - embedding labeling with 10-nm gold granules on a vertical section of normal stratum corneum compactum as visualized by transmission electron microscopy ( arrows corneodesmosomes present both at the lateral and at the ventral / dorsal faces of the cells , sg stratum granulosum , sc1 , sc2 successive horny layers ) . bars 50 m ( a ) , 20 m ( b ) , 200 nm ( c ) topical application of sodium lauryl sulfate ( sls ) detergent to the skin is known to disrupt sc function and is used as a reference in irritation tests in vivo . skin challenged with 1 % sls in occlusive patch reacts with early changes of mrna expression reflecting the up - regulation of pro - barrier elements such as involucrin and transglutaminase 1 and the down - regulation of serine proteases involved in corneodesmosome degradation ( trm et al . 2008 ) . in soap - induced xerosis , non - peripheral corneodesmosomes also remain undegraded in the upper sc ( rawlings et al . human skin chronically exposed to low temperatures and dry air develops sc dryness characterized by roughness and a papyraceous appearance of the surface , the presence of raised squames and/or scales and irritation , commonly called winter xerosis . in this reactive condition , the persistence of both peripheral and non - peripheral corneodesmosomes in the upper sc has been observed ( simon et al . epidermal differentiation in palmar / plantar ridged skin represents a particular case , because here corneodesmosomes are not degraded in a pattern known from normal interfollicular epidermis but persist all around the corneocytes up to the surface ( mils et al . this occurs together with corneocyte accumulation in the ridged horny layer conferring it with more physical resistance . in the case of repeated mechanical stress , the palmar / plantar epidermis reacts with an additional accumulation of the sc , leading to the clinical appearance of calluses . these physiological features well demonstrate the importance of corneodesmosome junctions in the response of the sc to the environment . impaired barrier function results in an increased cutaneous penetration of environmental allergens and can lead to eczematous reactions . links between skin inflammation , protease / inhibitor balance , filaggrin processing , the composition and molecular arrangement of the extracellular lipid matrix , corneodesmosome - degradation

corneodesmosomes are modified desmosomes present in the stratum corneum ( sc ) . they are crucial for sc cohesion and , thus , constitute one of the pivotal elements of the functional protective barrier of human skin . expression of corneodesmosomes and , notably , the process of their degradation are probably altered during several dermatoses leading to the disruption of the permeability barrier or to abnormal , often compensative , sc accumulation . these different situations are reviewed in the present paper .

Introduction Inherited forms of corneodesmosome dysfunction and their impact on the SC barrier Primary defects of corneodesmosomes Protease and protease inhibitor dysfunctions Impact of filaggrin mutations and changes attributable to abnormal epidermal differentiation Acquired forms of corneodesmosome dysfunction and their impact on the SC barrier Concluding remarks

the final product of epidermal differentiation , the stratum corneum ( sc ) , constitutes a barrier that efficiently separates the body s internal milieu from the terrestrial environment . the physical barrier of sc is highly interactive in terms of its constant response to changing environmental conditions and insults . a secondary barrier composed of epidermal tight junctions is located in the stratum granulosum and appears to play an important role in sc formation , notably in the case of the acute abrogation of the principal sc fence ( abdayem et al . the latter are constituted by a monolayer of ceramides that replace plasma membranes of the living cells . this layered lipid structure is essential for providing an adequate degree of waterproofing and the sc permeability barrier ( van smeden et al . corneocytes remain connected via cell - cell junctions persisting in the sc and their desquamation at the top of the skin depends on the gradual degradation of these cell attachments ( haftek et al . the principal mechanical junctions of the sc , namely the corneodesmosomes , are modified desmosomes from the uppermost nucleated epidermal layers . they retain the molecular composition of the stratum granulosum junctions , notably desmosomal cadherins characteristic of differentiated keratinocytes , i.e. the keratinocytes of the granular layer synthesize and excrete into the extracellular spaces a new glycoprotein , namely corneodesmosin , which spontaneously embeds within the intercellular portions of the stratum granulosum desmosomes occupied by cadherins ( serre et al . a complex interplay of serine proteases ( kallikreins ) and cysteine proteases ( cathepsins ) with their respective inhibitors ( all excreted through the same vesiculo - tubular system of lamellar granules as the intercellular lipids ) is orchestrated by the modifications of sc ph and hydration to result in the progressive digestion of the corneodesmosomes ( haftek et al . this results in the subdivision of the sc into a highly cohesive part , the sc compactum , with corneodesmosomes all around the cells and the sc disjunctum , with side - to - side cell connexions only . indeed , such energy values diminish together with the lowering of corneodesmosome density from the deeper parts of the sc towards the surface ( wu et al . all these interactions are crucial for the maintenance of epidermal homeostasis and stratum corneum barrier function interplay of proteolytic enzymes with their inhibitors taking place in human epidermis . all these interactions are crucial for the maintenance of epidermal homeostasis and stratum corneum barrier function homozygous nonsense mutations in the corneodesmosin ( cdsn ) gene leading to the complete absence of the encoded protein or to residual expression of small non - functional fragments result in peeling skin disease ( psd ) , a generalized form of peeling skin syndrome , classified as inflammatory form b ( mim270300 ; oji et al . rupture of the permeability barrier results , in turn , in cytokine production by keratinocytes ( wood et al . most probably , it comprises lamellar granule / lipid release , as suggested by the mouse experiments of the elias group ( menon et al . 1992 ) and the up - regulation of the tight junction structures , as documented by a significant increase in the tight junction remnants persisting in the sc of psd ( haftek et al . although the main structural changes , such as irregular desmosome distribution , hypergranulosis with focal absence of the granular layer and widespread acantholysis within the stratum spinosum and granulosum , result in subcorneal and intragranular separation , a modified sc is also observed showing mixed ortho- and parakeratosis . as can be logically predicted , analogical sc defects involving the other desmosomal cadherin engaged in the process of sc cohesion , desmocollin 1 , the protease - antiprotease system efficiently regulates the normal process of sc formation and desquamation ( egelrud 2000 ; rawlings and voegeli 2013 ) . however , whether a common pathway and a coordinated regulation of their activity are involved in the terminal differentiation of epidermal keratinocytes remains unclear . it is caused by mutations in the spink5 gene encoding lekti 1 , a serine protease inhibitor ( hovnanian 2013 ) . specific and measured neutralization of kallikreins 5 , 7 and 14 by lekti is necessary for the limitation of corneodesmosome degradation and hence , in netherton syndrome , premature desquamation occurs associated with inflammatory reaction and severe barrier impairment leading to multiple allergies ( deraison et al . an inverse pathomechanism takes place in recessive x - linked ichthyosis ( xli , mim 308100 ) in which deletions in the steroid sulfatase ( sts ) gene result in insufficiency of the enzyme and sc retention ( elias et al . in xli , reduction in the cholesterol molecules within the horny layer and the accumulation of cholesterol sulfate provoke disequilibrium in the extracellular lipid species leading to phase separation and suboptimal barrier function . moreover , the accumulation of cholesterol sulfate substrate ( up to 20-fold that of normal values ) has been revealed to be a potent inhibitor of sc kallikreins in vitro ( sato et al . other mechanisms leading to corneodesmosome retention in xli include ( 1 ) low sc ph , out of the neutral to basic operating optima of kallikreins ( ohman and vahlquist 1998 ) and ( 2 ) the increased presence of ca in the intercellular domains of the lower part of ichthyotic sc possibly contributing to the stabilization of corneodesmosome attachments ( elias et al . autosomal recessive ichthyosis with hypotrichosis ( arih , omim 610765 ) , an inherited disorder linked to mutations in the st14 gene coding for a serine protease matriptase , is characterized by the absence of the proteolytic activity of this type ii transmembrane enzyme ( chen et al . another inherited disease linked to various kinds of mutation of the matriptase gene and resulting in the total loss of the expression of the protein is ifah ( ichthyosis , follicular atrophoderma , hypotrichosis and hypohidrosis ; omim 602400 ; chen et al . clinical differences in phenotype between arih and ifah can possibly be explained by the presence of modified matriptase fragments in the former , in the light of evidence that reciprocal cross - activation of zymogen forms of matriptase and its downstream partner prostasin / prss8/cap-1 occurs and is independent of the activation state of the enzymes ( friis et al . because such enzymes have no obvious roles in the processing of profilaggrin , defects in the epidermal differentiation processes , even at early stages of differentiation , could affect downstream filaggrin processing . free amino acids originating from filaggrin degradation are the main contributors to the nmf ( natural moisturizing factor ) of the sc . therefore , the absence or largely reduced presence of filaggrin should impact nmf quantities and , consequently , sc hydration ( dapic et al . 1996 ) , one can hypothesize that low filaggrin levels influence the quality of these structures and , in this way , modify the fate of the intercellular lipid lamellae and corneodesmosomes . this is in stark contrast to the strips from normal subjects in which staining is present only at the lateral rims of corneocytes ( oyama et al . patterns of corneodesmosome distribution assessed with immunofluorescence by using antibodies to corneodesmosin , desmoglein 1 and desmocollin 1 are similar in atopic dermatitis and in ichthyosis vulgaris , although no information has been provided concerning the eventual existence of flg mutations in the former . this has confirmed the presence of wide - ranging defects in the cornification occurring in atopic dermatitis ( guttman - yassky et al . by the way , in netherton syndrome , which also presents a defective sc barrier function and atopy , the staining pattern is similar , although the corneocytes were stripped in an irregular way . these authors found a diffuse pattern of desmoglein 1 distribution all over the surface of corneocytes from the psoriatic scale , reminiscent of the above - described findings in ichthyosis vulgaris , suggesting that corneodesmosome retention at the ventral / dorsal surfaces of cells might not be pathognomonic but rather related to the relative this point of view seems to be strengthened by the biochemical analysis of the pattern of the cleavage of corneodesmosome proteins in psoriasis , as presented by simon et al . studies of dandruff have shown that the persistence of non - peripheral corneodesmosomes is a characteristic feature of the perturbed desquamation seen in this scalp affliction ( singh et al . the reported observations of the concomitant increased expression of lekti-1 and scca1 serine protease inhibitors are consistent with the view that the dandruff condition is characterized by an imbalance in protease / protease inhibitor interaction in the sc.fig . c post - embedding labeling with 10-nm gold granules on a vertical section of normal stratum corneum compactum as visualized by transmission electron microscopy ( arrows corneodesmosomes present both at the lateral and at the ventral / dorsal faces of the cells , sg stratum granulosum , sc1 , sc2 successive horny layers ) . c post - embedding labeling with 10-nm gold granules on a vertical section of normal stratum corneum compactum as visualized by transmission electron microscopy ( arrows corneodesmosomes present both at the lateral and at the ventral / dorsal faces of the cells , sg stratum granulosum , sc1 , sc2 successive horny layers ) . bars 50 m ( a ) , 20 m ( b ) , 200 nm ( c ) homozygous nonsense mutations in the corneodesmosin ( cdsn ) gene leading to the complete absence of the encoded protein or to residual expression of small non - functional fragments result in peeling skin disease ( psd ) , a generalized form of peeling skin syndrome , classified as inflammatory form b ( mim270300 ; oji et al . the extracellular portions of the corneodesmosomes prove less resistant to mechanical stress and are easily cleaved , especially at the bottom of the sc , at the interface with the granular layer . rupture of the permeability barrier results , in turn , in cytokine production by keratinocytes ( wood et al . a rescue response of the uppermost viable epidermal layers aimed at the re - establishment of the sc barrier is also induced . 1992 ) and the up - regulation of the tight junction structures , as documented by a significant increase in the tight junction remnants persisting in the sc of psd ( haftek et al . homozygous bi - allelic mutations of the desmoglein 1 gene ( dsg1 ) observed in rare consanguineous families also impact corneodesmosome function . although the main structural changes , such as irregular desmosome distribution , hypergranulosis with focal absence of the granular layer and widespread acantholysis within the stratum spinosum and granulosum , result in subcorneal and intragranular separation , a modified sc is also observed showing mixed ortho- and parakeratosis . corneodesmosome distribution also remains uneven and the disorder is characterized by compromised barrier function , which may expose the immune system to abnormal stimulation and lead to multiple allergies . as can be logically predicted , analogical sc defects involving the other desmosomal cadherin engaged in the process of sc cohesion , desmocollin 1 , the protease - antiprotease system efficiently regulates the normal process of sc formation and desquamation ( egelrud 2000 ; rawlings and voegeli 2013 ) . however , whether a common pathway and a coordinated regulation of their activity are involved in the terminal differentiation of epidermal keratinocytes remains unclear . it is caused by mutations in the spink5 gene encoding lekti 1 , a serine protease inhibitor ( hovnanian 2013 ) . an inverse pathomechanism takes place in recessive x - linked ichthyosis ( xli , mim 308100 ) in which deletions in the steroid sulfatase ( sts ) gene result in insufficiency of the enzyme and sc retention ( elias et al . steroid sulfatase is necessary for the conversion of cholesterol sulfate to cholesterol , a fundamental building brick of the intercellular lipid lamellae of sc . in xli , reduction in the cholesterol molecules within the horny layer and the accumulation of cholesterol sulfate provoke disequilibrium in the extracellular lipid species leading to phase separation and suboptimal barrier function . other mechanisms leading to corneodesmosome retention in xli include ( 1 ) low sc ph , out of the neutral to basic operating optima of kallikreins ( ohman and vahlquist 1998 ) and ( 2 ) the increased presence of ca in the intercellular domains of the lower part of ichthyotic sc possibly contributing to the stabilization of corneodesmosome attachments ( elias et al . autosomal recessive ichthyosis with hypotrichosis ( arih , omim 610765 ) , an inherited disorder linked to mutations in the st14 gene coding for a serine protease matriptase , is characterized by the absence of the proteolytic activity of this type ii transmembrane enzyme ( chen et al . another inherited disease linked to various kinds of mutation of the matriptase gene and resulting in the total loss of the expression of the protein is ifah ( ichthyosis , follicular atrophoderma , hypotrichosis and hypohidrosis ; omim 602400 ; chen et al . clinical differences in phenotype between arih and ifah can possibly be explained by the presence of modified matriptase fragments in the former , in the light of evidence that reciprocal cross - activation of zymogen forms of matriptase and its downstream partner prostasin / prss8/cap-1 occurs and is independent of the activation state of the enzymes ( friis et al . although the epidermal distribution of human and rodent matriptase diverges significantly ( chen et al . because such enzymes have no obvious roles in the processing of profilaggrin , defects in the epidermal differentiation processes , even at early stages of differentiation , could affect downstream filaggrin processing . free amino acids originating from filaggrin degradation are the main contributors to the nmf ( natural moisturizing factor ) of the sc . therefore , the absence or largely reduced presence of filaggrin should impact nmf quantities and , consequently , sc hydration ( dapic et al . 1996 ) , one can hypothesize that low filaggrin levels influence the quality of these structures and , in this way , modify the fate of the intercellular lipid lamellae and corneodesmosomes . this is in stark contrast to the strips from normal subjects in which staining is present only at the lateral rims of corneocytes ( oyama et al . this has confirmed the presence of wide - ranging defects in the cornification occurring in atopic dermatitis ( guttman - yassky et al . by the way , in netherton syndrome , which also presents a defective sc barrier function and atopy , the staining pattern is similar , although the corneocytes were stripped in an irregular way . these authors found a diffuse pattern of desmoglein 1 distribution all over the surface of corneocytes from the psoriatic scale , reminiscent of the above - described findings in ichthyosis vulgaris , suggesting that corneodesmosome retention at the ventral / dorsal surfaces of cells might not be pathognomonic but rather related to the relative this point of view seems to be strengthened by the biochemical analysis of the pattern of the cleavage of corneodesmosome proteins in psoriasis , as presented by simon et al . studies of dandruff have shown that the persistence of non - peripheral corneodesmosomes is a characteristic feature of the perturbed desquamation seen in this scalp affliction ( singh et al . the reported observations of the concomitant increased expression of lekti-1 and scca1 serine protease inhibitors are consistent with the view that the dandruff condition is characterized by an imbalance in protease / protease inhibitor interaction in the sc.fig . c post - embedding labeling with 10-nm gold granules on a vertical section of normal stratum corneum compactum as visualized by transmission electron microscopy ( arrows corneodesmosomes present both at the lateral and at the ventral / dorsal faces of the cells , sg stratum granulosum , sc1 , sc2 successive horny layers ) . c post - embedding labeling with 10-nm gold granules on a vertical section of normal stratum corneum compactum as visualized by transmission electron microscopy ( arrows corneodesmosomes present both at the lateral and at the ventral / dorsal faces of the cells , sg stratum granulosum , sc1 , sc2 successive horny layers ) . bars 50 m ( a ) , 20 m ( b ) , 200 nm ( c ) topical application of sodium lauryl sulfate ( sls ) detergent to the skin is known to disrupt sc function and is used as a reference in irritation tests in vivo . in soap - induced xerosis , non - peripheral corneodesmosomes also remain undegraded in the upper sc ( rawlings et al . human skin chronically exposed to low temperatures and dry air develops sc dryness characterized by roughness and a papyraceous appearance of the surface , the presence of raised squames and/or scales and irritation , commonly called winter xerosis . in this reactive condition , the persistence of both peripheral and non - peripheral corneodesmosomes in the upper sc has been observed ( simon et al . epidermal differentiation in palmar / plantar ridged skin represents a particular case , because here corneodesmosomes are not degraded in a pattern known from normal interfollicular epidermis but persist all around the corneocytes up to the surface ( mils et al . this occurs together with corneocyte accumulation in the ridged horny layer conferring it with more physical resistance . in the case of repeated mechanical stress , the palmar / plantar epidermis reacts with an additional accumulation of the sc , leading to the clinical appearance of calluses . these physiological features well demonstrate the importance of corneodesmosome junctions in the response of the sc to the environment . links between skin inflammation , protease / inhibitor balance , filaggrin processing , the composition and molecular arrangement of the extracellular lipid matrix , corneodesmosome - degradation

bacterial pathogens such as pseudomonas aeruginosa and staphylococcus aureus employ cell - to - cell communication or quorum sensing ( qs ) systems for coordinating collective activities that depend on the actions of one or more chemically distinct signal molecules . such molecules largely operate as effectors of qs - dependent gene expression through direct activation of membrane associated sensor kinases or via cytoplasmically located dna - binding proteins . they are also emerging as multifunctional signals that can influence interactions between different bacterial species and impact significantly the outcome of host pathogen interactions by also acting directly on the host . in p. aeruginosa , n-(3-oxododecanoyl)-l - homoserine lactone ( 3-oxo - c12-hsl , 1 ; table 1 ) activates the transcriptional regulator lasr to drive the expression of multiple qs target genes involved in exotoxin , exoenzyme and secondary metabolite production , as well as biofilm development . 3-oxo - c12-hsl also exerts a wide spectrum of other biological activities , for example , acting as a modulator of immune and inflammatory responses , impacting on the cardiovascular system , and disrupting epithelial barriers . as yet , the direct target(s ) for 3-oxo - c12-hsl in eukaryotic cells has not been identified . in lymphocytes , optimal immune suppressive activity is dependent on a c11c13 acyl chain containing a 3-oxo or a 3-hydroxy group . compounds lacking the l - configuration or with polar substituents were essentially devoid of activity . further modification of the 3-oxo - c12-hsl structure to generate compounds retaining the c12 acyl chain length while replacing the chiral homoserine lactone with more stable achiral heteroaryl moieties yielded compounds that possess similar immune suppressive activity to 3-oxo - c12-hsl but that inhibit rather than activate lasr in p. aeruginosa , thus indicating that at the molecular level it is possible to separate immune suppressive activity from qs activation . 3-oxo - c12-hsl also acts on other microorganisms , inhibiting filamentation in candida albicans(7 ) and the growth of gram - positive bacteria . at subgrowth inhibitory concentrations , 3-oxo - c12-hsl antagonizes the production of s. aureus exotoxins ( including -hemolysin , -hemolysin , and toxic shock syndrome toxin ) while enhancing cell wall protein biosynthesis including the fibronectin- and immunoglobulin - binding proteins . the mode of action of 3-oxo - c12-hsl in s. aureus appears to involve inhibition of agr - dependent qs , which reciprocally regulates exotoxins and cell wall colonization factors . the agr locus consists of two divergent transcriptional units , the p2 and p3 operons . the p2 operon consists of four genes , agrbdca , which are required for the activation of transcription from the p2 and p3 promoters , while the untranslated p3 transcript rnaiii is itself the effector for the agr response . agra and agrc constitute a two - component system in which agrc is the sensor kinase and agra is the response regulator . the system is activated by the interaction of agrc with a 7- to 9-mer macrocyclic - containing peptide termed the autoinducing peptide ( aip ) generated from the agrd gene product by agrb . since 3-oxo - c12-hsl binds to the s. aureus cytoplasmic membrane in a specific saturable manner , such membrane interactions may account for the agr inhibitory properties of 3-oxo - c12-hsl given the membrane localization of the agrb and agrc proteins . under aqueous alkaline conditions , 3-oxo - c12-hsl undergoes lactonolysis to form the corresponding ring - opened homoserine compound or an intramolecular rearrangement reaction to afford a vinylogous acid product , 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione [ ( s)-5-hydroxyethyl-3-decanoyltetramic acid ; 5-he - c10-tma , 5 ] ( see table 2 ) . this compound belongs to the tma class of compounds , such as reutericycline which display antibacterial properties . it is primarily active against gram - positive bacteria including bacillus , clostridium , and staphylococcus(8,13 ) where growth inhibition involves the dissipation of bacterial membrane potential and ph gradient . the physiological function of 5 in p. aeruginosa is not known , but it is capable of weakly inhibiting the lasr/3-oxo - c12-hsl - dependent activation of the elastase ( lasb ) gene and reducing p. aeruginosa viability . in contrast to 3-oxo - c12-hsl , 5 is also a ferric ion chelator . however , while it does not function as a siderophore for p. aeruginosa , iron was reported to abolish the antibacterial activity of 5 toward p. aeruginosa(15 ) and clostridium difficile . in common with 3-oxo - c12-hsl , 5 also exhibits immune suppressive activity in a murine peripheral blood lymphocyte proliferation assay and is cytotoxic toward jurkat but not bone - marrow - derived macrophage cells . given the threat posed by the emergence of bacterial strains resistant to conventional growth inhibitory antibiotics , there is renewed interest in the discovery of agents that control infection through the attenuation of bacterial virulence . this latter strategy has the potential advantage of expanding the repertoire of bacterial targets and exerting reduced selective pressure which , in turn , may retard the development of resistance . in this context , the development of strategies for inhibiting qs - dependent regulation of virulence has received significant attention particularly for problematic multiantibiotic resistant human pathogens such as s. aureus . here major efforts have been directed toward the inhibition of aip / agrc interactions to attenuate staphylococcal virulence largely through the synthesis and evaluation of aip mimetics . however , such aips have been reported to be susceptible to inactivation by host innate defenses . given our desire to discover new nonpeptidic agents that are capable of modulating the aip / agrc interaction , we have considered simple heterocyclic compounds . in this context , although a number of small molecules have been reported to cause blockade of the staphylococcal agr system , none of these compounds are known to directly modulate ligand / cognate receptor interactions . since we have previously shown that 3-oxo - c12-hsl can inhibit agr - dependent qs in s. aureus , a series of 3-oxo - c12-hsl , tma , and related toa analogues were synthesized and evaluated for their agr- and growth - inhibitory properties , structure in addition , the efficacy of the most potent toa analogue 17 was investigated in a mouse staphylococcal infection model . n - acyl - l - homoserine lactones ( ahls ) ( table 1 , 1 and 2 ; supporting information table s1 , s1s16 ) were synthesized using well - established methodologies . the synthesis of 3-oxo - c12-hsl 1 and its analogues has been previously reported by chhabra et al . and jadhav et al . similarly , the 3-acyl-5-(2-hydroxyethyl)tetramic acids 313 listed in table 2 were prepared from their respective n-3-oxoacyl - l - homoserine lactones by the method previously described by kaufmann et al . ( s)-3-decanoyl-5-methyltetramic acid 12 and 3-decanoyltetramic acid 13 were constructed by applying the strategy outlined in scheme s1 . thus , the appropriate n - boc--aminoacylated meldrum s acid , prepared by c - acylation of meldrum s acid with carbodiimide - activated n - boc - amino acid , was subjected to thermal cyclative elimination of me2co and co2 to yield the required 5-substituted n - boc - tetramic acid . subsequent acylation with activated decanoic acid followed by trifluoroacetic acid mediated acidolysis delivered ( s)-3-acyl-5-methyltetramic acid 12 . the 3-acyltetramic acid 13 was similarly obtained by using n - boc - glycine as the starting reagent . the 3-acyltetronic acids 1418 listed in table 3 were prepared by the general method of c - acylation of commercially available tetronic acid . to gain insights into the 3-oxo - c12-hsl structural requirements for staphylococcal agr inhibition and to discover quorum sensing inhibitors that do not impact on staphylococcal growth , systematic modification of 3-oxo - c12-hsl was carried out , focusing initially on the homoserine lactone ( i ) , 3-oxo substituent ( ii ) , acyl side chain ( iii ) , and amide ( iv ) structural units of the molecule ( figure s1 ) . seventeen analogues of 3-oxo - c12-hsl were synthesized and evaluated for inhibition of agr and bacterial growth ( table 1 and table s1 ) . while the l - isomer of 3-oxo - c12-hsl 1 inhibited agr with an ic50 of 22 6 m , the d - isomer 2 was approximately 2-fold less active ( ic50 of 37 9 m ) . however , neither the corresponding ring opened n-(3-oxododecanoyl)-l - homoserine s1 nor the heteroring truncated 3-oxododecanamide s2 inhibited agr . moreover , analogues in which the homoserine lactone moiety was modified by substitution with different heteroatoms or replaced with alternative heterocyclic ring systems failed to inhibit agr . removal or reduction of the 3-oxo substituent also abolished agr inhibitory activity ( table s1 ) . modification of the acyl chain by the incorporation of a double bond or partial replacement with phenyl or cyclohexyl substituents all resulted in the loss of agr inhibitory properties ( table s1 ) . apart from the two 3-oxo - c12-hsl isomers 1 and 2 , none of the other analogues examined inhibited bacterial growth at 100 m . taken together , these data show that subtle changes in 3-oxo - c12-hsl are sufficient to abolish qs and growth inhibitory properties . since 1 undergoes a base - catalyzed rearrangement to the tma 5 , we explored the agr inhibitory activities of tma analogues 313 ( table 2 ) by varying the 3-acyl chain length 38 , stereochemistry 9 , and substitution at the 5-position of the heterocyclic ring 12 and 13 . each of the tma analogues examined apart from 3 , 10 , and 11 inhibited agr ( table 2 ) , with the most active compound being 6 ( ic50 = 10 3 m ) . switching the c5 stereochemistry from ( s)-5 to ( r)-9 improved agr inhibitory activity by 1.5-fold , and replacement with me ( 12 ) or removal ( 13 ) of the 5-(2-hydroxyethyl ) substituent in 5 also resulted in enhanced agr inhibitory activity ( table 2 ) , thus indicating that the 5-position can withstand alteration . the asterisks indicate the following : , no growth inhibition up to 100 m ; , no inhibition of agr observed at concentrations up to 100 m . since tmas such as 5 are known to inhibit bacterial growth and since growth inhibition is an undesirable property for agents that attenuate virulence , we evaluated the growth inhibitory properties of each of the tma compounds synthesized . table 2 shows that the mic for the 3-oxo - c12-hsl - derived tma 5 was 100 m . while analogues with shorter 3-acyl chains ( 3 and 4 ) did not inhibit growth at this concentration , extension of the chain by one , two , or four carbons , 68 , respectively , substantially increased potency such that the c14 analogue 8 exhibited an 8-fold lower mic ( 12.5 m ) . these data are broadly in agreement with the staphylococcal mics for 5 and 7 reported by lowery et al . interestingly , the shorter chain compound 5-he - c4-tma 3 , which we found was not active against s. aureus , has been reported to kill p. aeruginosa(15 ) but not c. difficile . tmas with 3-acyl chains of 1014 carbons inhibited growth and agr with differences between the mic and ic50 values ranging from little different ( e.g. , 5-he - c14-tma 8) to 5-fold ( e.g. , 5-he - c11-tma 6 ) . compound 4 , 5-he - c8-tma , retained reasonable agr inhibitory activity ( 42 13 m ) and completely inhibited production of the agr - dependent exotoxin -hemolysin at 100 m without affecting staphylococcal growth ( figure s2 ) . it therefore offers a platform for the further improvement of agr antagonism independent of growth inhibition . the asterisk ( ) indicates no growth inhibition up to 100 m . tmas such as 5 are ferric ion chelators , and so it is possible that the ability to inhibit staphylococcal growth is , at least in part , a consequence of its ability to restrict the availability of an essential bacterial nutrient . since iron has been reported to abolish the antibacterial activity of 5 toward c. difficile and p. aeruginosa , we synthesized toa ( table 3 , 1418 ; table s2 , s17s24 ) variants of the tmas in which the ring nitrogen is replaced with oxygen . we predicted that the tetronic acid structure would not chelate iron , as it largely exists as a 4-enolic tautomer as opposed to the tma structure where the 3-exoenolic tautomer in ( z)-configuration predominates . the latter is in syn orientation with the 2-oxo group and thus can serve as a bidentate ligand for fe(iii ) binding . this was confirmed experimentally , and figure 1a shows that in contrast to the tma 5 , the toas 1416 with acyl chains ranging from c6 to c12 do not chelate ferric iron . when assayed for staphylococcal agr and growth inhibitory properties ( table 3 and table s2 ) , each of the toas apart from the c6-toa 14 exhibited similar activities to the tmas with potency increasing as a function of acyl chain length . for agr inhibition , c14-toa 17 was the most potent compound with an ic50 of 3 1 m , approximately 8 times lower than the mic ( 25 m ) . in fact , when s. aureus usa300 was exposed to 10 m c14-toa 17 , production of the agr - dependent exotoxin -hemolysin was substantially reduced but growth was unaffected ( figure s3 ) . this toa was also the most active agr inhibitor among the 3-oxo - c12-hsl , tma , and toa compounds evaluated in the present investigation . two additional toa analogues s18 and s19 incorporating an unsaturated acyl chain were also synthesized . while the presence of a trans double bond in s18 had little effect on activity ( cf . , 15 ) , it was almost abolished in the cis analogue s19 ( cf . , 16 ) . further modifications of the 3-acyl chain by introducing aryl substituents or 3-oxo substituent did not yield active analogues ( table s2 ) . taken together , these data demonstrate that toas can also be effective agr inhibitors and that iron chelation is not required for growth or agr inhibition . ( a ) iron - chelating properties of 5-he - c10-tma 5 and the c6- , c10- , and c12-toas 14 , 15 , 16 ( at 50 m ) as determined using the cas assay : positive control , desferrioxamine ( 10 m ) ; solvent control , mecn . changes in dipole potential were determined using di-8-anepps to measure the variation in the fluorescence ratio r(460/520 ) as a function of concentration . the binding profiles for 5-he - c10-tma 5 ( ) , c10-toa 15 ( ) , 5-he - c12-tma 7 ( ) , c12-toa 16 ( ) , 5-he - c14-tma 8 ( ) , and c14-toa 17 ( ) compared with 3-oxo - c12-hsl 1 ( ) are shown . since two of the major proteins involved in agr - dependent qs ( agrc and agrb ) are located in the cytoplasmic membrane , we sought to determine whether the tmas and toas bind directly to staphylococcal membranes . this was achieved using the fluorescent probe di-8-anepps in a dual - wavelength ratiometric method that detects changes in membrane dipole potential and yields binding information , such as affinity and overall binding capacity . figure 1b shows the binding profiles for the 5-he - c10- , 5-he - c12- , and 5-he - c14-tma and the c10- , c12 , and c14-toa compared with 3-oxo - c12-hsl 1 . for each of the 5-he - tmas 5 , 7 , 8 and toas 1517 , the data fit a hyperbolic function that is consistent with a noncooperative , single - site binding model ( figure 1b ) . the tmas and toas bind to the staphylococcal membrane with higher affinity than 3-oxo - c12-hsl with affinity increasing as chain length increases . in these experiments , kd values of 222 , 23 , and 2 m were estimated for the 5-he - c10 5 , 5-he - c12 7 , and 5-he - c14 8 tmas , respectively , compared with 270 m for 3-oxo - c12-hsl 1 . for the c10- , c12- , and c14-toas 1517 , the dissociation constants were similar to those of the 5-he - tmas , i.e. , 258 , 72 , and 4 m , respectively . from these tmas and toas , it is clear that the higher the membrane affinity , the more active is the compound against agr . 5-he - c10-tma 5 , and c12-toa 16 do not compete with aip-1 for cognate agrc . response curves showing the inhibition of a blaz - based agrp3 reporter by ( a ) 3-oxo - c12-hsl 1 , ( b ) 5-he - c10-tma 5 , ( c ) c12-toa 16 , and ( d ) the competitive antagonist ( ala5)aip-1 . consequently , we sought to determine whether 3-oxo - c12-hsl 1 , 5-he - c10-tma 5 , and c12-toa 16 are competitive inhibitors of the interaction between the s. aureus aip-1 and its cognate receptor agrc-1 . figure 2 shows that in contrast to the rightward shift in the concentration response curve for aip-1 ( i.e. , the ec50 of aip-1 increased by about 200-fold , from 5 nm to 1.10 m ) in the presence of increasing concentrations of the competitive agr inhibitor , ( ala5)aip-1 , neither 3-oxo - c12-hsl 1 , 5-he - c10-tma 5 , nor c12-toa 16 competitively inhibits the activation of agrc-1 by aip-1 . consequently , agr inhibition by these compounds is likely to involve an allosteric interaction with agrc , preventing efficient activation by the aip signal molecule . it is worth noting that c12-toa 16 is at least 5-fold more potent than 3-oxo - c12-hsl 1 and 5-he - c10-tma 5 in preventing the aip - mediated activation of agrc ( figure 2 ) . significantly , the three classes of negative allosteric modulators appeared to act by altering the efficacy of the natural ligand aip-1 with no noticeable effect on the affinity of aip-1 to cognate agrc receptor . for example , in the presence of increasing concentrations of 5-he - c10-tma 5 ( from 0 to 100 m ) , the maximum level of activation was decreased by over 10-fold while the ec50 of aip-1 remained broadly in the 24 nm range ( figure 2b ) . the consequence(s ) of the allosteric interaction mediated by these agr inhibitors could be prevention of agrc receptor dimerization or interference with the interactions between the agrc cytoplasmic domain and the response regulator protein agra which activates the agr p2 and p3 promoters . to date , we have not been able to express and purify functional recombinant agrc protein , and therefore , further elucidation of this inhibitory mechanism and/or binding mode awaits resolution of this technical hurdle . to determine whether the tmas and toas have potential as antistaphylococcal agents , we first examined the ability of the naturally produced p. aeruginosa tma , 5-he - c10-tma 5 , and the most potent toa c14-toa 17 to reduce the adherence of s. aureus to human nasal squamous cells , since a key risk factor for staphylococcal disease is nasal carriage . figure 3a shows that c14-toa 17 and 5-he - c10-tma 5 at 1 and 10 m , respectively , substantially reduced attachment to human squamous cells . impact of c14-toa 17 on adherence of s. aureus to desquamated human nasal epithelial cells and experimental infection . ( a ) binding of s. aureus to squamous epithelial cells before or after treatment with 5-he - c10-tma 5 ( a , 10 m ) or c14-toa 17 ( b , 1 m ) . ( b ) frequency of arthritis and ( c ) arthritic index of mice treated with 17 and challenged with s. aureus . white bars represent data from the control animals ( pbs treated ) and gray bars animals treated with 17 ( 10 mg / kg body weight ) . data are presented as median ( horizontal lines ) , interquartile ranges ( bars ) , and ranges ( error bars ) . fischer exact probability test was used to calculate statistical differences in the frequency of arthritis . to investigate the in vivo activity of c14-toa 17 and 5-he - c10-tma 5 , we used the established murine arthritis infection model . in these experiments , mice treated with c14-toa 17 the frequency and severity of induced arthritis were assessed over a 10 day period . the compound c14-toa 17 significantly reduced the frequency of arthritis and the arthritic index compared with the control group over the first 37 days ( figure 3b , c ) . while a reduction in synovitis and joint destruction was noted ( figure s4 ) , treatment with c14-toa 17 did not reduce weight loss or the numbers of viable staphylococci in the kidneys ( figure s4 ) . these results suggest that although 17 did not reduce bacterial growth in vivo at the dose tested , it exhibits antivirulence and/or anti - inflammatory activities in vivo which impact the course of staphylococcal disease . in contrast , 5-he - c10-tma 5 was inactive in this murine infection model ( data not shown ) . given the global health threat posed by multiantibiotic resistant bacteria , there is considerable interest in the discovery of antibacterial compounds that attenuate bacterial virulence rather than growth . in s. aureus , cyclic peptide mimetics of the native qs signal molecules have shown promise as agr - inhibitory virulence attenuators . these findings stimulated our search for simplified chemical structures capable of blocking agr - dependent qs . previously , we discovered that the p. aeruginosa signal molecule 3-oxo - c12-hsl 1 inhibited agr , and so we utilized this compound as a starting point to investigate the key structural features involved , mechanism of action , and in vivo efficacy . these subtle structural changes in 1 that resulted in the loss of qs inhibitory properties require further investigation and may involve differential access via the cell wall to the cytoplasmic membrane . fatty acids such as oleate ( nontoxic ) and palmitoleate ( growth inhibitor ) , for example , show structure - specific antistaphylococcal activity that depends on the cell wall teichoic acids . furthermore , we have identified two new related nonpeptidic classes of compounds , the tmas and toas , as noncompetitive agrc inhibitors that are more potent than 3-oxo - c12-hsl 1 . the most potent compound identified , c14-toa 17 , reduced colonization of human nasal epithelial cells and showed efficacy in a staphylococcal experimental mouse infection model without obvious toxicity . consequently , our findings offer additional opportunities to exploit this chemical architecture for the discovery of more potent analogues for probing agr - dependent qs and for evaluation as antivirulent agents . the chemical structures of the presented compounds were verified by h and c nmr and high - resolution mass spectrometry . the purity ( > 95% ) was established by rp - hplc and h nmr spectrometry . h nmr spectra were recorded in cdcl3 or dmso - d6 on a bruker avance-400 operating at 400 mhz . c nmr spectra were recorded on a bruker avance-400 or bruker avance(iii)-500 operating at 100 or 125 mhz , respectively . high - resolution electrospray ( es ) mass spectra were recorded using waters micromass lct spectrometer . analytical rp - hplc was used to establish purity and was performed using a waters setup comprising two 510 pumps , a 2487 dual absorbance detector , and millennium software . the separation was performed using a phenomenex onyx monolithic c18 column ( 4.6 mm 100 mm ) and a linear gradient of 3070% solvent b in 16.0 min , then 70100% b in 1.0 min at a flow rate of 3.0 ml / min . solvent a was 0.06% tfa in h2o , and solvent b was 0.06% tfa in mecn / h2o ( 90:10 ) . a solution of sodium methoxide in meoh ( 0.5 m , 2.0 ml , 1.0 mmol ) was added to a stirred solution of n-(3-oxoacyl)-l - homoserine lactone ( 1.0 mmol ) in meoh ( 3 ml ) under nitrogen . the mixture was stirred for 3 h at 55 c and then overnight at 50 c . the mixture was cooled to rt and then passed through an acidic ion - exchange resin ( dowex 50 wx2 - 200 ) . the eluents were combined and concentrated in vacuo to afford the tetramic acids as solids . using the above general procedure and n-(3-oxododecanoyl)-l - homoserine lactone gave the desired compound as a cream solid ( 68% ) . h nmr ( cdcl3 ) 0.90 ( 3h , t , me ) , 1.281.41 ( 12h , m , ( ch2)6me ) , 1.68 ( 2h , m , ch2(ch2)6me ) , 1.81 and 2.14 ( 2h , 2 m , ch2ch2oh ) , 2.87 ( 2h , m , ch2(ch2)7me ) , 3.844.01 ( 3h , m , ring ch and ch2ch2oh ) , 6.33 ( 1h , b , nh ) . c nmr ( cdcl3 ) 14.11 , 22.66 , 25.91 , 29.25 , 29.27 , 29.33 , 29.40 , 31.85 , 33.00 , 34.11 , 60.91 , 61.81 , 100.53 , 175.13 , 189.98 , 195.51 . es - ms m / z 298.2002 [ m + h ] , c16h28no4 requires 298.2018 . using the above procedure and n-(3-oxotetradecanoyl)-l - homoserine lactone gave 7 as an off - white solid ( 75% ) . h nmr ( cdcl3 ) 0.90 ( 3h , t , me ) , 1.281.40 ( 16h , m , ( ch2)8me ) , 1.68 ( 2h , m , ch2(ch2)8me ) , 1.83 and 2.15 ( 2h , 2 m , ch2ch2oh ) , 2.86 ( 2h , m , ch2(ch2)9me ) , 3.844.01 ( 3h , m , ring ch and ch2ch2oh ) , 6.33 ( 1h , b , nh ) . c nmr ( cdcl3 ) 14.12 , 22.69 , 23.36 , 25.91 , 28.99 , 29.28 , 29.33 , 29.44 , 29.53 , 29.60 , 29.80 , 31.91 , 32.96 , 34.09 , 60.96 , 61.85 , 65.95 , 100.57 , 175.10 , 189.94 , 195.47 . es - ms m / z 326.2345 [ m + h ] , c18h32no4 requires 326.2331 . n , n-dicyclohexylcarbodiimide ( 2.3 mmol ) was added to a stirred solution of an alkanoic acid ( 2.0 mmol ) and 4-(dimethylamino)pyridine ( 3.0 mmol ) in dry ch2cl2 ( 20 ml ) at rt under nitrogen atmosphere . tetronic acid ( 2.1 mmol ) was then added , and the mixture was stirred overnight at rt . the mixture was filtered , and the filtrate was extracted with 1 m aq hcl ( 2 10 ml ) . the organic extract was dried over mgso4 and concentrated in vacuo to dryness to give , following crystallization , the desired 3-acyltetronic acids . the use of dodecanoic acid in the above procedure gave 16 as a cream solid in 64% yield . 1.281.41 ( 16h , m , ( ch2)8me ) , 1.72 ( 2h , m , ch2(ch2)8me ) , 2.95 ( 2h , m , ch2(ch2)9me ) , 4.58 and 4.70 ( 2h , 2s , ring ch2 ) . c nmr ( cdcl3 ) 14.11 , 22.65 , 25.22 , 29.19 , 29.32 , 29.38 , 29.49 , 29.56 , 29.58 , 31.90 , 49.25 , 68.80 , 99.96 , 168.34 , 192.67 , 197.99 . es - ms m / z 281.1743 [ m h ] , c16h25o4 requires 281.1753 . the use of tetradecanoic acid in the above procedure gave 17 as a pale gray solid in 73% yield . h nmr ( cdcl3 ) 0.90 ( 3h , t , me ) , 1.281.41 ( 20h , m , ( ch2)10me ) , 1.72 ( 2h , m , ch2(ch2)10me ) , 2.95 ( 2h , m , ch2(ch2)11me ) , 4.58 and 4.70 ( 2h , 2s , ring ch2 ) . c nmr ( cdcl3 ) 14.12 , 22.69 , 24.92 , 25.59 , 29.19 , 29.22 , 29.35 , 29.39 , 29.57 , 29.64 , 29.66 , 31.92 , 38.73 , 68.16 , 128.81 , 177.17 , 192.31 , 197.88 . es - ms m / z 309.2061 [ m h ] , c18h29o4 requires 309.2066 . staphylococci were cultured in lb or mueller hinton ( mh ) or cygp broth and incubated with shaking at 37 c . for agr inhibition assays , the agrp3::blaz reporter strain s. aureus 6390b ( prn6683 ) was employed . growth curves in the presence or absence of test compounds were derived by determining optical density at 600 nm ( od600 ) over 18 h. the agr inhibition assays were carried out as described previously . briefly , s. aureus rn6390b ( prn6683 ) was grown at 37 c overnight in cygp containing 5 g / ml chloramphenicol . the culture was diluted 1/100 into fresh cygp and grown at 37 c with shaking to log phase ( od600 0.4 ) and used to inoculate a 96-well microtiter plate containing a range of concentrations of the agr activator aip-1 ( 0.01 nm to 10 m ) and the test compounds ( 0100 m ) . plates were incubated at 37 c for 55 min , and the reaction was quenched by the addition of 50 l of a 5 mm sodium azide solution in cygp broth . -lactamase activity was determined by adding 50 l of a 125 g / ml solution of the chromogenic nitrocefin . data were analyzed using the prism2 ( graphpad ) program to obtain ic50 values . bacteria were grown overnight with shaking ( 200 rpm ) at 37 c , diluted with fresh mh broth to 1 10 cells / ml , and dispensed into 96-well microtiter plates . each compound was evaluated in triplicate at concentrations from 0 to 100 m and each experiment repeated at least three times . after incubation at 37 c overnight , mics were determined by visual inspection and by measurement of od600 .

a series of 3-oxo - c12-hsl , tetramic acid , and tetronic acid analogues were synthesized to gain insights into the structural requirements for quorum sensing inhibition in staphylococcus aureus . compounds active against agr were noncompetitive inhibitors of the autoinducing peptide ( aip ) activated agrc receptor , by altering the activation efficacy of the cognate aip-1 . they appeared to act as negative allosteric modulators and are exemplified by 3-tetradecanoyltetronic acid 17 , which reduced nasal cell colonization and arthritis in a murine infection model .

Introduction Results and Discussion Conclusions Experimental Section

bacterial pathogens such as pseudomonas aeruginosa and staphylococcus aureus employ cell - to - cell communication or quorum sensing ( qs ) systems for coordinating collective activities that depend on the actions of one or more chemically distinct signal molecules . in p. aeruginosa , n-(3-oxododecanoyl)-l - homoserine lactone ( 3-oxo - c12-hsl , 1 ; table 1 ) activates the transcriptional regulator lasr to drive the expression of multiple qs target genes involved in exotoxin , exoenzyme and secondary metabolite production , as well as biofilm development . 3-oxo - c12-hsl also exerts a wide spectrum of other biological activities , for example , acting as a modulator of immune and inflammatory responses , impacting on the cardiovascular system , and disrupting epithelial barriers . as yet , the direct target(s ) for 3-oxo - c12-hsl in eukaryotic cells has not been identified . further modification of the 3-oxo - c12-hsl structure to generate compounds retaining the c12 acyl chain length while replacing the chiral homoserine lactone with more stable achiral heteroaryl moieties yielded compounds that possess similar immune suppressive activity to 3-oxo - c12-hsl but that inhibit rather than activate lasr in p. aeruginosa , thus indicating that at the molecular level it is possible to separate immune suppressive activity from qs activation . 3-oxo - c12-hsl also acts on other microorganisms , inhibiting filamentation in candida albicans(7 ) and the growth of gram - positive bacteria . at subgrowth inhibitory concentrations , 3-oxo - c12-hsl antagonizes the production of s. aureus exotoxins ( including -hemolysin , -hemolysin , and toxic shock syndrome toxin ) while enhancing cell wall protein biosynthesis including the fibronectin- and immunoglobulin - binding proteins . the mode of action of 3-oxo - c12-hsl in s. aureus appears to involve inhibition of agr - dependent qs , which reciprocally regulates exotoxins and cell wall colonization factors . the p2 operon consists of four genes , agrbdca , which are required for the activation of transcription from the p2 and p3 promoters , while the untranslated p3 transcript rnaiii is itself the effector for the agr response . the system is activated by the interaction of agrc with a 7- to 9-mer macrocyclic - containing peptide termed the autoinducing peptide ( aip ) generated from the agrd gene product by agrb . since 3-oxo - c12-hsl binds to the s. aureus cytoplasmic membrane in a specific saturable manner , such membrane interactions may account for the agr inhibitory properties of 3-oxo - c12-hsl given the membrane localization of the agrb and agrc proteins . under aqueous alkaline conditions , 3-oxo - c12-hsl undergoes lactonolysis to form the corresponding ring - opened homoserine compound or an intramolecular rearrangement reaction to afford a vinylogous acid product , 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione [ ( s)-5-hydroxyethyl-3-decanoyltetramic acid ; 5-he - c10-tma , 5 ] ( see table 2 ) . it is primarily active against gram - positive bacteria including bacillus , clostridium , and staphylococcus(8,13 ) where growth inhibition involves the dissipation of bacterial membrane potential and ph gradient . the physiological function of 5 in p. aeruginosa is not known , but it is capable of weakly inhibiting the lasr/3-oxo - c12-hsl - dependent activation of the elastase ( lasb ) gene and reducing p. aeruginosa viability . in contrast to 3-oxo - c12-hsl , 5 is also a ferric ion chelator . in common with 3-oxo - c12-hsl , 5 also exhibits immune suppressive activity in a murine peripheral blood lymphocyte proliferation assay and is cytotoxic toward jurkat but not bone - marrow - derived macrophage cells . since we have previously shown that 3-oxo - c12-hsl can inhibit agr - dependent qs in s. aureus , a series of 3-oxo - c12-hsl , tma , and related toa analogues were synthesized and evaluated for their agr- and growth - inhibitory properties , structure in addition , the efficacy of the most potent toa analogue 17 was investigated in a mouse staphylococcal infection model . n - acyl - l - homoserine lactones ( ahls ) ( table 1 , 1 and 2 ; supporting information table s1 , s1s16 ) were synthesized using well - established methodologies . the synthesis of 3-oxo - c12-hsl 1 and its analogues has been previously reported by chhabra et al . thus , the appropriate n - boc--aminoacylated meldrum s acid , prepared by c - acylation of meldrum s acid with carbodiimide - activated n - boc - amino acid , was subjected to thermal cyclative elimination of me2co and co2 to yield the required 5-substituted n - boc - tetramic acid . to gain insights into the 3-oxo - c12-hsl structural requirements for staphylococcal agr inhibition and to discover quorum sensing inhibitors that do not impact on staphylococcal growth , systematic modification of 3-oxo - c12-hsl was carried out , focusing initially on the homoserine lactone ( i ) , 3-oxo substituent ( ii ) , acyl side chain ( iii ) , and amide ( iv ) structural units of the molecule ( figure s1 ) . seventeen analogues of 3-oxo - c12-hsl were synthesized and evaluated for inhibition of agr and bacterial growth ( table 1 and table s1 ) . while the l - isomer of 3-oxo - c12-hsl 1 inhibited agr with an ic50 of 22 6 m , the d - isomer 2 was approximately 2-fold less active ( ic50 of 37 9 m ) . removal or reduction of the 3-oxo substituent also abolished agr inhibitory activity ( table s1 ) . modification of the acyl chain by the incorporation of a double bond or partial replacement with phenyl or cyclohexyl substituents all resulted in the loss of agr inhibitory properties ( table s1 ) . apart from the two 3-oxo - c12-hsl isomers 1 and 2 , none of the other analogues examined inhibited bacterial growth at 100 m . taken together , these data show that subtle changes in 3-oxo - c12-hsl are sufficient to abolish qs and growth inhibitory properties . since 1 undergoes a base - catalyzed rearrangement to the tma 5 , we explored the agr inhibitory activities of tma analogues 313 ( table 2 ) by varying the 3-acyl chain length 38 , stereochemistry 9 , and substitution at the 5-position of the heterocyclic ring 12 and 13 . each of the tma analogues examined apart from 3 , 10 , and 11 inhibited agr ( table 2 ) , with the most active compound being 6 ( ic50 = 10 3 m ) . switching the c5 stereochemistry from ( s)-5 to ( r)-9 improved agr inhibitory activity by 1.5-fold , and replacement with me ( 12 ) or removal ( 13 ) of the 5-(2-hydroxyethyl ) substituent in 5 also resulted in enhanced agr inhibitory activity ( table 2 ) , thus indicating that the 5-position can withstand alteration . table 2 shows that the mic for the 3-oxo - c12-hsl - derived tma 5 was 100 m . interestingly , the shorter chain compound 5-he - c4-tma 3 , which we found was not active against s. aureus , has been reported to kill p. aeruginosa(15 ) but not c. difficile . tmas such as 5 are ferric ion chelators , and so it is possible that the ability to inhibit staphylococcal growth is , at least in part , a consequence of its ability to restrict the availability of an essential bacterial nutrient . since iron has been reported to abolish the antibacterial activity of 5 toward c. difficile and p. aeruginosa , we synthesized toa ( table 3 , 1418 ; table s2 , s17s24 ) variants of the tmas in which the ring nitrogen is replaced with oxygen . this was confirmed experimentally , and figure 1a shows that in contrast to the tma 5 , the toas 1416 with acyl chains ranging from c6 to c12 do not chelate ferric iron . when assayed for staphylococcal agr and growth inhibitory properties ( table 3 and table s2 ) , each of the toas apart from the c6-toa 14 exhibited similar activities to the tmas with potency increasing as a function of acyl chain length . in fact , when s. aureus usa300 was exposed to 10 m c14-toa 17 , production of the agr - dependent exotoxin -hemolysin was substantially reduced but growth was unaffected ( figure s3 ) . this toa was also the most active agr inhibitor among the 3-oxo - c12-hsl , tma , and toa compounds evaluated in the present investigation . further modifications of the 3-acyl chain by introducing aryl substituents or 3-oxo substituent did not yield active analogues ( table s2 ) . ( a ) iron - chelating properties of 5-he - c10-tma 5 and the c6- , c10- , and c12-toas 14 , 15 , 16 ( at 50 m ) as determined using the cas assay : positive control , desferrioxamine ( 10 m ) ; solvent control , mecn . the binding profiles for 5-he - c10-tma 5 ( ) , c10-toa 15 ( ) , 5-he - c12-tma 7 ( ) , c12-toa 16 ( ) , 5-he - c14-tma 8 ( ) , and c14-toa 17 ( ) compared with 3-oxo - c12-hsl 1 ( ) are shown . this was achieved using the fluorescent probe di-8-anepps in a dual - wavelength ratiometric method that detects changes in membrane dipole potential and yields binding information , such as affinity and overall binding capacity . figure 1b shows the binding profiles for the 5-he - c10- , 5-he - c12- , and 5-he - c14-tma and the c10- , c12 , and c14-toa compared with 3-oxo - c12-hsl 1 . for each of the 5-he - tmas 5 , 7 , 8 and toas 1517 , the data fit a hyperbolic function that is consistent with a noncooperative , single - site binding model ( figure 1b ) . the tmas and toas bind to the staphylococcal membrane with higher affinity than 3-oxo - c12-hsl with affinity increasing as chain length increases . in these experiments , kd values of 222 , 23 , and 2 m were estimated for the 5-he - c10 5 , 5-he - c12 7 , and 5-he - c14 8 tmas , respectively , compared with 270 m for 3-oxo - c12-hsl 1 . for the c10- , c12- , and c14-toas 1517 , the dissociation constants were similar to those of the 5-he - tmas , i.e. , 258 , 72 , and 4 m , respectively . 5-he - c10-tma 5 , and c12-toa 16 do not compete with aip-1 for cognate agrc . response curves showing the inhibition of a blaz - based agrp3 reporter by ( a ) 3-oxo - c12-hsl 1 , ( b ) 5-he - c10-tma 5 , ( c ) c12-toa 16 , and ( d ) the competitive antagonist ( ala5)aip-1 . consequently , we sought to determine whether 3-oxo - c12-hsl 1 , 5-he - c10-tma 5 , and c12-toa 16 are competitive inhibitors of the interaction between the s. aureus aip-1 and its cognate receptor agrc-1 . , the ec50 of aip-1 increased by about 200-fold , from 5 nm to 1.10 m ) in the presence of increasing concentrations of the competitive agr inhibitor , ( ala5)aip-1 , neither 3-oxo - c12-hsl 1 , 5-he - c10-tma 5 , nor c12-toa 16 competitively inhibits the activation of agrc-1 by aip-1 . it is worth noting that c12-toa 16 is at least 5-fold more potent than 3-oxo - c12-hsl 1 and 5-he - c10-tma 5 in preventing the aip - mediated activation of agrc ( figure 2 ) . significantly , the three classes of negative allosteric modulators appeared to act by altering the efficacy of the natural ligand aip-1 with no noticeable effect on the affinity of aip-1 to cognate agrc receptor . the consequence(s ) of the allosteric interaction mediated by these agr inhibitors could be prevention of agrc receptor dimerization or interference with the interactions between the agrc cytoplasmic domain and the response regulator protein agra which activates the agr p2 and p3 promoters . to date , we have not been able to express and purify functional recombinant agrc protein , and therefore , further elucidation of this inhibitory mechanism and/or binding mode awaits resolution of this technical hurdle . to determine whether the tmas and toas have potential as antistaphylococcal agents , we first examined the ability of the naturally produced p. aeruginosa tma , 5-he - c10-tma 5 , and the most potent toa c14-toa 17 to reduce the adherence of s. aureus to human nasal squamous cells , since a key risk factor for staphylococcal disease is nasal carriage . in contrast , 5-he - c10-tma 5 was inactive in this murine infection model ( data not shown ) . previously , we discovered that the p. aeruginosa signal molecule 3-oxo - c12-hsl 1 inhibited agr , and so we utilized this compound as a starting point to investigate the key structural features involved , mechanism of action , and in vivo efficacy . furthermore , we have identified two new related nonpeptidic classes of compounds , the tmas and toas , as noncompetitive agrc inhibitors that are more potent than 3-oxo - c12-hsl 1 . the most potent compound identified , c14-toa 17 , reduced colonization of human nasal epithelial cells and showed efficacy in a staphylococcal experimental mouse infection model without obvious toxicity . the chemical structures of the presented compounds were verified by h and c nmr and high - resolution mass spectrometry . analytical rp - hplc was used to establish purity and was performed using a waters setup comprising two 510 pumps , a 2487 dual absorbance detector , and millennium software . solvent a was 0.06% tfa in h2o , and solvent b was 0.06% tfa in mecn / h2o ( 90:10 ) . tetronic acid ( 2.1 mmol ) was then added , and the mixture was stirred overnight at rt . the mixture was filtered , and the filtrate was extracted with 1 m aq hcl ( 2 10 ml ) . c nmr ( cdcl3 ) 14.12 , 22.69 , 24.92 , 25.59 , 29.19 , 29.22 , 29.35 , 29.39 , 29.57 , 29.64 , 29.66 , 31.92 , 38.73 , 68.16 , 128.81 , 177.17 , 192.31 , 197.88 . the culture was diluted 1/100 into fresh cygp and grown at 37 c with shaking to log phase ( od600 0.4 ) and used to inoculate a 96-well microtiter plate containing a range of concentrations of the agr activator aip-1 ( 0.01 nm to 10 m ) and the test compounds ( 0100 m ) . plates were incubated at 37 c for 55 min , and the reaction was quenched by the addition of 50 l of a 5 mm sodium azide solution in cygp broth . -lactamase activity was determined by adding 50 l of a 125 g / ml solution of the chromogenic nitrocefin . bacteria were grown overnight with shaking ( 200 rpm ) at 37 c , diluted with fresh mh broth to 1 10 cells / ml , and dispensed into 96-well microtiter plates .

entrainment in nature is accomplished by exposure of animals to the cycles of day and night , which consist of three powerful photic cues : the daylight , twilight transitions and the natural dim scotopic illumination . although the dim illumination failed to evoke any phase shifts or suppress the melatonin secretion in nocturnal rodents , it was regarded as a persuasive photic contributor capable of altering the basic properties of the circadian pacemaker . for example , the dimly lit nights modified the waveform of activity rhythms of the white - footed mice , bats and owl - monkeys . dim nocturnal illumination in hamsters extended the range of entrainment , increased the incident of dissociation of locomotor activity rhythm and showed the phase - dependent effects on reentrainment and masking . moreover , the continuous dim green light in hamsters lengthened the period of free - running rhythm and increased the duration of activity by ~3 h when compared with the hamsters in continuous complete darkness . the dim nocturnal irradiance of ~0.2 lx was reported to accelerate the reentrainment in the young and old hamsters following simulated advance and delay jetlags of 4 and 8 h. dimly - lit nights modified the basic properties of the pacemakers controlling the pupal eclosion and adult locomotor activity rhythms of a few insects too . for example , the dim natural and artificial nighttime illuminations altered the attributes of entrainment and free - running rhythm of the pupal eclosion of d. jambulina . in the same species , the nocturnal illumination at 0.0006 lx advanced the activity onset by several hours , extended the activity duration , enhanced the activity level and shortened the period of free - running rhythm . varying durations ( 915 h ) of the dim scotophase at 0.0006 lx modulated the fundamental properties of the circadian pacemakers that controlled the adult locomotor activity rhythm of d. jambulina . dim nocturnal irradiance in d. melanogaster also advanced the morning peak but delayed the evening peak of activity that rendered the flies completely nocturnal moreover , it also altered the clock protein rhythms in the pacemaker neurons of the brain . dim illumination even during the daytime had profound influence on the locomotor activity rhythms of a few insects . for instance , the dim photophase at 1 lx delayed the onset of the locomotor activity rhythm of the onion fly , delia antiqua and lengthened the period of free - running rhythm in continuous dim light . the dim photophase contrary to the bright one , enhanced the activity level and stimulated the behavioral components like the resting , grooming and feeding in d. melanogaster . thus , these studies revealed that the dim naturalistic light during the night or even during the day had multitude of effects on the circadian features of several mammals and insects . nevertheless , the influence of the dimly lit nights in accelerating the reentrainment following simulated jetlags has not been reported yet in any diurnal animal model . the objective of the present study was to examine the efficacy of the dimly lit nights in accelerating the reentrainment of the circadian rhythm of the adult locomotor activity of drosophila biarmipes , which is predominantly a diurnal insect in nature . this report is the part of ongoing investigation in the ability of the photic zeitgebers to influence the reentrainment following the simulated jetlags in d. biarmipes . recently , the authors have demonstrated that the bright photophase at 300 lx as compared with the dim photophase at 30 lx , speeded up the reentrainment in this species following the phase advance and delay of light - dark cycles . even the duration of photophase influenced the rate of reentrainment in d. biarmipes , however , it was dependent on the direction of the shifts in light - dark cycles . the short photophase of 9 h and the long photophase of 15 h accelerated the reentrainment following simulated advance and delay jetlags , respectively . the stock culture of the wild - type strain designated as md-101 of drosophila biarmipes was maintained at 21 0.5c and ~60% relative humidity ( r.h . ) in light - dark ( ld ) cycles of 12 h of white light at 300 lx and 12 h of complete darkness which are regarded as the standard ld cycles . the broad spectrum ( 365810 nm ) white light obtained from the uv - free white light emitting diodes ( 6 vdc-0.02w , the angle of light emission 2030 , ligitek electronics co. ) was used during the photophase at 300 lx as well as during the dim scotophase at 0.03 lx . the computerized photoelectric method for recording the adult locomotor activity rhythm of drosophila has been described in details . however , the method in brief is as follows . three units of the chronobiology kit ( stanford software systems , release version 1c , 19982004 - 62-channel ) were used for recording the activity rhythm and for the analysis of the circadian parameters . one - day old males were individually introduced in an activity recording glass tube ( 100 mm long 7 mm outer diameter ) . one end of the tube was inserted in 5 g of culture medium kept in a plastic container ( vol . 10 ml ) which served as the source of food and water for the fly . the other end of the tube was closed with a loose cotton - plug for aeration . this arrangement allowed the uninterrupted recording of the activity for ~10 d. thereafter , the fly was transferred to a fresh tube to continue the data recording . each tube was placed in the path of an infrared beam ( peak transmission of 738 nm ) of an activity recording device . twenty such activity recording devices were housed in each of 35 light - proof wooden - enclosures ( 60 80 90 cm ) kept in a room maintained at 21 0.5c and ~60% r.h . efficacy of the dim nocturnal illumination in accelerating the reentrainment was examined by subjecting the flies to two types of lighting schedules wherein the 12 h photophase was at 300 lx for all flies but the 12 h scotophase was at 0 l ( d ) for the control flies and 0.03 lx , i.e. , the artificial starlight ( s ) for the experimental flies . flies were maintained for two generations in each lighting schedule to avoid the after - effects of the standard ld cycles in which they were bred for several generations . two experiments were performed on the one - day old males ( n = 49 ) of the third generation in the same lighting regime in which they were maintained . the first experiment examined whether the synchronization of the control and experimental flies by ld 12:12 and ls 12:12 cycles , respectively , was the circadian entrainment or the masking effects of the scotopic illumination . this was accomplished by subjecting the flies to each lighting schedule for 10 d and thereafter , transferring them to continuous darkness ( dd ) that commenced at the lights - on phase of the day 11 . five parameters of entrainment were evaluated : the phase - angle difference between the activity onset ( aon ) phase of the morning ( m ) peak and the lights - on transition ( o ) , the phase - angle difference between the activity offset ( aoff ) phase of the evening ( e ) peak and the lights - off transition ( e ) , the phase - relationship between the aoff phase of the m peak and the aon phase of the e peak ( m - e ) , the duration of activity ( ) , and the duration of the rest ( ) . the mean activity profile during entrainment of 10 d in each lighting regime was presented in 0.25 h bins ( mean sd , n = 49 flies ) to distinguish the circadian peak ( the expected peak appearing before the lights - on or lights - off transition ) from the masking peak ( the short activity burst in response to the lights - on or lights - off transition ) . the entrainment was comprehensively determined by considering the three forms of stable phase relationships ( the o , m - e and e ) in each lighting schedule and the predictable phase ( the aon phase of the m peak of the last day of the entrainment ) from which the free - run commenced upon transfer to dd . activity level ( al ) was defined as the average number of activity passes per fly per day from the pooled data of 49 flies during the entrainment and the stable free - running state in dd . the period of free - running rhythm ( ) in dd was computed by the chi - square periodogram analysis ( step - size = 5 min ) . the second experiment examined the efficacy of the dim nocturnal irradiance in influencing the reentrainment . initially , the control and experimental flies were entrained for 10 d by ld 12:12 and ls 12:12 cycles , respectively . on day 11 , the lighting regime was abruptly phase advanced or phase delayed by shortening and lengthening of the scotophase by 8 h , respectively . each shifted lighting schedule was continued for 1823 d until the stable reentrainment was instituted . it took several days for the flies to re - entrain and the reentrainment was regarded to have accomplished when the three forms of the previous phase relationships ( the o , m - e and the e ) were reestablished in the shifted lighting schedule . transients were the temporary oscillatory states of the aon and aoff phases between the initial entrainment and the subsequent stable reentrainment . advancing and/or delaying transients endured by the aon and aoff phases were individually counted to distinguish the different rate of reentrainment of these two phase markers . there was always a single activity bout during 14 transients immediately following the advance or delay shift in the lighting schedule , therefore the offset of such single activity bout was regarded as the aoff phase of the prospective e peak of the rhythm . furthermore , two forms of transients were distinguished : the orthodromic form in which the transients were in a direction of the shifted ld cycle , and the antidromic form in which the transients were in a direction opposite to the shifted ld cycle . values of the parameters of entrainment and free - running rhythmicity were the means ( sd , n = 49 flies ) . the wilcoxon - mann - whitney test was performed on the activity data of both experiments to see any significant effect of the dim scotopic irradiance . the figure 1a and c show the actograms of the representative control and experimental flies of d. biarmipes entrained by the ld 12:12 and ls 12:12 cycles , respectively . all flies exhibited the bimodal activity pattern with the major m peak and the minor e peak as shown in their activity profiles ( fig . the control flies were characterized by the circadian m and e peaks as well as the masking peaks at the lights - on and lights - off transitions ( fig . the experimental flies too showed the circadian m and e peaks , in addition to the lights - on induced masking peak , nonetheless , the lights - off induced masking peak was perpetually missing ( fig . moreover , the aoff phase of the e peak always occurred just before the lights - off transitions since the activity was never extended in the early part of the dimly lit night . after - effects of the nocturnal illumination on were examined by transferring the flies from each lighting regime to dd that initiated the robust free - running rhythmicity following 2.4 0.8 delaying and 4.4 1.1 advancing transients in the control and experimental flies , respectively ( n = 49 flies in each lighting schedule ) . the bimodal activity pattern was never observed in any group of the flies once transferred to dd . the free - running rhythmicity was initiated from the aon phase of the last m peak of the entrained rhythm and not from the lights - on phase of the lighting schedule . moreover , the free - running rhythmicity was generated by the participation of the m peak only , as the e peak was clearly left out . all parameters of the entrainment and free - running rhythmicity of the experimental flies were significantly different from that of the control flies ( p < 0.001 ) ( table 1 ) . the actograms of the representative control and experimental males of d. biarmipes which were entrained by ld 12:12 ( a ) and ls 12:12 ( c ) cycles , respectively , and then transferred to dd on the day 11 ( oblique arrow ) . the mean activity profiles of 49 control ( b ) and experimental ( d ) males during the entrainment are showing the circadian peaks ( dark arrows ) , the masking peaks ( open arrows ) and the phase relationship between the aoff of the m peak and aon of the e peak ( m - e ) . the dark and open time bars denote the photophase ( 300 lx ) and scotophase ( 0 lx ) , respectively , the long dark time bars denote dd and the hatched portion of the time - bars denotes the dim nocturnal irradiance at 0.03 lx . values are given in hours ( mean sd , n = 49 flies ) . o , the phase angle difference between the activity onset and lights - on ; e , the phase angle difference between the activity offset and lights - off ; m - e , the phase relationship ( i.e. , the interval ) between the morning and evening activity peaks ; -ent , duration of activity during entrainment ; -ent , duration of rest during entrainment ; dd , duration of activity during free - run in dd ; -dd , duration of rest during free - run in dd ; , the period of free - running rhythm in dd ; al - dd , the activity level in dd . the reentrainment in both groups of the flies following the phase advance as well as phase delay of the lighting schedules was also mediated by the m peak only as the e peak was never involved ( fig . 2 ) . the actograms of the representative control and experimental flies following the phase advance of 8 h are presented in the figure 2a and b , respectively . the reentrainment of the aon and aoff phases of both groups of the flies differed with respect to the direction and number of transients ( fig . 3a ) . in both groups of the flies , the aon phase reentrained consistently by the orthodromic advancing transients , whereas the aoff phase initially endured the orthodromic advancing transients , and then the antidromic delaying transients ( fig . the actograms of the four representative males of d. biarmipes which were subjected to 8 h phase advance and delay of ld 12:12 cycles ( a and c , respectively ) and ls 12:12 cycles ( b and d , respectively ) . transients after advance and delay jetlags . advancing and delaying transients ( mean s.d . , n = 49 flies ) endured by the activity onsets ( aon - ld ) and offsets ( aoff - ld ) of the control males and that of the experimental males ( aon - ls and aoff - ls ) of d. biarmipes following the advance ( a ) and delay shift ( b ) in the lighting schedules . the asterisks denote the statistically significant effects of the dim nocturnal illumination ( p < 0.001 ) . the actograms of the representative control and experimental flies after the phase delay of 8 h are present in the figure 2c and d , respectively . both , the aon and aoff phases of the control and experimental flies reentrained consistently by the orthodromic delaying transients ( fig . the decompression of in all flies occurred when the morning activity bout was dissociated into two components . the first component reestablished its customary phase relationship with the lights - on phase , whereas the second component was delayed until it achieved the usual phase position with the lights - off event ( fig . the transients experienced by the aon and aoff phases of the experimental flies after the advance as well as the delay shifts in the lighting schedules were significantly less than that of the control flies ( p < 0.001 ) ( fig . moreover , the total number of transients endured by the aon phases of all flies of each group were always significantly less than the total number of transients endured by the aoff phases after the advance or delay shift in the lighting regime ( p < 0.001 ) . the synchronization of the activity rhythm of the control and experimental flies of d. biarmipes by the 24 h lighting schedules with completely dark nights ( fig . 1c ) , was the circadian entrainment indeed and not the masking effects of the scotopic lighting conditions . this could be corroborated by the distinctive o and e in each lighting regime , the origin of the free - running rhythmicity from the aon phase of the rhythm and not from the lights - on phase of the light - dark cycles , the undeniable presence of the transients following the transfer from the lighting regime to dd , and the characteristic in dd as the after - effect of the different photic ambience of the prior scotophase . the authors have also demonstrated such influence of the extremely dim nocturnal illumination at 0.0006 lx on the attributes of entrainment and the free - running rhythmicity of the locomotor activity of d. jambulina . although such nocturnal illumination was 50-times dimmer than that used in the present study , it advanced the activity onset in the ls 12:12 cycles and shortened the in subsequent dd . it would be interesting to examine the efficacy of such extremely dim nocturnal illumination in influencing the process of reentrainment in d. biarmipes , d. jambulina or a diurnal mammalian experimental model like the indian palm - squirrel . the present study comprehensively assessed the reentrainment by using five basic but often overlooked components of the entrainment : the aon and aoff phases as the rhythm markers , the m - e , and . except for the two studies on reentrainment , the activity onset phase had been used as a sole rhythm marker to assess the reentrainment of locomotor activity rhythm , however , this approach fell short of projecting the accurate behavior of the underlying pacemakers during the reentrainment process . these studies apparently overlooked the complex organization of the waveform of the locomotor activity rhythm with five indispensable components , which are prone to be modified by various features of the lighting regime . if the aon phase is regarded as the exclusive phase maker in the present study , then the reentrainment would be much faster than demonstrated here ( figs . 2 and 3 ) . melatonin ameliorates the jetlag - symptoms only after the phase advance of the ld cycle , whereas , the dim nocturnal illumination indisputably accelerates the reentrainment in d. biarmipes following both , the phase advance and delay of the lighting schedules ( fig . 2 ) . these results are in agreement with the acceleration of reentrainment in hamsters by the dim nocturnal irradiance . the possible mechanism for the acceleration of reentrainment in d. biarmipes could be the photic or non - photic . the photic mechanism envisaged the direct effect of the dimly lit nights on the pacemakers . dim scotopic illumination is documented to alter the circadian waveform of the activity rhythm by modulating the and thereby changing the coupling mechanism between the m and e oscillators . the dim nighttime illumination unequivocally altered the waveform of the activity rhythm of d. biarmipes as the of the experimental flies during both , the entrained and free - running states was considerably longer than that of the control flies ( table 1 ) . such change in the waveform of the activity rhythm apparently augmented the flexibility of the underlying pacemakers resulting in minimizing the desynchrony among the constituent oscillators following the abrupt shifts in the ls cycle . that obviously reduced the number of transients endured by the aon and aoff phases of the experimental flies . the existence of such a desynchrony among molecular components of the pacemakers controlling the locomotor activity rhythm of rodents was shown to prolong the reentrainment . the non - photic mechanism for accelerating the reentrainment in d. biarmipes would involve the indirect effects of the dim nights on the pacemakers . a remarkable increment in the activity level caused by the dimly lit nights might act as the potential non - photic input to the pacemaker . such a non - photic input although not induced by the dim light speeded up the resynchronization in rodents too . for instance , the activity increment immediately following the phase advance of the ld cycles resulted in an impressive acceleration of reentrainment in the hamsters . an increment in the activity level caused by the dim scotopic irradiance was demonstrated to act through a non - photic mechanism too that influenced the entrainment and phase resetting in hamsters . the dim scotopic illumination apparently promoted the dissociation of the activity rhythm by facilitating the non - photic mechanism as well as the photic mechanism in the hamsters subjected to the ldld 7:5:7:5 cycles . furthermore , the dim nocturnal irradiance influenced the reentrainment in the hamsters by altering the magnitude of the phase resetting caused either by the bright light or the non - photic input such as the cage - changes . the dim nocturnal illumination also augmented the activity level of the young hamsters but the possibility of its role as a non - photic zeitgeber to speed up the resynchronization was ruled out . since the old hamsters exposed to the dimly - lit nights reentrained always faster than those exposed to the dark - nights , despite the both groups of hamsters had a comparable activity level . that prompted the authors to investigate the influence of the activity increment caused by the physical exercise schedule on the rate of reentrainment in d. biarmipes ( sinam et al . . an impressive increment of about 29% in the activity level was observed in the dark - night exposed males ( n = 69 ) of d. biarmipes when subjected to 1 h bout of physical exercise immediately following the 6 h advance shift in the ld cycles . however , such increment in the activity level failed to accelerate the reentrainment . therefore , the effectiveness of the activity increment induced by the dim - nights as a non - photic input to the pacemakers to accelerate the reentrainment in d. biarmipes should be negated in the present study . other features of the photic zeitgeber were documented to accelerate the reentrainment of d. biarmipes , too . for instance , the bright photophase akin to the dim nocturnal illumination in the present study , indiscriminately accelerated the reentrainment in d. biarmipes , following shifts in ld cycles in both the directions . these results were ascribed to the enhanced zeitgeber strength of the bright photophase that reinforced the coupling mechanism of the aon and aoff phases with the lights - on and lights - off transitions , respectively . the duration of photophase also influenced the reentrainment in this species , nevertheless , it depended on the direction of the ld cycle shift . the short photophase accelerated the reentrainment only after the phase advance , whereas the long photophase accelerated the reentrainment only after the phase delay of the ld cycles . these results were attributed to the early aon phase and short of the flies exposed to the short photophase , and the opposite was true for the flies exposed to the long photophase . such studies may have the practical application as a non - drug treatment to minimize the jetlag problems , especially , among the frequent flyers like the cabin - crew , sportspersons , etc .

jetlag results from the misalignment between the endogenous circadian timing and the civil timing after a transmeridian flight . efficacy of the dim nocturnal illumination ( 0.03 lx ) in accelerating the reentrainment following simulated jetlags in drosophila biarmipes was examined by subjecting the flies to 24 h light - dark cycles in which the 12 h photophase was at 300 lx for all flies but the scotophase was at 0 and 0.03 lx for the control and experimental flies , respectively . reentrainment was always faster in the experimental flies than the control ones . moreover , unlike melatonin , the dimly lit nights accelerated the reentrainment following both , the phase advance and delay of the light - dark cycles . this study might have potential application as a non - drug jetlag treatment .

Introduction Methods Results Discussion

although the dim illumination failed to evoke any phase shifts or suppress the melatonin secretion in nocturnal rodents , it was regarded as a persuasive photic contributor capable of altering the basic properties of the circadian pacemaker . for example , the dimly lit nights modified the waveform of activity rhythms of the white - footed mice , bats and owl - monkeys . dim nocturnal illumination in hamsters extended the range of entrainment , increased the incident of dissociation of locomotor activity rhythm and showed the phase - dependent effects on reentrainment and masking . moreover , the continuous dim green light in hamsters lengthened the period of free - running rhythm and increased the duration of activity by ~3 h when compared with the hamsters in continuous complete darkness . the dim nocturnal irradiance of ~0.2 lx was reported to accelerate the reentrainment in the young and old hamsters following simulated advance and delay jetlags of 4 and 8 h. dimly - lit nights modified the basic properties of the pacemakers controlling the pupal eclosion and adult locomotor activity rhythms of a few insects too . for example , the dim natural and artificial nighttime illuminations altered the attributes of entrainment and free - running rhythm of the pupal eclosion of d. jambulina . in the same species , the nocturnal illumination at 0.0006 lx advanced the activity onset by several hours , extended the activity duration , enhanced the activity level and shortened the period of free - running rhythm . varying durations ( 915 h ) of the dim scotophase at 0.0006 lx modulated the fundamental properties of the circadian pacemakers that controlled the adult locomotor activity rhythm of d. jambulina . dim nocturnal irradiance in d. melanogaster also advanced the morning peak but delayed the evening peak of activity that rendered the flies completely nocturnal moreover , it also altered the clock protein rhythms in the pacemaker neurons of the brain . for instance , the dim photophase at 1 lx delayed the onset of the locomotor activity rhythm of the onion fly , delia antiqua and lengthened the period of free - running rhythm in continuous dim light . nevertheless , the influence of the dimly lit nights in accelerating the reentrainment following simulated jetlags has not been reported yet in any diurnal animal model . the objective of the present study was to examine the efficacy of the dimly lit nights in accelerating the reentrainment of the circadian rhythm of the adult locomotor activity of drosophila biarmipes , which is predominantly a diurnal insect in nature . this report is the part of ongoing investigation in the ability of the photic zeitgebers to influence the reentrainment following the simulated jetlags in d. biarmipes . recently , the authors have demonstrated that the bright photophase at 300 lx as compared with the dim photophase at 30 lx , speeded up the reentrainment in this species following the phase advance and delay of light - dark cycles . even the duration of photophase influenced the rate of reentrainment in d. biarmipes , however , it was dependent on the direction of the shifts in light - dark cycles . the short photophase of 9 h and the long photophase of 15 h accelerated the reentrainment following simulated advance and delay jetlags , respectively . the stock culture of the wild - type strain designated as md-101 of drosophila biarmipes was maintained at 21 0.5c and ~60% relative humidity ( r.h . ) in light - dark ( ld ) cycles of 12 h of white light at 300 lx and 12 h of complete darkness which are regarded as the standard ld cycles . the broad spectrum ( 365810 nm ) white light obtained from the uv - free white light emitting diodes ( 6 vdc-0.02w , the angle of light emission 2030 , ligitek electronics co. ) was used during the photophase at 300 lx as well as during the dim scotophase at 0.03 lx . three units of the chronobiology kit ( stanford software systems , release version 1c , 19982004 - 62-channel ) were used for recording the activity rhythm and for the analysis of the circadian parameters . this arrangement allowed the uninterrupted recording of the activity for ~10 d. thereafter , the fly was transferred to a fresh tube to continue the data recording . efficacy of the dim nocturnal illumination in accelerating the reentrainment was examined by subjecting the flies to two types of lighting schedules wherein the 12 h photophase was at 300 lx for all flies but the 12 h scotophase was at 0 l ( d ) for the control flies and 0.03 lx , i.e. , the artificial starlight ( s ) for the experimental flies . flies were maintained for two generations in each lighting schedule to avoid the after - effects of the standard ld cycles in which they were bred for several generations . two experiments were performed on the one - day old males ( n = 49 ) of the third generation in the same lighting regime in which they were maintained . the first experiment examined whether the synchronization of the control and experimental flies by ld 12:12 and ls 12:12 cycles , respectively , was the circadian entrainment or the masking effects of the scotopic illumination . this was accomplished by subjecting the flies to each lighting schedule for 10 d and thereafter , transferring them to continuous darkness ( dd ) that commenced at the lights - on phase of the day 11 . five parameters of entrainment were evaluated : the phase - angle difference between the activity onset ( aon ) phase of the morning ( m ) peak and the lights - on transition ( o ) , the phase - angle difference between the activity offset ( aoff ) phase of the evening ( e ) peak and the lights - off transition ( e ) , the phase - relationship between the aoff phase of the m peak and the aon phase of the e peak ( m - e ) , the duration of activity ( ) , and the duration of the rest ( ) . the entrainment was comprehensively determined by considering the three forms of stable phase relationships ( the o , m - e and e ) in each lighting schedule and the predictable phase ( the aon phase of the m peak of the last day of the entrainment ) from which the free - run commenced upon transfer to dd . the second experiment examined the efficacy of the dim nocturnal irradiance in influencing the reentrainment . initially , the control and experimental flies were entrained for 10 d by ld 12:12 and ls 12:12 cycles , respectively . on day 11 , the lighting regime was abruptly phase advanced or phase delayed by shortening and lengthening of the scotophase by 8 h , respectively . it took several days for the flies to re - entrain and the reentrainment was regarded to have accomplished when the three forms of the previous phase relationships ( the o , m - e and the e ) were reestablished in the shifted lighting schedule . transients were the temporary oscillatory states of the aon and aoff phases between the initial entrainment and the subsequent stable reentrainment . there was always a single activity bout during 14 transients immediately following the advance or delay shift in the lighting schedule , therefore the offset of such single activity bout was regarded as the aoff phase of the prospective e peak of the rhythm . furthermore , two forms of transients were distinguished : the orthodromic form in which the transients were in a direction of the shifted ld cycle , and the antidromic form in which the transients were in a direction opposite to the shifted ld cycle . the figure 1a and c show the actograms of the representative control and experimental flies of d. biarmipes entrained by the ld 12:12 and ls 12:12 cycles , respectively . the experimental flies too showed the circadian m and e peaks , in addition to the lights - on induced masking peak , nonetheless , the lights - off induced masking peak was perpetually missing ( fig . moreover , the aoff phase of the e peak always occurred just before the lights - off transitions since the activity was never extended in the early part of the dimly lit night . after - effects of the nocturnal illumination on were examined by transferring the flies from each lighting regime to dd that initiated the robust free - running rhythmicity following 2.4 0.8 delaying and 4.4 1.1 advancing transients in the control and experimental flies , respectively ( n = 49 flies in each lighting schedule ) . the bimodal activity pattern was never observed in any group of the flies once transferred to dd . the free - running rhythmicity was initiated from the aon phase of the last m peak of the entrained rhythm and not from the lights - on phase of the lighting schedule . moreover , the free - running rhythmicity was generated by the participation of the m peak only , as the e peak was clearly left out . all parameters of the entrainment and free - running rhythmicity of the experimental flies were significantly different from that of the control flies ( p < 0.001 ) ( table 1 ) . the actograms of the representative control and experimental males of d. biarmipes which were entrained by ld 12:12 ( a ) and ls 12:12 ( c ) cycles , respectively , and then transferred to dd on the day 11 ( oblique arrow ) . the mean activity profiles of 49 control ( b ) and experimental ( d ) males during the entrainment are showing the circadian peaks ( dark arrows ) , the masking peaks ( open arrows ) and the phase relationship between the aoff of the m peak and aon of the e peak ( m - e ) . the dark and open time bars denote the photophase ( 300 lx ) and scotophase ( 0 lx ) , respectively , the long dark time bars denote dd and the hatched portion of the time - bars denotes the dim nocturnal irradiance at 0.03 lx . o , the phase angle difference between the activity onset and lights - on ; e , the phase angle difference between the activity offset and lights - off ; m - e , the phase relationship ( i.e. , the interval ) between the morning and evening activity peaks ; -ent , duration of activity during entrainment ; -ent , duration of rest during entrainment ; dd , duration of activity during free - run in dd ; -dd , duration of rest during free - run in dd ; , the period of free - running rhythm in dd ; al - dd , the activity level in dd . the reentrainment in both groups of the flies following the phase advance as well as phase delay of the lighting schedules was also mediated by the m peak only as the e peak was never involved ( fig . the actograms of the representative control and experimental flies following the phase advance of 8 h are presented in the figure 2a and b , respectively . the reentrainment of the aon and aoff phases of both groups of the flies differed with respect to the direction and number of transients ( fig . in both groups of the flies , the aon phase reentrained consistently by the orthodromic advancing transients , whereas the aoff phase initially endured the orthodromic advancing transients , and then the antidromic delaying transients ( fig . the actograms of the four representative males of d. biarmipes which were subjected to 8 h phase advance and delay of ld 12:12 cycles ( a and c , respectively ) and ls 12:12 cycles ( b and d , respectively ) . , n = 49 flies ) endured by the activity onsets ( aon - ld ) and offsets ( aoff - ld ) of the control males and that of the experimental males ( aon - ls and aoff - ls ) of d. biarmipes following the advance ( a ) and delay shift ( b ) in the lighting schedules . the asterisks denote the statistically significant effects of the dim nocturnal illumination ( p < 0.001 ) . the actograms of the representative control and experimental flies after the phase delay of 8 h are present in the figure 2c and d , respectively . both , the aon and aoff phases of the control and experimental flies reentrained consistently by the orthodromic delaying transients ( fig . the transients experienced by the aon and aoff phases of the experimental flies after the advance as well as the delay shifts in the lighting schedules were significantly less than that of the control flies ( p < 0.001 ) ( fig . moreover , the total number of transients endured by the aon phases of all flies of each group were always significantly less than the total number of transients endured by the aoff phases after the advance or delay shift in the lighting regime ( p < 0.001 ) . the synchronization of the activity rhythm of the control and experimental flies of d. biarmipes by the 24 h lighting schedules with completely dark nights ( fig . this could be corroborated by the distinctive o and e in each lighting regime , the origin of the free - running rhythmicity from the aon phase of the rhythm and not from the lights - on phase of the light - dark cycles , the undeniable presence of the transients following the transfer from the lighting regime to dd , and the characteristic in dd as the after - effect of the different photic ambience of the prior scotophase . the authors have also demonstrated such influence of the extremely dim nocturnal illumination at 0.0006 lx on the attributes of entrainment and the free - running rhythmicity of the locomotor activity of d. jambulina . although such nocturnal illumination was 50-times dimmer than that used in the present study , it advanced the activity onset in the ls 12:12 cycles and shortened the in subsequent dd . it would be interesting to examine the efficacy of such extremely dim nocturnal illumination in influencing the process of reentrainment in d. biarmipes , d. jambulina or a diurnal mammalian experimental model like the indian palm - squirrel . the present study comprehensively assessed the reentrainment by using five basic but often overlooked components of the entrainment : the aon and aoff phases as the rhythm markers , the m - e , and . except for the two studies on reentrainment , the activity onset phase had been used as a sole rhythm marker to assess the reentrainment of locomotor activity rhythm , however , this approach fell short of projecting the accurate behavior of the underlying pacemakers during the reentrainment process . if the aon phase is regarded as the exclusive phase maker in the present study , then the reentrainment would be much faster than demonstrated here ( figs . melatonin ameliorates the jetlag - symptoms only after the phase advance of the ld cycle , whereas , the dim nocturnal illumination indisputably accelerates the reentrainment in d. biarmipes following both , the phase advance and delay of the lighting schedules ( fig . the possible mechanism for the acceleration of reentrainment in d. biarmipes could be the photic or non - photic . the photic mechanism envisaged the direct effect of the dimly lit nights on the pacemakers . dim scotopic illumination is documented to alter the circadian waveform of the activity rhythm by modulating the and thereby changing the coupling mechanism between the m and e oscillators . the dim nighttime illumination unequivocally altered the waveform of the activity rhythm of d. biarmipes as the of the experimental flies during both , the entrained and free - running states was considerably longer than that of the control flies ( table 1 ) . such change in the waveform of the activity rhythm apparently augmented the flexibility of the underlying pacemakers resulting in minimizing the desynchrony among the constituent oscillators following the abrupt shifts in the ls cycle . that obviously reduced the number of transients endured by the aon and aoff phases of the experimental flies . the existence of such a desynchrony among molecular components of the pacemakers controlling the locomotor activity rhythm of rodents was shown to prolong the reentrainment . the non - photic mechanism for accelerating the reentrainment in d. biarmipes would involve the indirect effects of the dim nights on the pacemakers . a remarkable increment in the activity level caused by the dimly lit nights might act as the potential non - photic input to the pacemaker . such a non - photic input although not induced by the dim light speeded up the resynchronization in rodents too . for instance , the activity increment immediately following the phase advance of the ld cycles resulted in an impressive acceleration of reentrainment in the hamsters . an increment in the activity level caused by the dim scotopic irradiance was demonstrated to act through a non - photic mechanism too that influenced the entrainment and phase resetting in hamsters . the dim scotopic illumination apparently promoted the dissociation of the activity rhythm by facilitating the non - photic mechanism as well as the photic mechanism in the hamsters subjected to the ldld 7:5:7:5 cycles . furthermore , the dim nocturnal irradiance influenced the reentrainment in the hamsters by altering the magnitude of the phase resetting caused either by the bright light or the non - photic input such as the cage - changes . the dim nocturnal illumination also augmented the activity level of the young hamsters but the possibility of its role as a non - photic zeitgeber to speed up the resynchronization was ruled out . since the old hamsters exposed to the dimly - lit nights reentrained always faster than those exposed to the dark - nights , despite the both groups of hamsters had a comparable activity level . however , such increment in the activity level failed to accelerate the reentrainment . therefore , the effectiveness of the activity increment induced by the dim - nights as a non - photic input to the pacemakers to accelerate the reentrainment in d. biarmipes should be negated in the present study . other features of the photic zeitgeber were documented to accelerate the reentrainment of d. biarmipes , too . for instance , the bright photophase akin to the dim nocturnal illumination in the present study , indiscriminately accelerated the reentrainment in d. biarmipes , following shifts in ld cycles in both the directions . these results were ascribed to the enhanced zeitgeber strength of the bright photophase that reinforced the coupling mechanism of the aon and aoff phases with the lights - on and lights - off transitions , respectively . the duration of photophase also influenced the reentrainment in this species , nevertheless , it depended on the direction of the ld cycle shift . the short photophase accelerated the reentrainment only after the phase advance , whereas the long photophase accelerated the reentrainment only after the phase delay of the ld cycles . these results were attributed to the early aon phase and short of the flies exposed to the short photophase , and the opposite was true for the flies exposed to the long photophase . such studies may have the practical application as a non - drug treatment to minimize the jetlag problems , especially , among the frequent flyers like the cabin - crew , sportspersons , etc .

we recently reported that concentrations of circulating tumor necrosis factor receptors 1 and 2 ( tnfr1 and tnfr2 ) are strong predictors of future progression to chronic kidney disease ( ckd ) 3 in patients with type 1 diabetes and microalbuminuria ( 1 ) . these biomarkers also predict the onset of end - stage renal disease ( esrd ) in patients with type 2 diabetes ( 2 ) . their effects are equivalent and are independent of traditional clinical characteristics measured at the beginning of follow - up such as estimated glomerular filtration rate ( egfr ) , urinary albumin / creatinine ratio ( acr ) , and glycated hemoglobin a1c ( hba1c ) . here we seek to examine the association of the concentration of circulating tnfr2 with risk of esrd in a different study group , namely patients with type 1 diabetes and persistent proteinuria , part of a previously described joslin proteinuria cohort ( 3 ) . in contrast to the previous studies where threshold - based outcomes such as ckd 3 or esrd were used , in this study we used rate of renal decline as the quantitative measure of intensity of disease process leading to esrd ( 4 ) . steeper rate of renal decline results in shorter time to onset of esrd . this quantitative approach helps us to overcome problems associated with variable renal function at entry and variable duration of follow - up in our cohort . esrd develops in 40% of type 1 diabetic patients with proteinuria after 15 years of follow - up ( 3 ) . as we recently demonstrated , the process of renal function loss leading to esrd is approximately linear and can be expressed as a constant rate of renal decline , or egfr slope ( 4 ) . thus , if the slope is known , we can estimate the time to esrd , conditionally on the level of renal function at the beginning of the follow - up ( 4 ) . in this study of the association between circulating tnfr2 and the rate of renal decline , we used serial creatinine based egfr obtained during follow - up together with information about time of onset of esrd . these two types of information ( longitudinal egfr data and time to esrd ) were combined in a joint longitudinal - survival model to estimate the rate of renal decline ( egfr loss ) and time to esrd , taking into account variable egfr at entry , variable duration of follow - up , and variable number of egfr estimates during follow - up ( 58 ) . the study group has been described previously ( 3 ) . in brief , the joslin proteinuria cohort ( n = 423 ) was ascertained between 1991 and 2004 in the population of adult type 1 diabetic patients receiving long - term care at the joslin clinic , boston , ma ( 3,500 adult patients ) ( 3 ) . all patients were caucasian with urinary albumin excretion within the proteinuria range in at least two out of the three consecutive determinations of the acr performed at the joslin clinic laboratory during a 2-year interval preceding enrollment into the study ( 3,4 ) . informed consent procedures and protocols for examinations were approved by the joslin institutional review board , as were methods for ascertaining dates of onset of esrd and death . descriptions of baseline and follow - up examinations , definitions of esrd , time of its onset , and natural history of esrd in this cohort have been reported previously ( 3,4 ) . out of 423 patients enrolled in the joslin proteinuria cohort , 349 patients were in ckd stage 13 at enrollment . in addition to collecting and storing baseline and follow - up serum samples , we retrieved 4,097 serum creatinine measurements performed in the joslin clinic laboratory for routine visits during follow - up . throughout the period of study , they were assayed by jaffe s modified picrate method on a ciba corning express plus chemistry analyzer ( medfield , ma ) . for 1,113 of these measurements ( 27% ) , serum obtained from the same blood draw as the sample submitted for assay in the joslin clinic laboratory was also submitted to the advanced research and diagnostic laboratory at the university of minnesota , where creatinine was measured with the roche enzymatic assay ( product 11775685 ) q8 on a roche / hitache mod p analyzer ( indianapolis , in ) , calibrated to be traceable to an isotope dilution mass spectrometry reference assay . these duplicate measurements were used to develop a conversion formula to calibrate the other joslin clinic laboratory results to the isotope dilution mass spectrometry traceable method , as previously described ( 4 ) . egfr was estimated using calibrated serum creatinine measurements and the ckd - epi formula ( 9 ) . biomarkers were measured in serum samples collected at the enrollment into the study and stored at 80c . tnfr2 was measured with an elisa assay ( catalog no . drt200 ; r&d systems , minneapolis , mn ) as described earlier ( 2 ) . in our previous study because serum concentrations of tnfr1 and tnfr2 are highly correlated and provide redundant information regarding prediction of ckd 3 or esrd , only the latter was measured in the current study . urinary albumin concentration was measured at the joslin clinic laboratory by immunonephelometry on a bn100 instrument with n albumin kits ( behring , somerville , nj ) and urinary creatinine concentration with jaffe 's modified picrate method on a ciba corning express plus chemistry analyzer , as previously described ( 4 ) . hba1c was measured at the joslin clinic laboratory in the 1990s with bio - rad hplc analyzer ( bio - rad laboratories , hercules , ca ) and in 20012004 with tosoh 2 + 2 hplc analyzer ( tosoh bioscience , south san francisco , ca ) . both methods were standardized to diabetes control and complications trial ( dcct ) and were consistent throughout the study period . first , we studied the association of serum tnfr2 with traditional clinical risk factors and egfr at study entry . we also estimated cumulative incidence of esrd during follow - up in quartiles of tnfr2 and tested differences between quartiles with a log - rank test . for descriptive purpose , we estimated renal function change as regression slope fitted to each patient s longitudinal egfr data and expressed it in ml / min/1.73 m / year . second , for measuring the strength of a covariate s association with renal function changes during follow - up , we applied a joint longitudinal - survival model ( described in detail in the supplementary data ) . in brief , this model overcomes the bias that would be present in overall estimates of the slopes in the cohort due to the truncated follow - up observations from patients with rapid egfr loss ( 11 ) . it uses both the longitudinal egfr data and observed times to esrd for the whole study group to obtain unbiased estimates of individual rates of renal decline ( egfr loss ) ( 58,11 ) . this is a novel approach to estimate the impact of a covariate ( such as tnfr2 ) on the rate of egfr loss , the most important manifestation of the underlying disease process leading to esrd . the approach also imputes a value of egfr at zero follow - up time ( intercept ) and a time to esrd ( whether observed or not ) . the method is an extension of mixed - effects models and involves two levels of data . first , longitudinal egfr data and observed times of onset of esrd serve to estimate patient - specific regression parameters . on the second level , statistical inferences are drawn about the magnitudes of the covariate associations with the rate of egfr loss . to minimize confusion between the least squares and joint model parameters , we refer to the least squares regression estimate as a slope of egfr and joint model estimate as a rate of renal decline . similarly , we refer to the observed egfr at entry to the study as entry egfr and refer to the joint model intercept as imputed baseline egfr . to assess performance of tnfr2 and other covariates in predicting time to esrd , we calculated c - index ( 12 ) using a score derived from the log times to event imputed by the joint model ( x ) with a sas macro developed in the mayo clinic ( 13 ) . to test interactions between tnfr2 and another risk factor , we used the joint model to compare the association of tnfr2 with renal outcomes across strata formed by the quartiles of the interacting variable using a likelihood ratio test . analyses were performed in sas for windows , version 9.3 ( sas institute , cary , nc ) . the study group has been described previously ( 3 ) . in brief , the joslin proteinuria cohort ( n = 423 ) was ascertained between 1991 and 2004 in the population of adult type 1 diabetic patients receiving long - term care at the joslin clinic , boston , ma ( 3,500 adult patients ) ( 3 ) . all patients were caucasian with urinary albumin excretion within the proteinuria range in at least two out of the three consecutive determinations of the acr performed at the joslin clinic laboratory during a 2-year interval preceding enrollment into the study ( 3,4 ) . informed consent procedures and protocols for examinations were approved by the joslin institutional review board , as were methods for ascertaining dates of onset of esrd and death . descriptions of baseline and follow - up examinations , definitions of esrd , time of its onset , and natural history of esrd in this cohort have been reported previously ( 3,4 ) . out of 423 patients enrolled in the joslin proteinuria cohort , 349 patients were in ckd stage 13 at enrollment . in addition to collecting and storing baseline and follow - up serum samples , we retrieved 4,097 serum creatinine measurements performed in the joslin clinic laboratory for routine visits during follow - up . throughout the period of study , they were assayed by jaffe s modified picrate method on a ciba corning express plus chemistry analyzer ( medfield , ma ) . for 1,113 of these measurements ( 27% ) , serum obtained from the same blood draw as the sample submitted for assay in the joslin clinic laboratory was also submitted to the advanced research and diagnostic laboratory at the university of minnesota , where creatinine was measured with the roche enzymatic assay ( product 11775685 ) q8 on a roche / hitache mod p analyzer ( indianapolis , in ) , calibrated to be traceable to an isotope dilution mass spectrometry reference assay . these duplicate measurements were used to develop a conversion formula to calibrate the other joslin clinic laboratory results to the isotope dilution mass spectrometry traceable method , as previously described ( 4 ) . egfr was estimated using calibrated serum creatinine measurements and the ckd - epi formula ( 9 ) . biomarkers were measured in serum samples collected at the enrollment into the study and stored at 80c . tnfr2 was measured with an elisa assay ( catalog no . drt200 ; r&d systems , minneapolis , mn ) as described earlier ( 2 ) . in our previous study , we found that storage time did not affect tnfr2 concentration ( 10 ) . because serum concentrations of tnfr1 and tnfr2 are highly correlated and provide redundant information regarding prediction of ckd 3 or esrd , only the latter was measured in the current study . urinary albumin concentration was measured at the joslin clinic laboratory by immunonephelometry on a bn100 instrument with n albumin kits ( behring , somerville , nj ) and urinary creatinine concentration with jaffe 's modified picrate method on a ciba corning express plus chemistry analyzer , as previously described ( 4 ) . hba1c was measured at the joslin clinic laboratory in the 1990s with bio - rad hplc analyzer ( bio - rad laboratories , hercules , ca ) and in 20012004 with tosoh 2 + 2 hplc analyzer ( tosoh bioscience , south san francisco , ca ) . both methods were standardized to diabetes control and complications trial ( dcct ) and were consistent throughout the study period . first , we studied the association of serum tnfr2 with traditional clinical risk factors and egfr at study entry . we also estimated cumulative incidence of esrd during follow - up in quartiles of tnfr2 and tested differences between quartiles with a log - rank test . for descriptive purpose , we estimated renal function change as regression slope fitted to each patient s longitudinal egfr data and expressed it in ml / min/1.73 m / year . second , for measuring the strength of a covariate s association with renal function changes during follow - up , we applied a joint longitudinal - survival model ( described in detail in the supplementary data ) . in brief , this model overcomes the bias that would be present in overall estimates of the slopes in the cohort due to the truncated follow - up observations from patients with rapid egfr loss ( 11 ) . it uses both the longitudinal egfr data and observed times to esrd for the whole study group to obtain unbiased estimates of individual rates of renal decline ( egfr loss ) ( 58,11 ) . this is a novel approach to estimate the impact of a covariate ( such as tnfr2 ) on the rate of egfr loss , the most important manifestation of the underlying disease process leading to esrd . the approach also imputes a value of egfr at zero follow - up time ( intercept ) and a time to esrd ( whether observed or not ) . the method is an extension of mixed - effects models and involves two levels of data . first , longitudinal egfr data and observed times of onset of esrd serve to estimate patient - specific regression parameters . on the second level , statistical inferences are drawn about the magnitudes of the covariate associations with the rate of egfr loss . to minimize confusion between the least squares and joint model parameters , we refer to the least squares regression estimate as a slope of egfr and joint model estimate as a rate of renal decline . entry egfr and refer to the joint model intercept as imputed baseline egfr . to assess performance of tnfr2 and other covariates in predicting time to esrd , we calculated c - index ( 12 ) using a score derived from the log times to event imputed by the joint model ( x ) with a sas macro developed in the mayo clinic ( 13 ) . to test interactions between tnfr2 and another risk factor , we used the joint model to compare the association of tnfr2 with renal outcomes across strata formed by the quartiles of the interacting variable using a likelihood ratio test . analyses were performed in sas for windows , version 9.3 ( sas institute , cary , nc ) . this study includes 349 patients with proteinuria and ckd stage 13 who were enrolled into the joslin proteinuria cohort ( 3 ) . median age was 38 years , diabetes duration 24 years , and bmi 25 kg / m . by design , their urinary albumin excretion was within the proteinuric range , and median entry egfr was 81 ml / min/1.73 m ( 55 , 104 ) . systolic and diastolic blood pressures were close to their therapeutic target ( medians 130 and 78 mmhg , respectively ) . ace inhibitors ( ace - i ) or angiotensin ii receptor blockers ( arb ) were already prescribed for 69% of them . this proportion is somewhat lower than is currently the case but reflects the fact that use of these medications was not widespread in the early 1990s when a large proportion of the study cohort entered follow - up . glycemic control was predominantly poor ; median hba1c was 8.9% ( 74 mmol / mol ) . the median of serum concentration of tnfr2 ( 25th and 75th percentiles ) was 4,415 pg / ml ( 3,497 , 5,777 ) at entry . clinical characteristics of the 349 patients in the joslin proteinuria cohort in ckd stages 13 at the study entry data are median ( 25th , 75th percentile ) or percent ( n ) unless otherwise indicated . * data are incidence rate per 100 person - years ( number of events ) . renal function changes during 518 years of follow - up were evaluated with the 4,097 creatinine determinations ( median seven per patient ) and assigned egfr = 10 ml / min/1.73 m at onset of esrd in 111 patients . using least square regression mean ( sd ) , egfr slope was 5.9 ( 8.2 ) ml / min/1.73 m / year . using the joint modeling , mean rate of renal decline was 5.2 ( 4.9 ) ml / min/1.73 m / year . the incidence rate of esrd was 3.9 per 100 person - years , and mortality rate unrelated to esrd was 0.9 per 100 person - years . neither hba1c nor blood pressure varied with increasing concentration of tnfr2 ; spearman correlation coefficients were 0.01 ( p = 0.87 ) and 0.10 ( p = 0.06 ) , respectively . the tnfr2 concentration was significantly ( p = 0.006 ) lower in the patients without renoprotective treatment ( medians : 3,883 vs. 4,574 pg / ml ) for reasons that are obscure because their physicians had no knowledge of their low tnfr2 . acr at entry increased ( r = 0.36 , p < 0.001 ) whereas entry egfr decreased ( r = 0.70 , p < 0.001 ) with increasing concentration of tnfr2 . 1 . after 12 years of follow - up , the risk was 14 , 30 , 35 , and 88% in the first through fourth quartiles , respectively ( p < 0.001 ) . subsequently , we used a joint longitudinal - survival model to examine the associations of clinical covariates at entry with the rate of egfr loss and with the imputed baseline egfr and time to esrd ( table 2 ) . in this analysis , serum tnfr2 was transformed in percentile ranks and scaled to one quartile units , as in our previous publications ( 1,2 ) , and acr was log transformed , such that 1 unit change corresponds to doubling of its value . estimates from univariate ( top ) and multivariate ( bottom ) joint models of the effects of tnfr2 and clinical characteristics on three components of renal decline : rate of egfr loss ( expressed in ml / min/1.73 m / year ) , egfr at baseline ( ml / min/1.73 m ) , and time to esrd ( percent change of time to esrd ) data are point estimates ( 95% cis ) . the primary outcome in the study , the rate of egfr loss , is in boldface . in univariate analysis , serum concentration of tnfr2 had a strong impact on the rate of renal decline . a one quartile increase in tnfr2 was associated with a 1.3 ml / min/1.73 m / year steeper rate of egfr loss and a 17.4 ml / min/1.73 m lower imputed baseline egfr ( p < 0.001 for each ) . together , the lower baseline and steeper rate of egfr loss shortened the imputed time to esrd by 38.4% ( p < 0.001 ) . doubling of acr was associated with a 1.7 ml / min/1.73 m / year steeper rate of egfr loss and a 6.9 ml / min/1.73 m lower imputed baseline egfr ( p < 0.001 for each ) . together , the lower baseline and steeper rate of egfr loss shortened the imputed time to esrd by 29.0% ( p < 0.001 ) . a 1% ( 11 mmol / mol ) increase in hba1c was associated with a 1.0 ml / min/1.73 m / year steeper rate of egfr loss ( p < 0.001 ) but was not associated with imputed baseline egfr . the result was a 10.4% shorter imputed time to esrd ( p < 0.001 ) . a counterintuitive effect of diabetes duration was observed on egfr loss . a 10-year increase in duration of diabetes was associated with a 0.8 ml / min/1.73 m / year ( p = 0.024 ) reduction in the rate of egfr loss , an 8.6 ml / min/1.73 m lower imputed baseline egfr ( p < 0.001 ) , and no reduction in imputed time to esrd . a 1 mmol / l increase in total serum cholesterol level was associated with a 1.0 ml / min/1.73 m faster rate of egfr loss ( p < 0.001 ) , no change in imputed baseline egfr , and 8.1% reduction in imputed time to esrd ( p = 0.026 ) . diastolic ( but not systolic ) blood pressure was also associated with a slightly steeper rate of egfr loss ( p = 0.014 ) but not with the imputed baseline egfr or time to esrd . renoprotective treatment was associated with diminished rate of egfr loss ( p = 0.021 ) but with significantly lower imputed baseline egfr ( p < 0.001 ) . other covariates measured at enrollment such as sex , age , bmi , and smoking did not have a significant impact on the rate of renal decline during follow - up ( data not shown ) . to help understand the results of the analyses described above , we use a simplified example with tnfr2 dichotomized by its median value . 2 . it illustrates relationships between imputed baseline egfr , rate of renal decline , and imputed time to esrd . the average rate of egfr loss in patients below median is 4.1 ml / min/1.73 m / year , and imputed baseline egfr and time to esrd are 96.4 ml / min/1.73 m and 20.2 years , respectively . in patients with tnfr2 above median , the rate of egfr loss is steeper ( 6.2 ml / min/1.73 m / year ) , their imputed baseline egfr lower ( 62.3 ml / min/1.73 m ) , and their imputed time to esrd substantially shorter ( 8.2 years ) . the differences in the joint model trajectories for patients above versus below the median tnfr2 are indicated in the figure with thin black lines . they are as follows : rate of egfr loss steeper by 2.1 ml / min/1.73 m / year , imputed baseline egfr lower by 34.1 ml / min/1.73 m ( p < 0.001 ) , and imputed time to esrd shortened by 59.3% , i.e. , 0.9 in log(time ) ( p in analyses from table 2 , the predictors are modeled as continuous variables ( except renoprotection at baseline ) . thick lines depict imputed egfr trajectories , and thin solid lines indicate a covariate s ( tnfr2 ) associations with imputed baseline egfr , rate of renal decline , and imputed time to esrd . thin interrupted line indicates ckd stage 5 . e , imputed time of esrd . in multivariate analysis , only three covariates were significantly associated with the rate of renal decline ( see lower part of table 2 ) . the changes of the rate of egfr loss associated with tnfr2 , acr , and hba1c were 0.8 , 1.3 , and 1.0 ml / min/1.73 m / year , respectively , and all were significant at p < 0.001 . tnfr2 was the only one of the three that remained significantly associated with imputed baseline egfr . the coefficient for its association remained unchanged in comparison with the univariate model ( 17.2 ml / min/1.73 m , p < 0.001 ) . the strong association of tnfr2 with the rate of egfr loss and imputed baseline egfr produced the largest change in imputed time to esrd , a reduction by 34.6% ( p < 0.001 ) . this was more than twice the magnitude of the associations of acr and hba1c with imputed time to esrd : reductions of 15.0 and 9.6% , respectively ( p < 0.001 for each ) . in addition to the analyses on the determinants of the rate of renal decline described above , we compared the predictive performances of tnfr2 , acr , and hba1c for discrimination between patients who developed and did not develop esrd during 518 years of follow - up . in this analysis , we used the c - index derived from the time to esrd component of the joint model . the highest discrimination in predicting time to esrd was provided by the serum concentration of tnfr2 ; the c - index from a single - variable model was 0.79 ( 95% ci 0.75 , 0.83 ) . the index was 0.72 ( 0.66 , 0.77 ) for acr and 0.62 ( 0.57 , 0.68 ) for hba1c . in the multivariate joint model presented in the lower portion of table 2 , tnfr2 , acr , and hba1c together had a c - index 0.86 ( 0.84 , 0.89 ) . the results of this analysis were comparable with our previous findings regarding determinants of progression to esrd in type 2 diabetes ( 2 ) . to evaluate whether the association of serum tnfr2 with the rate of egfr loss depended upon the value of egfr at entry , we tested the association for heterogeneity across strata of the study group defined by ckd stage . similarly , we tested for its heterogeneity across quartiles of other covariates ( hba1c and acr ) . the only statistically significant interaction ( p = 0.030 ) was that between tnfr2 and hba1c . 3 , where the study group was subdivided according to quartiles of tnfr2 and quartiles of hba1c . in the first quartile of hba1c , the relationship between tnfr2 and the rate egfr loss was weak ; the difference in mean rates of egfr loss between first and fourth quartiles of tnfr2 was only 1.9 ml / min/1.73 m / year . this difference increased in three successive quartiles to 3.0 , 5.4 , and 5.4 ml / min/1.73 m / year , respectively . expressed as linear trend across tnfr2 quartiles , the coefficients were 0.8 ( p = 0.157 ) , 1.1 ( p = 0.051 ) , 1.7 ( p = 0.001 ) , and 1.9 ( p < 0.001 ) in the first , second , third , and fourth quartiles of hba1c , respectively . equivalent results were obtained when we examined the effect of hba1c within quartiles of tnfr2 ( reading down the columns in fig . 2 rather than across the rows ) . mean and standard error of the rate of egfr loss ( joint model ) according to quartiles of tnfr2 within quartiles of hba1c . it is important to note that baseline tnfr2 and hba1c are independent ( spearman correlation coefficient 0.01 , p = 0.87 ) . the first , second , and third quartiles of the distribution of tnfr2 in patients with hba1c < 8.9% or < 74 mmol / mol ( median ) were 3,542 , 4,439 , and 5,725 pg / ml , nearly the same as in patients with hba1c 8.9% or 74 mmol / mol ( 3,481 , 4,409 , and 5,887 this study includes 349 patients with proteinuria and ckd stage 13 who were enrolled into the joslin proteinuria cohort ( 3 ) . median age was 38 years , diabetes duration 24 years , and bmi 25 kg / m . by design , their urinary albumin excretion was within the proteinuric range , and median entry egfr was 81 ml / min/1.73 m ( 55 , 104 ) . systolic and diastolic blood pressures were close to their therapeutic target ( medians 130 and 78 mmhg , respectively ) . ace inhibitors ( ace - i ) or angiotensin ii receptor blockers ( arb ) were already prescribed for 69% of them . this proportion is somewhat lower than is currently the case but reflects the fact that use of these medications was not widespread in the early 1990s when a large proportion of the study cohort entered follow - up . glycemic control was predominantly poor ; median hba1c was 8.9% ( 74 mmol / mol ) . the median of serum concentration of tnfr2 ( 25th and 75th percentiles ) was 4,415 pg / ml ( 3,497 , 5,777 ) at entry . clinical characteristics of the 349 patients in the joslin proteinuria cohort in ckd stages 13 at the study entry data are median ( 25th , 75th percentile ) or percent ( n ) unless otherwise indicated . * data are incidence rate per 100 person - years ( number of events ) . renal function changes during 518 years of follow - up were evaluated with the 4,097 creatinine determinations ( median seven per patient ) and assigned egfr = 10 ml / min/1.73 m at onset of esrd in 111 patients . using least square regression mean ( sd ) , egfr slope was 5.9 ( 8.2 ) ml / min/1.73 m / year . using the joint modeling , mean rate of renal decline was 5.2 ( 4.9 ) ml / min/1.73 m / year . the incidence rate of esrd was 3.9 per 100 person - years , and mortality rate unrelated to esrd was 0.9 per 100 person - years . neither hba1c nor blood pressure varied with increasing concentration of tnfr2 ; spearman correlation coefficients were 0.01 ( p = 0.87 ) and 0.10 ( p = 0.06 ) , respectively . the tnfr2 concentration was significantly ( p = 0.006 ) lower in the patients without renoprotective treatment ( medians : 3,883 vs. 4,574 pg / ml ) for reasons that are obscure because their physicians had no knowledge of their low tnfr2 . acr at entry increased ( r = 0.36 , p < 0.001 ) whereas entry egfr decreased ( r = 0.70 , p < 0.001 ) with increasing concentration of tnfr2 . , the risk was 14 , 30 , 35 , and 88% in the first through fourth quartiles , respectively ( p < 0.001 ) . subsequently , we used a joint longitudinal - survival model to examine the associations of clinical covariates at entry with the rate of egfr loss and with the imputed baseline egfr and time to esrd ( table 2 ) . in this analysis , serum tnfr2 was transformed in percentile ranks and scaled to one quartile units , as in our previous publications ( 1,2 ) , and acr was log transformed , such that 1 unit change corresponds to doubling of its value . estimates from univariate ( top ) and multivariate ( bottom ) joint models of the effects of tnfr2 and clinical characteristics on three components of renal decline : rate of egfr loss ( expressed in ml / min/1.73 m / year ) , egfr at baseline ( ml / min/1.73 m ) , and time to esrd ( percent change of time to esrd ) data are point estimates ( 95% cis ) . the primary outcome in the study , the rate of egfr loss , is in boldface . in univariate analysis , serum concentration of tnfr2 had a strong impact on the rate of renal decline . a one quartile increase in tnfr2 was associated with a 1.3 ml / min/1.73 m / year steeper rate of egfr loss and a 17.4 ml / min/1.73 m lower imputed baseline egfr ( p < 0.001 for each ) . together , the lower baseline and steeper rate of egfr loss shortened the imputed time to esrd by 38.4% ( p < 0.001 ) . doubling of acr was associated with a 1.7 ml / min/1.73 m / year steeper rate of egfr loss and a 6.9 ml / min/1.73 m lower imputed baseline egfr ( p < 0.001 for each ) . together , the lower baseline and steeper rate of egfr loss shortened the imputed time to esrd by 29.0% ( p a 1% ( 11 mmol / mol ) increase in hba1c was associated with a 1.0 ml / min/1.73 m / year steeper rate of egfr loss ( p < 0.001 ) but was not associated with imputed baseline egfr . the result was a 10.4% shorter imputed time to esrd ( p < 0.001 ) . a counterintuitive effect of diabetes duration was observed on egfr loss . a 10-year increase in duration of diabetes was associated with a 0.8 ml / min/1.73 m / year ( p = 0.024 ) reduction in the rate of egfr loss , an 8.6 ml / min/1.73 m lower imputed baseline egfr ( p < 0.001 ) , and no reduction in imputed time to esrd . a 1 mmol / l increase in total serum cholesterol level was associated with a 1.0 ml / min/1.73 m faster rate of egfr loss ( p < 0.001 ) , no change in imputed baseline egfr , and 8.1% reduction in imputed time to esrd ( p = 0.026 ) . diastolic ( but not systolic ) blood pressure was also associated with a slightly steeper rate of egfr loss ( p = 0.014 ) but not with the imputed baseline egfr or time to esrd . renoprotective treatment was associated with diminished rate of egfr loss ( p = 0.021 ) but with significantly lower imputed baseline egfr ( p < 0.001 ) . other covariates measured at enrollment such as sex , age , bmi , and smoking did not have a significant impact on the rate of renal decline during follow - up ( data not shown ) . to help understand the results of the analyses described above , we use a simplified example with tnfr2 dichotomized by its median value . 2 . it illustrates relationships between imputed baseline egfr , rate of renal decline , and imputed time to esrd . the average rate of egfr loss in patients below median is 4.1 ml / min/1.73 m / year , and imputed baseline egfr and time to esrd are 96.4 ml / min/1.73 m and 20.2 years , respectively . in patients with tnfr2 above median , the rate of egfr loss is steeper ( 6.2 ml / min/1.73 m / year ) , their imputed baseline egfr lower ( 62.3 ml / min/1.73 m ) , and their imputed time to esrd substantially shorter ( 8.2 years ) . the differences in the joint model trajectories for patients above versus below the median tnfr2 are indicated in the figure with thin black lines . they are as follows : rate of egfr loss steeper by 2.1 ml / min/1.73 m / year , imputed baseline egfr lower by 34.1 ml / min/1.73 m ( p < 0.001 ) , and imputed time to esrd shortened by 59.3% , i.e. , 0.9 in log(time ) ( p < 0.001 ) . in analyses from table 2 , thick lines depict imputed egfr trajectories , and thin solid lines indicate a covariate s ( tnfr2 ) associations with imputed baseline egfr , rate of renal decline , and imputed time to esrd . in multivariate analysis , only three covariates were significantly associated with the rate of renal decline ( see lower part of table 2 ) . the changes of the rate of egfr loss associated with tnfr2 , acr , and hba1c were 0.8 , 1.3 , and 1.0 ml / min/1.73 m / year , respectively , and all were significant at p < 0.001 . tnfr2 was the only one of the three that remained significantly associated with imputed baseline egfr . the coefficient for its association remained unchanged in comparison with the univariate model ( 17.2 ml / min/1.73 m , p < 0.001 ) . the strong association of tnfr2 with the rate of egfr loss and imputed baseline egfr produced the largest change in imputed time to esrd , a reduction by 34.6% ( p < 0.001 ) . this was more than twice the magnitude of the associations of acr and hba1c with imputed time to esrd : reductions of 15.0 and 9.6% , respectively ( p < 0.001 for each ) . in addition to the analyses on the determinants of the rate of renal decline described above , we compared the predictive performances of tnfr2 , acr , and hba1c for discrimination between patients who developed and did not develop esrd during 518 years of follow - up . in this analysis , we used the c - index derived from the time to esrd component of the joint model . the highest discrimination in predicting time to esrd was provided by the serum concentration of tnfr2 ; the c - index from a single - variable model was 0.79 ( 95% ci 0.75 , 0.83 ) . the index was 0.72 ( 0.66 , 0.77 ) for acr and 0.62 ( 0.57 , 0.68 ) for hba1c . in the multivariate joint model presented in the lower portion of table 2 , tnfr2 , acr , and hba1c together had a c - index 0.86 ( 0.84 , 0.89 ) . the results of this analysis were comparable with our previous findings regarding determinants of progression to esrd in type 2 diabetes ( 2 ) . to evaluate whether the association of serum tnfr2 with the rate of egfr loss depended upon the value of egfr at entry , we tested the association for heterogeneity across strata of the study group defined by ckd stage . similarly , we tested for its heterogeneity across quartiles of other covariates ( hba1c and acr ) . the only statistically significant interaction ( p = 0.030 ) was that between tnfr2 and hba1c . 3 , where the study group was subdivided according to quartiles of tnfr2 and quartiles of hba1c . in the first quartile of hba1c , the relationship between tnfr2 and the rate egfr loss was weak ; the difference in mean rates of egfr loss between first and fourth quartiles of tnfr2 was only 1.9 ml / min/1.73 m / year . this difference increased in three successive quartiles to 3.0 , 5.4 , and 5.4 ml / min/1.73 m / year , respectively . expressed as linear trend across tnfr2 quartiles , the coefficients were 0.8 ( p = 0.157 ) , 1.1 ( p = 0.051 ) , 1.7 ( p = 0.001 ) , and 1.9 ( p < 0.001 ) in the first , second , third , and fourth quartiles of hba1c , respectively . equivalent results were obtained when we examined the effect of hba1c within quartiles of tnfr2 ( reading down the columns in fig . 2 rather than across the rows ) . mean and standard error of the rate of egfr loss ( joint model ) according to quartiles of tnfr2 within quartiles of hba1c . it is important to note that baseline tnfr2 and hba1c are independent ( spearman correlation coefficient 0.01 , p = 0.87 ) . the first , second , and third quartiles of the distribution of tnfr2 in patients with hba1c < 8.9% or < 74 mmol / mol ( median ) were 3,542 , 4,439 , and 5,725 pg / ml , nearly the same as in patients with hba1c 8.9% or 74 mmol / mol ( 3,481 , 4,409 , and 5,887 the current study showed a strong association between a single baseline measurement of serum concentration of tnfr2 and the future rate of renal function decline in type 1 diabetic patients with proteinuria . it replicates and expands our previous findings of a strong association of circulating tnfrs with the risk of ckd stage 3 in patients with type 1 diabetes and microalbuminuria , and risk of esrd in patients with type 2 diabetes ( 1,2 ) . in contrast to the previous studies , this study focused on serum tnfr2 as a determinant of the rate of egfr loss , the most direct measure available of the intensity of the underlying disease process that leads to esrd . our analytical method , joint longitudinal - survival analysis , estimates the rate of egfr loss together with two other characteristics of the trajectory of renal decline during follow - up : the intercept ( imputed baseline egfr ) and the imputed time to onset of esrd . in both univariate and multivariate analyses , the serum concentration of tnfr2 is strongly associated with the rate of egfr loss , secondarily with the imputed baseline egfr ( a consequence of what damage has already accumulated ) and ultimately the imputed time to esrd . the strength of this association is unchanged by inclusion of acr and other clinical risk factors in the multivariate model , indicating that its role is independent of them . the mechanisms through which circulating levels of tnfrs might impact renal decline have been discussed in our previous publications ( 1,2 ) . urinary albumin excretion ( measured as acr ) is also associated with the rate of egfr loss , and although that association is attenuated in multivariate analysis with tnfr2 , it is not fully accounted for . regarding the respective roles of serum tnfrs and urinary albumin excretion , several aspects are known . in type 1 diabetic patients , circulating tnfr2 predicts the development of proteinuria , as shown in the dcct / epidemiology of diabetes interventions and complications ( edic ) study ( 14 ) . although that study has not yet examined its effect on renal decline , our recent study has shown that tnfr 1 and 2 are major determinants of early renal decline in type 1 diabetic patients with normoalbuminuria , as well as in those with microalbuminuria ( 15 ) and those with proteinuria ( current study ) . thus , one may conclude that a high serum concentration of tnfrs increases both urinary albumin excretion and risk of renal decline . regarding precedence , early renal decline is demonstrable before the onset of microalbuminuria and its progression to proteinuria ( 15 ) . therefore , the level of urinary albumin excretion can be considered more of an intermediate or accompanying phenotype rather than an independent determinant of renal decline . the effect of hba1c on the rate of egfr loss is strong and independent of other clinical risk factors . the fact that , unlike tnfr2 , it has no impact on the imputed baseline egfr suggests that these two determinants play different roles in the disease process leading to esrd . one plausible hypothesis accounting for this difference is that hba1c is an environmental exposure that comes into play only at specific stages of diabetic nephropathy . the decline itself is perhaps a consequence of a genetic susceptibility that is reflected in the serum concentration of tnfr2 . an exposure , such as hba1c , may vary over time , and the single baseline measurement may not reflect well its prior level or that level s influence on baseline egfr . in contrast , a constitutive susceptibility , such as tnfr2 , may be more constant and the baseline egfr will reflect its prior effect on the disease process . for example , patients with a high serum tnfr2 will have a lower baseline egfr resulting from the renal decline that took place before the study entry . despite different associations with the imputed baseline egfr , hba1c and tnfr2 had strong and similar associations with the rate of egfr loss , and these effects were synergistic . in patients with good and moderate glycemic control , the association of tnfr2 with egfr loss was much less than in patients with poor glycemic control . by the same token , the association of poor glycemic control with the rate of egfr loss was blunted in patients with low serum tnfr2 . in the remaining patients , i.e. , in those with poor glycemic control and high serum levels of tnfr2 , egfr loss was rapid and might lead to esrd within a short follow - up time . despite this strong finding , the biological interpretation of this synergism is not clear . changes in clinical covariates ( such as blood pressure , treatment with renoprotective drugs , and other characteristics ) that went unrecognized during follow - up may have contributed to egfr trajectories and limit the biological interpretation of our findings . nevertheless , this study provides an assessment that does not need qualification of the predictive ability of a one - time measurement of tnfr2 and hba1c together for identifying patients at high risk of rapid renal decline and rapid progression to esrd . our study design resembles the typical patient visit during which endocrinologists or nephrologists try to predict the future from laboratory results without knowing what pathogenetic mechanisms might be involved or what changes might occur before the outcome ( in this case esrd ) is reached many years in the future . tnfr2 is a strong determinant of renal decline in patients with type 1 diabetes and proteinuria . by combining this measurement with levels of hba1c patients at highest risk should be enrolled in therapeutic programs to retard the rapid rate of renal function loss . although such programs need to be developed , there is some evidence that in patients with proteinuria and high hba1c , significantly improved glycemic control maintained for 4 or more years can slow the rate of egfr loss and postpone the onset of esrd after a several - year lag time ( 16 ) . an important strength of our study and its novelty is the focus on the annual rate of egfr loss as the renal outcome measure . the joint longitudinal - survival model simultaneously uses information about temporal changes in egfr and the observed times to esrd to estimate this quantitative outcome . traditionally , observational studies and clinical trials have used threshold - based outcomes such as time to ckd stage 3 , time to a doubling of serum creatinine , or time to onset of esrd ( 17,18 ) . within the typical time horizon of a study ( several years ) , only a fraction , the highest - risk patients or those closest to the threshold , develop the outcome . they depend on measurements of single serum creatinine values , whose assay has considerable random variation . in addition , the precision of measurements of event times is a function of the frequency of study visits and missed appointments . the rate of egfr loss , on the other hand , is estimated from multiple egfr observations and is less sensitive to biologic and assay variation in serum creatinine , missed appointments , and frequency of study visits . first , we used serum creatinine based estimates of gfr , which are less accurate than direct measurements or estimates based on serum cystatin c. direct gfr measurements might increase the strength of the associations . second , we assessed exposure to hyperglycemia by the value of hba1c at entry to the study only . similarly , as for other risk markers , we considered only their value at entry . accounting for possible changes in these covariates before or after the start of follow - up , most likely , might have improved their strength as predictors of the rate of renal decline .

objectivewe studied the serum concentration of tumor necrosis factor receptor 2 ( tnfr2 ) and the rate of renal decline , a measure of the intensity of the disease process leading to end - stage renal disease ( esrd).research design and methodsa cohort of 349 type 1 diabetic patients with proteinuria was followed for 518 years . serum tnfr2 , glycated hemoglobin a1c ( hba1c ) , and other characteristics were measured at enrollment . we used a novel analytic approach , a joint longitudinal - survival model , fitted to serial estimates of glomerular filtration rate ( egfr ) based on serum creatinine ( median seven per patient ) and time to onset of esrd ( 112 patients ) to estimate the rate of renal decline ( egfr loss).resultsat enrollment , all patients had chronic kidney disease stage 13 . the mean ( sd ) rate of egfr loss during 518 years of follow - up was 5.2 ( 4.9 ) ml / min/1.73 m2/year . serum tnfr2 was the strongest determinant of renal decline and esrd risk ( c - index 0.79 ) . the rate of egfr loss became steeper with rising concentration of tnfr2 , and elevated hba1c augmented the strength of this association ( p = 0.030 for interaction ) . in patients with hba1c 10.1% ( 87 mmol / mol ) , the difference in the rate of egfr loss between the first and fourth quartiles of tnfr2 was 5.4 ml / min/1.73 m2/year , whereas it was only 1.9 in those with hba1c < 7.9% ( 63 mmol / mol).conclusionscirculating tnfr2 is a major determinant of renal decline in patients with type 1 diabetes and proteinuria . elevated hba1c magnifies its effect . although the mechanisms of this synergism are unknown , our findings allow us to stratify patients according to risk of esrd .

Introduction Research Design and Methods Study Group Longitudinal Observation of Renal Decline Biomarker Assays Statistical Analysis Results Clinical Characteristics of the Study Group Association of Serum Levels of TNFR2 With Clinical Characteristics and Risk of ESRD Univariate Joint Longitudinal-Survival Analysis of Serum Level of TNFR2 and Other Covariates on Rate of Renal Decline Multivariate Joint Longitudinal-Survival Analysis of Serum Level of TNFR2 and Other Covariates on Rate of Renal Decline Search for Modifiers of the Association of TNFR2 With the Rate of eGFR Loss Conclusions Supplementary Material

we recently reported that concentrations of circulating tumor necrosis factor receptors 1 and 2 ( tnfr1 and tnfr2 ) are strong predictors of future progression to chronic kidney disease ( ckd ) 3 in patients with type 1 diabetes and microalbuminuria ( 1 ) . these biomarkers also predict the onset of end - stage renal disease ( esrd ) in patients with type 2 diabetes ( 2 ) . their effects are equivalent and are independent of traditional clinical characteristics measured at the beginning of follow - up such as estimated glomerular filtration rate ( egfr ) , urinary albumin / creatinine ratio ( acr ) , and glycated hemoglobin a1c ( hba1c ) . here we seek to examine the association of the concentration of circulating tnfr2 with risk of esrd in a different study group , namely patients with type 1 diabetes and persistent proteinuria , part of a previously described joslin proteinuria cohort ( 3 ) . in contrast to the previous studies where threshold - based outcomes such as ckd 3 or esrd were used , in this study we used rate of renal decline as the quantitative measure of intensity of disease process leading to esrd ( 4 ) . steeper rate of renal decline results in shorter time to onset of esrd . esrd develops in 40% of type 1 diabetic patients with proteinuria after 15 years of follow - up ( 3 ) . as we recently demonstrated , the process of renal function loss leading to esrd is approximately linear and can be expressed as a constant rate of renal decline , or egfr slope ( 4 ) . thus , if the slope is known , we can estimate the time to esrd , conditionally on the level of renal function at the beginning of the follow - up ( 4 ) . in this study of the association between circulating tnfr2 and the rate of renal decline , we used serial creatinine based egfr obtained during follow - up together with information about time of onset of esrd . these two types of information ( longitudinal egfr data and time to esrd ) were combined in a joint longitudinal - survival model to estimate the rate of renal decline ( egfr loss ) and time to esrd , taking into account variable egfr at entry , variable duration of follow - up , and variable number of egfr estimates during follow - up ( 58 ) . second , for measuring the strength of a covariate s association with renal function changes during follow - up , we applied a joint longitudinal - survival model ( described in detail in the supplementary data ) . this is a novel approach to estimate the impact of a covariate ( such as tnfr2 ) on the rate of egfr loss , the most important manifestation of the underlying disease process leading to esrd . second , for measuring the strength of a covariate s association with renal function changes during follow - up , we applied a joint longitudinal - survival model ( described in detail in the supplementary data ) . in brief , this model overcomes the bias that would be present in overall estimates of the slopes in the cohort due to the truncated follow - up observations from patients with rapid egfr loss ( 11 ) . this is a novel approach to estimate the impact of a covariate ( such as tnfr2 ) on the rate of egfr loss , the most important manifestation of the underlying disease process leading to esrd . renal function changes during 518 years of follow - up were evaluated with the 4,097 creatinine determinations ( median seven per patient ) and assigned egfr = 10 ml / min/1.73 m at onset of esrd in 111 patients . using least square regression mean ( sd ) , egfr slope was 5.9 ( 8.2 ) ml / min/1.73 m / year . using the joint modeling , mean rate of renal decline was 5.2 ( 4.9 ) ml / min/1.73 m / year . neither hba1c nor blood pressure varied with increasing concentration of tnfr2 ; spearman correlation coefficients were 0.01 ( p = 0.87 ) and 0.10 ( p = 0.06 ) , respectively . after 12 years of follow - up , the risk was 14 , 30 , 35 , and 88% in the first through fourth quartiles , respectively ( p < 0.001 ) . subsequently , we used a joint longitudinal - survival model to examine the associations of clinical covariates at entry with the rate of egfr loss and with the imputed baseline egfr and time to esrd ( table 2 ) . estimates from univariate ( top ) and multivariate ( bottom ) joint models of the effects of tnfr2 and clinical characteristics on three components of renal decline : rate of egfr loss ( expressed in ml / min/1.73 m / year ) , egfr at baseline ( ml / min/1.73 m ) , and time to esrd ( percent change of time to esrd ) data are point estimates ( 95% cis ) . a one quartile increase in tnfr2 was associated with a 1.3 ml / min/1.73 m / year steeper rate of egfr loss and a 17.4 ml / min/1.73 m lower imputed baseline egfr ( p < 0.001 for each ) . doubling of acr was associated with a 1.7 ml / min/1.73 m / year steeper rate of egfr loss and a 6.9 ml / min/1.73 m lower imputed baseline egfr ( p < 0.001 for each ) . a 1% ( 11 mmol / mol ) increase in hba1c was associated with a 1.0 ml / min/1.73 m / year steeper rate of egfr loss ( p < 0.001 ) but was not associated with imputed baseline egfr . a 10-year increase in duration of diabetes was associated with a 0.8 ml / min/1.73 m / year ( p = 0.024 ) reduction in the rate of egfr loss , an 8.6 ml / min/1.73 m lower imputed baseline egfr ( p < 0.001 ) , and no reduction in imputed time to esrd . a 1 mmol / l increase in total serum cholesterol level was associated with a 1.0 ml / min/1.73 m faster rate of egfr loss ( p < 0.001 ) , no change in imputed baseline egfr , and 8.1% reduction in imputed time to esrd ( p = 0.026 ) . other covariates measured at enrollment such as sex , age , bmi , and smoking did not have a significant impact on the rate of renal decline during follow - up ( data not shown ) . the average rate of egfr loss in patients below median is 4.1 ml / min/1.73 m / year , and imputed baseline egfr and time to esrd are 96.4 ml / min/1.73 m and 20.2 years , respectively . in patients with tnfr2 above median , the rate of egfr loss is steeper ( 6.2 ml / min/1.73 m / year ) , their imputed baseline egfr lower ( 62.3 ml / min/1.73 m ) , and their imputed time to esrd substantially shorter ( 8.2 years ) . they are as follows : rate of egfr loss steeper by 2.1 ml / min/1.73 m / year , imputed baseline egfr lower by 34.1 ml / min/1.73 m ( p < 0.001 ) , and imputed time to esrd shortened by 59.3% , i.e. thick lines depict imputed egfr trajectories , and thin solid lines indicate a covariate s ( tnfr2 ) associations with imputed baseline egfr , rate of renal decline , and imputed time to esrd . the changes of the rate of egfr loss associated with tnfr2 , acr , and hba1c were 0.8 , 1.3 , and 1.0 ml / min/1.73 m / year , respectively , and all were significant at p < 0.001 . the strong association of tnfr2 with the rate of egfr loss and imputed baseline egfr produced the largest change in imputed time to esrd , a reduction by 34.6% ( p < 0.001 ) . in addition to the analyses on the determinants of the rate of renal decline described above , we compared the predictive performances of tnfr2 , acr , and hba1c for discrimination between patients who developed and did not develop esrd during 518 years of follow - up . the highest discrimination in predicting time to esrd was provided by the serum concentration of tnfr2 ; the c - index from a single - variable model was 0.79 ( 95% ci 0.75 , 0.83 ) . in the first quartile of hba1c , the relationship between tnfr2 and the rate egfr loss was weak ; the difference in mean rates of egfr loss between first and fourth quartiles of tnfr2 was only 1.9 ml / min/1.73 m / year . expressed as linear trend across tnfr2 quartiles , the coefficients were 0.8 ( p = 0.157 ) , 1.1 ( p = 0.051 ) , 1.7 ( p = 0.001 ) , and 1.9 ( p < 0.001 ) in the first , second , third , and fourth quartiles of hba1c , respectively . mean and standard error of the rate of egfr loss ( joint model ) according to quartiles of tnfr2 within quartiles of hba1c . the first , second , and third quartiles of the distribution of tnfr2 in patients with hba1c < 8.9% or < 74 mmol / mol ( median ) were 3,542 , 4,439 , and 5,725 pg / ml , nearly the same as in patients with hba1c 8.9% or 74 mmol / mol ( 3,481 , 4,409 , and 5,887 this study includes 349 patients with proteinuria and ckd stage 13 who were enrolled into the joslin proteinuria cohort ( 3 ) . renal function changes during 518 years of follow - up were evaluated with the 4,097 creatinine determinations ( median seven per patient ) and assigned egfr = 10 ml / min/1.73 m at onset of esrd in 111 patients . using least square regression mean ( sd ) , egfr slope was 5.9 ( 8.2 ) ml / min/1.73 m / year . using the joint modeling , mean rate of renal decline was 5.2 ( 4.9 ) ml / min/1.73 m / year . neither hba1c nor blood pressure varied with increasing concentration of tnfr2 ; spearman correlation coefficients were 0.01 ( p = 0.87 ) and 0.10 ( p = 0.06 ) , respectively . subsequently , we used a joint longitudinal - survival model to examine the associations of clinical covariates at entry with the rate of egfr loss and with the imputed baseline egfr and time to esrd ( table 2 ) . estimates from univariate ( top ) and multivariate ( bottom ) joint models of the effects of tnfr2 and clinical characteristics on three components of renal decline : rate of egfr loss ( expressed in ml / min/1.73 m / year ) , egfr at baseline ( ml / min/1.73 m ) , and time to esrd ( percent change of time to esrd ) data are point estimates ( 95% cis ) . a one quartile increase in tnfr2 was associated with a 1.3 ml / min/1.73 m / year steeper rate of egfr loss and a 17.4 ml / min/1.73 m lower imputed baseline egfr ( p < 0.001 for each ) . together , the lower baseline and steeper rate of egfr loss shortened the imputed time to esrd by 29.0% ( p a 1% ( 11 mmol / mol ) increase in hba1c was associated with a 1.0 ml / min/1.73 m / year steeper rate of egfr loss ( p < 0.001 ) but was not associated with imputed baseline egfr . a 10-year increase in duration of diabetes was associated with a 0.8 ml / min/1.73 m / year ( p = 0.024 ) reduction in the rate of egfr loss , an 8.6 ml / min/1.73 m lower imputed baseline egfr ( p < 0.001 ) , and no reduction in imputed time to esrd . a 1 mmol / l increase in total serum cholesterol level was associated with a 1.0 ml / min/1.73 m faster rate of egfr loss ( p < 0.001 ) , no change in imputed baseline egfr , and 8.1% reduction in imputed time to esrd ( p = 0.026 ) . diastolic ( but not systolic ) blood pressure was also associated with a slightly steeper rate of egfr loss ( p = 0.014 ) but not with the imputed baseline egfr or time to esrd . other covariates measured at enrollment such as sex , age , bmi , and smoking did not have a significant impact on the rate of renal decline during follow - up ( data not shown ) . the average rate of egfr loss in patients below median is 4.1 ml / min/1.73 m / year , and imputed baseline egfr and time to esrd are 96.4 ml / min/1.73 m and 20.2 years , respectively . in patients with tnfr2 above median , the rate of egfr loss is steeper ( 6.2 ml / min/1.73 m / year ) , their imputed baseline egfr lower ( 62.3 ml / min/1.73 m ) , and their imputed time to esrd substantially shorter ( 8.2 years ) . they are as follows : rate of egfr loss steeper by 2.1 ml / min/1.73 m / year , imputed baseline egfr lower by 34.1 ml / min/1.73 m ( p < 0.001 ) , and imputed time to esrd shortened by 59.3% , i.e. in analyses from table 2 , thick lines depict imputed egfr trajectories , and thin solid lines indicate a covariate s ( tnfr2 ) associations with imputed baseline egfr , rate of renal decline , and imputed time to esrd . the changes of the rate of egfr loss associated with tnfr2 , acr , and hba1c were 0.8 , 1.3 , and 1.0 ml / min/1.73 m / year , respectively , and all were significant at p < 0.001 . the strong association of tnfr2 with the rate of egfr loss and imputed baseline egfr produced the largest change in imputed time to esrd , a reduction by 34.6% ( p < 0.001 ) . in addition to the analyses on the determinants of the rate of renal decline described above , we compared the predictive performances of tnfr2 , acr , and hba1c for discrimination between patients who developed and did not develop esrd during 518 years of follow - up . the highest discrimination in predicting time to esrd was provided by the serum concentration of tnfr2 ; the c - index from a single - variable model was 0.79 ( 95% ci 0.75 , 0.83 ) . in the first quartile of hba1c , the relationship between tnfr2 and the rate egfr loss was weak ; the difference in mean rates of egfr loss between first and fourth quartiles of tnfr2 was only 1.9 ml / min/1.73 m / year . expressed as linear trend across tnfr2 quartiles , the coefficients were 0.8 ( p = 0.157 ) , 1.1 ( p = 0.051 ) , 1.7 ( p = 0.001 ) , and 1.9 ( p < 0.001 ) in the first , second , third , and fourth quartiles of hba1c , respectively . mean and standard error of the rate of egfr loss ( joint model ) according to quartiles of tnfr2 within quartiles of hba1c . the first , second , and third quartiles of the distribution of tnfr2 in patients with hba1c < 8.9% or < 74 mmol / mol ( median ) were 3,542 , 4,439 , and 5,725 pg / ml , nearly the same as in patients with hba1c 8.9% or 74 mmol / mol ( 3,481 , 4,409 , and 5,887 the current study showed a strong association between a single baseline measurement of serum concentration of tnfr2 and the future rate of renal function decline in type 1 diabetic patients with proteinuria . it replicates and expands our previous findings of a strong association of circulating tnfrs with the risk of ckd stage 3 in patients with type 1 diabetes and microalbuminuria , and risk of esrd in patients with type 2 diabetes ( 1,2 ) . in contrast to the previous studies , this study focused on serum tnfr2 as a determinant of the rate of egfr loss , the most direct measure available of the intensity of the underlying disease process that leads to esrd . our analytical method , joint longitudinal - survival analysis , estimates the rate of egfr loss together with two other characteristics of the trajectory of renal decline during follow - up : the intercept ( imputed baseline egfr ) and the imputed time to onset of esrd . in both univariate and multivariate analyses , the serum concentration of tnfr2 is strongly associated with the rate of egfr loss , secondarily with the imputed baseline egfr ( a consequence of what damage has already accumulated ) and ultimately the imputed time to esrd . although that study has not yet examined its effect on renal decline , our recent study has shown that tnfr 1 and 2 are major determinants of early renal decline in type 1 diabetic patients with normoalbuminuria , as well as in those with microalbuminuria ( 15 ) and those with proteinuria ( current study ) . by the same token , the association of poor glycemic control with the rate of egfr loss was blunted in patients with low serum tnfr2 . tnfr2 is a strong determinant of renal decline in patients with type 1 diabetes and proteinuria . although such programs need to be developed , there is some evidence that in patients with proteinuria and high hba1c , significantly improved glycemic control maintained for 4 or more years can slow the rate of egfr loss and postpone the onset of esrd after a several - year lag time ( 16 ) . first , we used serum creatinine based estimates of gfr , which are less accurate than direct measurements or estimates based on serum cystatin c. direct gfr measurements might increase the strength of the associations . accounting for possible changes in these covariates before or after the start of follow - up , most likely , might have improved their strength as predictors of the rate of renal decline .

digital photography has simplified the process of capturing and utilizing digital images for health care professionals . the process of taking high - quality digital photographs has been recognized as efficient , timely , and cost - effective.13 in particular , the evolution of smartphone and comparable technologies has enabled digital photography to become a vital component of teaching and learning in the health care setting.4,5 consultation and documentation , clinical education , patient and family education , and publications are four key domains where digital images are frequently utilized within the clinical setting.6 this can be attributed to the minimal cost involved , user friendliness , and the ability to produce high - quality images that are easily stored , downloaded , and distributed via a multitude of mediums.2 however , ethical standards in relation to digital photography for teaching and learning have not always been of the highest standard . the inappropriate utilization of digital images within the health care setting has the capacity to compromise patient confidentiality and increase the risk of litigation.7 in particular , health care personnel need to consider the ethical implications of digital technology within the health care setting.8,9 the very simplicity of modern digital photography has resulted , in some cases , in a relaxation of the usually applied guidelines of informed consent.9 an unequal power balance could exist between health care professionals and patients who may feel coerced into consenting to digital photography.10 taking photographs of at - risk and vulnerable populations requires greater ethical responsibility.11 ensuring that the patient s identity and privacy is not sacrificed for the reason of ease and convenience is of prime importance . furthermore , misuse of digital images obtained in clinical settings is becoming an area of concern10,12 and brings into question and highlights issues surrounding privacy and confidentiality . consent , when obtained , is often verbal and may not include an explanation conveying photography is not only for treatment - related purposes , but also for education.9,10,13,14 consequently , there is a heightened need for guidance in relation to the use of digital photography within the clinical setting where such technology plays an increasingly prominent role.15 the application of digital photography in the clinical setting for the purpose of teaching and learning is poorly reported in the health care literature,16 and the authors have been unable to identify a single review that investigates digital photography in teaching and learning of health professionals . therefore , the aim of this integrative review was to investigate the current literature concerning the ethical implications of digital photography for teaching and learning purposes within the health care environment . the framework guiding this integrative review is based on whittemore and knafls17 five stages : problem identification , literature search , data evaluation , data analysis , and presentation . a systematic search was conducted using pubmed , embase , cumulative index to nursing and allied health literature , educational resources information center , and scopus . the references of all potential papers retrieved were examined to identify any additional papers fulfilling the inclusion criteria that may have been missed by the electronic searching strategy . boolean connectors were used to combine search terms such as health care , medical , image * , smartphone * , digital , photography , ethic * , informed consent , privacy , confidential , education , guideline * , policy , teaching , and learning . the search strategy undertaken yielded 514 articles after the duplicates were removed ( figure 1 ) . the inclusion criteria included : peer - reviewed reports of original research;literature published in the english language within the last 10 years ; andexploration of the use of identifiable digital imagery for teaching and learning in the health care setting . peer - reviewed reports of original research ; literature published in the english language within the last 10 years ; and exploration of the use of identifiable digital imagery for teaching and learning in the health care setting . the 10-year range was chosen due to the advancements made in the area of digital photography , imaging , and smartphone technology in the past decade.18,19 studies that examined histopathology specimens were excluded as these are unidentified specimens . although integrative reviews allow the use of theoretical pieces , review articles , commentaries , editorials , gray literature , and narrative opinion,17 they were excluded . these abstracts were read by all authors ( rk , vb , and rb ) based on inclusion / exclusion criteria , yielding nine studies . reading the full text of these studies resulted in the exclusion of four more articles as they did not meet the inclusion criteria , resulting in five studies deemed appropriate for inclusion in the review ( table 1 ) . any discrepancies experienced were resolved by active discussion until consensus was attained by all the parties . this integrative review incorporated the key aspects of ethical implications of utilizing digital photography in the clinical setting for purposes of teaching and learning . the domains of the clinical practice settings included plastic surgery , dermatology , emergency medicine , and surgical / medical wards . all included studies were surveys / questionnaires with a response rate ranging from 22.6% to 78% . the studies reported the use of digital photography / imaging in the clinical setting for the purpose of teaching and learning , including the issues of consent and utilization of technology . surprisingly , out of the five studies , only one reported policies / guidelines in relation to digital photography in their results.20 the five studies were synthesized , and their findings categorized into three themes : knowledge deficit , consent and beyond , and standards driving scope of practice . it was clearly identified that having a poor understanding of the process of consent when capturing digital images and a lack of familiarity with the available policies had implications for ethical compliance and the potential for patient identification and harm . burns and belton10 highlighted that ethical compliance may potentially be compromised when there is a deficit in comprehension and understanding . a knowledge deficit may lead to inappropriate and unsafe practice(s ) in the clinical area , placing the patient at further risk of harm by compromising his / her privacy and identity.6 a knowledge deficit also renders health care professional at risk of breaching ethical standards and guidelines within their institutions , a finding reported by both burns and belton10 and taylor et al.2 this demonstrates the need for educational workshops and further training within the clinical setting . an excerpt from an interview with a participant from the study of burns and belton10 clearly indicates a lack of awareness surrounding digital photography and the necessary process one must follow to comply with an ethical standard : everyone has phones that can take photos these days but i was like where do you put that information ? i do not fancy having it sitting around on a hard drive i have no control over . everyone has phones that can take photos these days but i was like where do you put that information ? i do not fancy having it sitting around on a hard drive i have no control over . hubbard et al1 identified that dermatological trainees sought consent from consultants when taking digital images of patients , rather than obtaining consent from patients themselves . this raises questions and concerns around their understanding and perception of ethical process of digital photography . it is critical for clinicians to be aware of policies and guidelines concerning the process of digital photography , the ethical implications , and the potential for harm . to highlight this point , hubbard et al,1 found that 33.6% of respondents were not aware of any available guidelines on the process of digital photography in their clinical area subsequently , despite a small number of facilities conducting an annual training program describing a protocol for obtaining , storing , and consenting digital images , bhangoo et al20 found that these sessions were predominately attended by senior nursing staff with poor attendance by medical staff . this is surprising and concerning considering that most digital images of patients are taken by medical staff . there remains an inadequacy of health care facilities to develop and implement policies and guidelines relevant to digital photography . consequently , this has resulted in a disparity that currently exists between the taking of digital photographs for use in teaching and learning and the obtaining of informed patient consent for this specified purpose . however , agencies guidelines such as the general medical council guidelines making and using visual and audio recordings of patients in the united kingdom21 and acts such as the health insurance portability and accountability act ( hipaa)6 provide clear guidance on when consent should be obtained and how it should be recorded and also address the taking of photographs for educational reasons , although this was not reflected in the studies included in this review . taylor et al2 identified that 25 of the 30 respondents in their study acquired digital images for the purpose of teaching and learning . of the 25 respondents , ten always gained consent with two rarely and one never . similarly , burns and belton10 found that 24 of 41 health care professionals reported verbal consent as the preferred method . this is problematic as there remains no data trail or evidence that the consent was sought or that the consent was obtained for the use of the images for purposes other than treatment , including teaching and learning . these concerns are echoed by a participant from burns and belton10 study stating : i do nt think people put significant enough emphasis on the consent process . i have often told people you know make sure you have got consent and they will go oh yeah , i have often told people you know make sure you have got consent and they will go oh yeah , this quote clearly emphasizes the lack of awareness concerning consent and the ethical implications of using the digital photography for educational purposes . furthermore , the absence of consent was similarly identified by hubbard et al1 who reported that 7.4% of respondents who used a digital camera to gain images for teaching and learning did so without consent . kunde et al9 revealed that only 54% of their respondents reported that they regularly informed the patients of any third parties who may potentially view their image . while it may not always be possible for health care professionals to be specific about such viewing in a clinical setting , it remains important that we differentiate between the clinical and educational settings . however , the philosophy of obtaining written valid informed consent , prior to the acquisition of digital images for educational purpose , seems lacking . taylor et al2 also emphasized the need for staff to inform patients that their consent may be withdrawn at any time , prior to images entering the public domain where they are irretrievable . however , withdrawing consent for the images regardless of the purpose of their acquisition may be a redundant notion given the speed of which data can be transferred and may well need to be addressed with the patient and their family . it is apparent from this review that within the clinical area , discrepancies exist around the availability of policies and guidelines related to digital photography . of those policies that are available , the awareness of their existence by health care professionals appears to range from limited to 0 , posing concerns that they may not comply with these guidelines . given that the utilization of digital photography was for the purpose of teaching and learning across all the five included studies , it is essential that policymakers , health care practitioners , and administrators become familiar with ethical regulations and guidelines related to digital images.10 this is clearly highlighted in bhangoo et als20 findings that only 36% of emergency departments had a written policy pertaining to the taking of images , and of those , only two departments had a written policy specifically focused on photography in clinical and educational settings . to further highlight this issue , taylor et al2 found that of the 25 surgeons in their study who took images for the purposes of teaching and learning , 17 took no extra measures to protect the anonymity of their patient . conversely , hubbard et al1 did identify that in total 181 ( 53.3% ) respondents were aware of guidelines for digital photography , although 40 ( 11.8% ) stated that there were none and 114 ( 33.6% ) did not know whether there were any guidelines . this raises ethical concerns as there appears to be considerable variation in the specific guidance provided for the use of digital photography . it is also clear that health care professionals are partaking in the process of digital photography without recognizing the existence of policies and guidelines that guide safe practice , ensuring the patient s privacy is protected and no harm is perpetrated . despite taylor et als2 study emphasizing the importance of health professionals being aware of guidelines pertaining to digital photography , burns and belton10 study illustrates that noncompliance in the domain of obtaining consent for digital photography was widespread . hence , it is evident that accessible , well - publicized policies and guidelines need to be developed to guide and safeguard those practicing digital photography within health care setting as well as the patients being photographed.20 this integrative review incorporated the key aspects of ethical implications of utilizing digital photography in the clinical setting for purposes of teaching and learning . the domains of the clinical practice settings included plastic surgery , dermatology , emergency medicine , and surgical / medical wards . all included studies were surveys / questionnaires with a response rate ranging from 22.6% to 78% . the studies reported the use of digital photography / imaging in the clinical setting for the purpose of teaching and learning , including the issues of consent and utilization of technology . surprisingly , out of the five studies , only one reported policies / guidelines in relation to digital photography in their results.20 the five studies were synthesized , and their findings categorized into three themes : knowledge deficit , consent and beyond , and standards driving scope of practice . it was clearly identified that having a poor understanding of the process of consent when capturing digital images and a lack of familiarity with the available policies had implications for ethical compliance and the potential for patient identification and harm . burns and belton10 highlighted that ethical compliance may potentially be compromised when there is a deficit in comprehension and understanding . a knowledge deficit may lead to inappropriate and unsafe practice(s ) in the clinical area , placing the patient at further risk of harm by compromising his / her privacy and identity.6 a knowledge deficit also renders health care professional at risk of breaching ethical standards and guidelines within their institutions , a finding reported by both burns and belton10 and taylor et al.2 this demonstrates the need for educational workshops and further training within the clinical setting . an excerpt from an interview with a participant from the study of burns and belton10 clearly indicates a lack of awareness surrounding digital photography and the necessary process one must follow to comply with an ethical standard : everyone has phones that can take photos these days but i was like where do you put that information ? i do not fancy having it sitting around on a hard drive i have no control over . everyone has phones that can take photos these days but i was like where do you put that information ? i do not fancy having it sitting around on a hard drive i have no control over . hubbard et al1 identified that dermatological trainees sought consent from consultants when taking digital images of patients , rather than obtaining consent from patients themselves . this raises questions and concerns around their understanding and perception of ethical process of digital photography . it is critical for clinicians to be aware of policies and guidelines concerning the process of digital photography , the ethical implications , and the potential for harm . to highlight this point , hubbard et al,1 found that 33.6% of respondents were not aware of any available guidelines on the process of digital photography in their clinical area subsequently , despite a small number of facilities conducting an annual training program describing a protocol for obtaining , storing , and consenting digital images , bhangoo et al20 found that these sessions were predominately attended by senior nursing staff with poor attendance by medical staff . this is surprising and concerning considering that most digital images of patients are taken by medical staff . there remains an inadequacy of health care facilities to develop and implement policies and guidelines relevant to digital photography . consequently , this has resulted in a disparity that currently exists between the taking of digital photographs for use in teaching and learning and the obtaining of informed patient consent for this specified purpose . however , agencies guidelines such as the general medical council guidelines making and using visual and audio recordings of patients in the united kingdom21 and acts such as the health insurance portability and accountability act ( hipaa)6 provide clear guidance on when consent should be obtained and how it should be recorded and also address the taking of photographs for educational reasons , although this was not reflected in the studies included in this review . taylor et al2 identified that 25 of the 30 respondents in their study acquired digital images for the purpose of teaching and learning . of the 25 respondents , ten always gained consent with two rarely and one never . similarly , burns and belton10 found that 24 of 41 health care professionals reported verbal consent as the preferred method . this is problematic as there remains no data trail or evidence that the consent was sought or that the consent was obtained for the use of the images for purposes other than treatment , including teaching and learning . these concerns are echoed by a participant from burns and belton10 study stating : i do nt think people put significant enough emphasis on the consent process . i have often told people you know make sure you have got consent and they will go oh yeah , yeah , but it s just for education . i do nt think people put significant enough emphasis on the consent process . i have often told people you know make sure you have got consent and they will go oh yeah , this quote clearly emphasizes the lack of awareness concerning consent and the ethical implications of using the digital photography for educational purposes . furthermore , the absence of consent was similarly identified by hubbard et al1 who reported that 7.4% of respondents who used a digital camera to gain images for teaching and learning did so without consent . kunde et al9 revealed that only 54% of their respondents reported that they regularly informed the patients of any third parties who may potentially view their image . while it may not always be possible for health care professionals to be specific about such viewing in a clinical setting , it remains important that we differentiate between the clinical and educational settings . . however , the philosophy of obtaining written valid informed consent , prior to the acquisition of digital images for educational purpose , seems lacking . taylor et al2 also emphasized the need for staff to inform patients that their consent may be withdrawn at any time , prior to images entering the public domain where they are irretrievable . however , withdrawing consent for the images regardless of the purpose of their acquisition may be a redundant notion given the speed of which data can be transferred and may well need to be addressed with the patient and their family . it is apparent from this review that within the clinical area , discrepancies exist around the availability of policies and guidelines related to digital photography . of those policies that are available , the awareness of their existence by health care professionals appears to range from limited to 0 , posing concerns that they may not comply with these guidelines . given that the utilization of digital photography was for the purpose of teaching and learning across all the five included studies , it is essential that policymakers , health care practitioners , and administrators become familiar with ethical regulations and guidelines related to digital images.10 this is clearly highlighted in bhangoo et als20 findings that only 36% of emergency departments had a written policy pertaining to the taking of images , and of those , only two departments had a written policy specifically focused on photography in clinical and educational settings . to further highlight this issue , taylor et al2 found that of the 25 surgeons in their study who took images for the purposes of teaching and learning , 17 took no extra measures to protect the anonymity of their patient . conversely , hubbard et al1 did identify that in total 181 ( 53.3% ) respondents were aware of guidelines for digital photography , although 40 ( 11.8% ) stated that there were none and 114 ( 33.6% ) did not know whether there were any guidelines . this raises ethical concerns as there appears to be considerable variation in the specific guidance provided for the use of digital photography . it is also clear that health care professionals are partaking in the process of digital photography without recognizing the existence of policies and guidelines that guide safe practice , ensuring the patient s privacy is protected and no harm is perpetrated . despite taylor et als2 study emphasizing the importance of health professionals being aware of guidelines pertaining to digital photography , burns and belton10 study illustrates that noncompliance in the domain of obtaining consent for digital photography was widespread . hence , it is evident that accessible , well - publicized policies and guidelines need to be developed to guide and safeguard those practicing digital photography within health care setting as well as the patients being photographed.20 the authors have explored ethical considerations associated with the use of digital photography for teaching and learning purposes in the health care environment . considering the fundamental importance that is directed toward ethics , informed consent , and patients privacy , there is a lack of appropriate ethical guidelines to govern the use of digital photography for clinical education . we found that even when guidelines were available as demonstrated with the general medical council s making and using visual and audio recordings of patients21 in the uk and the hipaa in the us,6 health care personnel were either unaware of their existence or loath to follow them . to compound this , the definition of protected health information under hipaa , although only relevant to the us , is very broad and generalized and therefore does not specifically address the use of full facial images and images that could potentially reveal the identity of the patient.22 however , the studies reviewed revealed that when consent was obtained , it was commonly verbal , seemingly rarely informed , and often omitted reference to digital photographs being used for teaching and learning purposes . finally , we found that those taking digital photographs had neither common understanding of how these photographs were to be stored securely nor with whom such images might be ethically shared . while digital photography has become a simple , useful , and readily available tool to assist in diagnosis , it is becoming increasingly used by clinicians in areas including wound management as it provides an accurate and objective method of assessment and assists in with monitoring the progress of treatment . however , the use of such photographs for other purposes raises significant ethical questions,23 although we do not wish to obstruct the proper use of such images and strongly believe that the following steps should be undertaken to improve ethical practices and protect patients and clinicians : professional bodies should develop guidelines to assist in the acquisition and use of digital photographs for health education purposes.training programs should then be developed to raise awareness of these guidelines and local institutional policies to raise awareness and promote ethical conduct.appropriate consent is critical to the management of digital photographs . consent should be informed , written , and obtained prior to any procedure and should include full disclosure of how images are to be taken , stored , and de - identified and how they are to be used and which audiences are likely to view them . professional bodies should develop guidelines to assist in the acquisition and use of digital photographs for health education purposes . training programs should then be developed to raise awareness of these guidelines and local institutional policies to raise awareness and promote ethical conduct . consent should be informed , written , and obtained prior to any procedure and should include full disclosure of how images are to be taken , stored , and de - identified and how they are to be used and which audiences are likely to view them . appropriately acquired consent will protect both patients and staff from ethical dilemmas surrounding the use of digital photography within the domain of teaching and learning . in order to comply with ethical standards in the face of ever - changing technology , the need for auditing of health care facilities on their practices around digital photography is critical and may shed light on the frequency and use of digital photography and the presence of policies and guidelines that reflect the current technological climate . health care institutions globally have been incorporating the use of smartphone technology into their daily practice.6 for example , apps such as picsafe and epic haiku ( epic systems corporation , verona , wi , usa ) allow clinicians to consolidate the consent , capture and means to retain the image within a single program10 and allows for integration into the medical record . apps such as these may also be a critical tool for those who deliver education to clinicians and could be incorporated into existing policies and guidelines . however , it is essential that any policy or guideline must reflect and comply with the existing legislation . this has become increasingly difficult as technology is far outpacing legislative and legal systems.6 as such , this has resulted in some health care institutions deeming smartphone cameras as a significant ethical dilemma for patient privacy , resulting in the development and implementation of smartphone policies.6 therefore , the development of policies and guidelines in accordance with the relevant legislation is warranted . strengths of this review include the use of three independent reviewers during the selection and extraction stages . no assumptions were made when methodology was unclear , which further strengthened the review process . similarly , the development of logic tables and a search strategy across five major relevant databases , including the educational resources information database ( educational resources information center ) , offer reassurance that the review is both rigorous and comprehensive . this review is limited by the small number of original papers that were identified for evaluation . all papers included in this review were questionnaires , which may limit the strength of the findings . however , given the methodological complexities of evaluating issues as ethical implications , surveys can still offer useful and practical evidence that has the potential to guide policy and for guideline development . as the majority of studies in this review involved voluntary participation in questionnaires , there is potential for bias within the results . likewise , small sample sizes and self - reporting may also impact data quality and the strength of our results . finally , this review incorporates studies from australia and the uk and as such may be perceived as limiting for it does not reflect a global perspective . despite the fact that the database search was extensive and inclusive , it was limited to only english language publications over the past 10 years and we did not review the gray literature , and therefore , may have overlooked some relevant studies . there is a lack of appropriate ethical guidelines to govern the use of digital photography for clinical education . we found that even when guidelines were available as demonstrated with the general medical council s making and using visual and audio recordings of patients21 in the uk and the hipaa in the us,6 health care personnel were either unaware of their existence or loath to follow them . to compound this , the definition of protected health information under hipaa , although only relevant to the us , is very broad and generalized and therefore does not specifically address the use of full facial images and images that could potentially reveal the identity of the patient.22 however , the studies reviewed revealed that when consent was obtained , it was commonly verbal , seemingly rarely informed , and often omitted reference to digital photographs being used for teaching and learning purposes . finally , we found that those taking digital photographs had neither common understanding of how these photographs were to be stored securely nor with whom such images might be ethically shared . while digital photography has become a simple , useful , and readily available tool to assist in diagnosis , it is becoming increasingly used by clinicians in areas including wound management as it provides an accurate and objective method of assessment and assists in with monitoring the progress of treatment . however , the use of such photographs for other purposes raises significant ethical questions,23 although we do not wish to obstruct the proper use of such images and strongly believe that the following steps should be undertaken to improve ethical practices and protect patients and clinicians : professional bodies should develop guidelines to assist in the acquisition and use of digital photographs for health education purposes.training programs should then be developed to raise awareness of these guidelines and local institutional policies to raise awareness and promote ethical conduct.appropriate consent is critical to the management of digital photographs . consent should be informed , written , and obtained prior to any procedure and should include full disclosure of how images are to be taken , stored , and de - identified and how they are to be used and which audiences are likely to view them . professional bodies should develop guidelines to assist in the acquisition and use of digital photographs for health education purposes . training programs should then be developed to raise awareness of these guidelines and local institutional policies to raise awareness and promote ethical conduct . consent should be informed , written , and obtained prior to any procedure and should include full disclosure of how images are to be taken , stored , and de - identified and how they are to be used and which audiences are likely to view them . appropriately acquired consent will protect both patients and staff from ethical dilemmas surrounding the use of digital photography within the domain of teaching and learning . in order to comply with ethical standards in the face of ever - changing technology , the need for auditing of health care facilities on their practices around digital photography is critical and may shed light on the frequency and use of digital photography and the presence of policies and guidelines that reflect the current technological climate . health care institutions globally have been incorporating the use of smartphone technology into their daily practice.6 for example , apps such as picsafe and epic haiku ( epic systems corporation , verona , wi , usa ) allow clinicians to consolidate the consent , capture and means to retain the image within a single program10 and allows for integration into the medical record . apps such as these may also be a critical tool for those who deliver education to clinicians and could be incorporated into existing policies and guidelines . however , it is essential that any policy or guideline must reflect and comply with the existing legislation . this has become increasingly difficult as technology is far outpacing legislative and legal systems.6 as such , this has resulted in some health care institutions deeming smartphone cameras as a significant ethical dilemma for patient privacy , resulting in the development and implementation of smartphone policies.6 therefore , the development of policies and guidelines in accordance with the relevant legislation is warranted . strengths of this review include the use of three independent reviewers during the selection and extraction stages . no assumptions were made when methodology was unclear , which further strengthened the review process . similarly , the development of logic tables and a search strategy across five major relevant databases , including the educational resources information database ( educational resources information center ) , offer reassurance that the review is both rigorous and comprehensive . this review is limited by the small number of original papers that were identified for evaluation . all papers included in this review were questionnaires , which may limit the strength of the findings . however , given the methodological complexities of evaluating issues as ethical implications , surveys can still offer useful and practical evidence that has the potential to guide policy and for guideline development . as the majority of studies in this review involved voluntary participation in questionnaires likewise , small sample sizes and self - reporting may also impact data quality and the strength of our results . finally , this review incorporates studies from australia and the uk and as such may be perceived as limiting for it does not reflect a global perspective . despite the fact that the database search was extensive and inclusive , it was limited to only english language publications over the past 10 years and we did not review the gray literature , and therefore , may have overlooked some relevant studies . this integrative review suggests that there is currently a deficit in knowledge pertaining to ethical considerations of clinicians in obtaining , storing , utilizing , and distributing digital photography within the clinical and teaching environment . these results have implications for informing the practices of those who utilize digital photography routinely in their practice , in particular , newly graduated health professionals , with the opportunity to lay the foundations and embed their future career in sound ethical practices that ensure the safety of their patients and themselves in a dynamic highly technological and litigious health care environment .

backgrounddigital photography has simplified the process of capturing and utilizing medical images . the process of taking high - quality digital photographs has been recognized as efficient , timely , and cost - effective . in particular , the evolution of smartphone and comparable technologies has become a vital component in teaching and learning of health care professionals . however , ethical standards in relation to digital photography for teaching and learning have not always been of the highest standard . the inappropriate utilization of digital images within the health care setting has the capacity to compromise patient confidentiality and increase the risk of litigation . therefore , the aim of this review was to investigate the literature concerning the ethical implications for health professionals utilizing digital photography for teaching and learning.methodsa literature search was conducted utilizing five electronic databases , pubmed , embase ( excerpta medica database ) , cumulative index to nursing and allied health literature , educational resources information center , and scopus , limited to english language . studies that endeavored to evaluate the ethical implications of digital photography for teaching and learning purposes in the health care setting were included.resultsthe search strategy identified 514 papers of which nine were retrieved for full review . four papers were excluded based on the inclusion criteria , leaving five papers for final analysis . three key themes were developed : knowledge deficit , consent and beyond , and standards driving scope of practice.conclusionthe assimilation of evidence in this review suggests that there is value for health professionals utilizing digital photography for teaching purposes in health education . however , there is limited understanding of the process of obtaining and storage and use of such mediums for teaching purposes . disparity was also highlighted related to policy and guideline identification and development in clinical practice . therefore , the implementation of policy to guide practice requires further research .

Introduction Methods Results Study characteristics Knowledge deficit Consent and beyond Standards driving scope of practice Discussion Implications for practice Limitations and strength of evidence Conclusion

digital photography has simplified the process of capturing and utilizing digital images for health care professionals . the process of taking high - quality digital photographs has been recognized as efficient , timely , and cost - effective.13 in particular , the evolution of smartphone and comparable technologies has enabled digital photography to become a vital component of teaching and learning in the health care setting.4,5 consultation and documentation , clinical education , patient and family education , and publications are four key domains where digital images are frequently utilized within the clinical setting.6 this can be attributed to the minimal cost involved , user friendliness , and the ability to produce high - quality images that are easily stored , downloaded , and distributed via a multitude of mediums.2 however , ethical standards in relation to digital photography for teaching and learning have not always been of the highest standard . the inappropriate utilization of digital images within the health care setting has the capacity to compromise patient confidentiality and increase the risk of litigation.7 in particular , health care personnel need to consider the ethical implications of digital technology within the health care setting.8,9 the very simplicity of modern digital photography has resulted , in some cases , in a relaxation of the usually applied guidelines of informed consent.9 an unequal power balance could exist between health care professionals and patients who may feel coerced into consenting to digital photography.10 taking photographs of at - risk and vulnerable populations requires greater ethical responsibility.11 ensuring that the patient s identity and privacy is not sacrificed for the reason of ease and convenience is of prime importance . consent , when obtained , is often verbal and may not include an explanation conveying photography is not only for treatment - related purposes , but also for education.9,10,13,14 consequently , there is a heightened need for guidance in relation to the use of digital photography within the clinical setting where such technology plays an increasingly prominent role.15 the application of digital photography in the clinical setting for the purpose of teaching and learning is poorly reported in the health care literature,16 and the authors have been unable to identify a single review that investigates digital photography in teaching and learning of health professionals . therefore , the aim of this integrative review was to investigate the current literature concerning the ethical implications of digital photography for teaching and learning purposes within the health care environment . a systematic search was conducted using pubmed , embase , cumulative index to nursing and allied health literature , educational resources information center , and scopus . boolean connectors were used to combine search terms such as health care , medical , image * , smartphone * , digital , photography , ethic * , informed consent , privacy , confidential , education , guideline * , policy , teaching , and learning . the inclusion criteria included : peer - reviewed reports of original research;literature published in the english language within the last 10 years ; andexploration of the use of identifiable digital imagery for teaching and learning in the health care setting . peer - reviewed reports of original research ; literature published in the english language within the last 10 years ; and exploration of the use of identifiable digital imagery for teaching and learning in the health care setting . the 10-year range was chosen due to the advancements made in the area of digital photography , imaging , and smartphone technology in the past decade.18,19 studies that examined histopathology specimens were excluded as these are unidentified specimens . although integrative reviews allow the use of theoretical pieces , review articles , commentaries , editorials , gray literature , and narrative opinion,17 they were excluded . reading the full text of these studies resulted in the exclusion of four more articles as they did not meet the inclusion criteria , resulting in five studies deemed appropriate for inclusion in the review ( table 1 ) . this integrative review incorporated the key aspects of ethical implications of utilizing digital photography in the clinical setting for purposes of teaching and learning . the studies reported the use of digital photography / imaging in the clinical setting for the purpose of teaching and learning , including the issues of consent and utilization of technology . surprisingly , out of the five studies , only one reported policies / guidelines in relation to digital photography in their results.20 the five studies were synthesized , and their findings categorized into three themes : knowledge deficit , consent and beyond , and standards driving scope of practice . it was clearly identified that having a poor understanding of the process of consent when capturing digital images and a lack of familiarity with the available policies had implications for ethical compliance and the potential for patient identification and harm . a knowledge deficit may lead to inappropriate and unsafe practice(s ) in the clinical area , placing the patient at further risk of harm by compromising his / her privacy and identity.6 a knowledge deficit also renders health care professional at risk of breaching ethical standards and guidelines within their institutions , a finding reported by both burns and belton10 and taylor et al.2 this demonstrates the need for educational workshops and further training within the clinical setting . it is critical for clinicians to be aware of policies and guidelines concerning the process of digital photography , the ethical implications , and the potential for harm . to highlight this point , hubbard et al,1 found that 33.6% of respondents were not aware of any available guidelines on the process of digital photography in their clinical area subsequently , despite a small number of facilities conducting an annual training program describing a protocol for obtaining , storing , and consenting digital images , bhangoo et al20 found that these sessions were predominately attended by senior nursing staff with poor attendance by medical staff . there remains an inadequacy of health care facilities to develop and implement policies and guidelines relevant to digital photography . consequently , this has resulted in a disparity that currently exists between the taking of digital photographs for use in teaching and learning and the obtaining of informed patient consent for this specified purpose . however , agencies guidelines such as the general medical council guidelines making and using visual and audio recordings of patients in the united kingdom21 and acts such as the health insurance portability and accountability act ( hipaa)6 provide clear guidance on when consent should be obtained and how it should be recorded and also address the taking of photographs for educational reasons , although this was not reflected in the studies included in this review . taylor et al2 identified that 25 of the 30 respondents in their study acquired digital images for the purpose of teaching and learning . this is problematic as there remains no data trail or evidence that the consent was sought or that the consent was obtained for the use of the images for purposes other than treatment , including teaching and learning . i have often told people you know make sure you have got consent and they will go oh yeah , i have often told people you know make sure you have got consent and they will go oh yeah , this quote clearly emphasizes the lack of awareness concerning consent and the ethical implications of using the digital photography for educational purposes . furthermore , the absence of consent was similarly identified by hubbard et al1 who reported that 7.4% of respondents who used a digital camera to gain images for teaching and learning did so without consent . while it may not always be possible for health care professionals to be specific about such viewing in a clinical setting , it remains important that we differentiate between the clinical and educational settings . however , the philosophy of obtaining written valid informed consent , prior to the acquisition of digital images for educational purpose , seems lacking . it is apparent from this review that within the clinical area , discrepancies exist around the availability of policies and guidelines related to digital photography . of those policies that are available , the awareness of their existence by health care professionals appears to range from limited to 0 , posing concerns that they may not comply with these guidelines . given that the utilization of digital photography was for the purpose of teaching and learning across all the five included studies , it is essential that policymakers , health care practitioners , and administrators become familiar with ethical regulations and guidelines related to digital images.10 this is clearly highlighted in bhangoo et als20 findings that only 36% of emergency departments had a written policy pertaining to the taking of images , and of those , only two departments had a written policy specifically focused on photography in clinical and educational settings . this raises ethical concerns as there appears to be considerable variation in the specific guidance provided for the use of digital photography . it is also clear that health care professionals are partaking in the process of digital photography without recognizing the existence of policies and guidelines that guide safe practice , ensuring the patient s privacy is protected and no harm is perpetrated . despite taylor et als2 study emphasizing the importance of health professionals being aware of guidelines pertaining to digital photography , burns and belton10 study illustrates that noncompliance in the domain of obtaining consent for digital photography was widespread . hence , it is evident that accessible , well - publicized policies and guidelines need to be developed to guide and safeguard those practicing digital photography within health care setting as well as the patients being photographed.20 this integrative review incorporated the key aspects of ethical implications of utilizing digital photography in the clinical setting for purposes of teaching and learning . the studies reported the use of digital photography / imaging in the clinical setting for the purpose of teaching and learning , including the issues of consent and utilization of technology . surprisingly , out of the five studies , only one reported policies / guidelines in relation to digital photography in their results.20 the five studies were synthesized , and their findings categorized into three themes : knowledge deficit , consent and beyond , and standards driving scope of practice . it was clearly identified that having a poor understanding of the process of consent when capturing digital images and a lack of familiarity with the available policies had implications for ethical compliance and the potential for patient identification and harm . a knowledge deficit may lead to inappropriate and unsafe practice(s ) in the clinical area , placing the patient at further risk of harm by compromising his / her privacy and identity.6 a knowledge deficit also renders health care professional at risk of breaching ethical standards and guidelines within their institutions , a finding reported by both burns and belton10 and taylor et al.2 this demonstrates the need for educational workshops and further training within the clinical setting . it is critical for clinicians to be aware of policies and guidelines concerning the process of digital photography , the ethical implications , and the potential for harm . to highlight this point , hubbard et al,1 found that 33.6% of respondents were not aware of any available guidelines on the process of digital photography in their clinical area subsequently , despite a small number of facilities conducting an annual training program describing a protocol for obtaining , storing , and consenting digital images , bhangoo et al20 found that these sessions were predominately attended by senior nursing staff with poor attendance by medical staff . there remains an inadequacy of health care facilities to develop and implement policies and guidelines relevant to digital photography . consequently , this has resulted in a disparity that currently exists between the taking of digital photographs for use in teaching and learning and the obtaining of informed patient consent for this specified purpose . however , agencies guidelines such as the general medical council guidelines making and using visual and audio recordings of patients in the united kingdom21 and acts such as the health insurance portability and accountability act ( hipaa)6 provide clear guidance on when consent should be obtained and how it should be recorded and also address the taking of photographs for educational reasons , although this was not reflected in the studies included in this review . taylor et al2 identified that 25 of the 30 respondents in their study acquired digital images for the purpose of teaching and learning . this is problematic as there remains no data trail or evidence that the consent was sought or that the consent was obtained for the use of the images for purposes other than treatment , including teaching and learning . i have often told people you know make sure you have got consent and they will go oh yeah , this quote clearly emphasizes the lack of awareness concerning consent and the ethical implications of using the digital photography for educational purposes . furthermore , the absence of consent was similarly identified by hubbard et al1 who reported that 7.4% of respondents who used a digital camera to gain images for teaching and learning did so without consent . while it may not always be possible for health care professionals to be specific about such viewing in a clinical setting , it remains important that we differentiate between the clinical and educational settings . however , the philosophy of obtaining written valid informed consent , prior to the acquisition of digital images for educational purpose , seems lacking . it is apparent from this review that within the clinical area , discrepancies exist around the availability of policies and guidelines related to digital photography . of those policies that are available , the awareness of their existence by health care professionals appears to range from limited to 0 , posing concerns that they may not comply with these guidelines . given that the utilization of digital photography was for the purpose of teaching and learning across all the five included studies , it is essential that policymakers , health care practitioners , and administrators become familiar with ethical regulations and guidelines related to digital images.10 this is clearly highlighted in bhangoo et als20 findings that only 36% of emergency departments had a written policy pertaining to the taking of images , and of those , only two departments had a written policy specifically focused on photography in clinical and educational settings . this raises ethical concerns as there appears to be considerable variation in the specific guidance provided for the use of digital photography . it is also clear that health care professionals are partaking in the process of digital photography without recognizing the existence of policies and guidelines that guide safe practice , ensuring the patient s privacy is protected and no harm is perpetrated . despite taylor et als2 study emphasizing the importance of health professionals being aware of guidelines pertaining to digital photography , burns and belton10 study illustrates that noncompliance in the domain of obtaining consent for digital photography was widespread . hence , it is evident that accessible , well - publicized policies and guidelines need to be developed to guide and safeguard those practicing digital photography within health care setting as well as the patients being photographed.20 the authors have explored ethical considerations associated with the use of digital photography for teaching and learning purposes in the health care environment . considering the fundamental importance that is directed toward ethics , informed consent , and patients privacy , there is a lack of appropriate ethical guidelines to govern the use of digital photography for clinical education . to compound this , the definition of protected health information under hipaa , although only relevant to the us , is very broad and generalized and therefore does not specifically address the use of full facial images and images that could potentially reveal the identity of the patient.22 however , the studies reviewed revealed that when consent was obtained , it was commonly verbal , seemingly rarely informed , and often omitted reference to digital photographs being used for teaching and learning purposes . while digital photography has become a simple , useful , and readily available tool to assist in diagnosis , it is becoming increasingly used by clinicians in areas including wound management as it provides an accurate and objective method of assessment and assists in with monitoring the progress of treatment . however , the use of such photographs for other purposes raises significant ethical questions,23 although we do not wish to obstruct the proper use of such images and strongly believe that the following steps should be undertaken to improve ethical practices and protect patients and clinicians : professional bodies should develop guidelines to assist in the acquisition and use of digital photographs for health education purposes.training programs should then be developed to raise awareness of these guidelines and local institutional policies to raise awareness and promote ethical conduct.appropriate consent is critical to the management of digital photographs . professional bodies should develop guidelines to assist in the acquisition and use of digital photographs for health education purposes . appropriately acquired consent will protect both patients and staff from ethical dilemmas surrounding the use of digital photography within the domain of teaching and learning . in order to comply with ethical standards in the face of ever - changing technology , the need for auditing of health care facilities on their practices around digital photography is critical and may shed light on the frequency and use of digital photography and the presence of policies and guidelines that reflect the current technological climate . this has become increasingly difficult as technology is far outpacing legislative and legal systems.6 as such , this has resulted in some health care institutions deeming smartphone cameras as a significant ethical dilemma for patient privacy , resulting in the development and implementation of smartphone policies.6 therefore , the development of policies and guidelines in accordance with the relevant legislation is warranted . similarly , the development of logic tables and a search strategy across five major relevant databases , including the educational resources information database ( educational resources information center ) , offer reassurance that the review is both rigorous and comprehensive . however , given the methodological complexities of evaluating issues as ethical implications , surveys can still offer useful and practical evidence that has the potential to guide policy and for guideline development . as the majority of studies in this review involved voluntary participation in questionnaires , there is potential for bias within the results . despite the fact that the database search was extensive and inclusive , it was limited to only english language publications over the past 10 years and we did not review the gray literature , and therefore , may have overlooked some relevant studies . there is a lack of appropriate ethical guidelines to govern the use of digital photography for clinical education . to compound this , the definition of protected health information under hipaa , although only relevant to the us , is very broad and generalized and therefore does not specifically address the use of full facial images and images that could potentially reveal the identity of the patient.22 however , the studies reviewed revealed that when consent was obtained , it was commonly verbal , seemingly rarely informed , and often omitted reference to digital photographs being used for teaching and learning purposes . while digital photography has become a simple , useful , and readily available tool to assist in diagnosis , it is becoming increasingly used by clinicians in areas including wound management as it provides an accurate and objective method of assessment and assists in with monitoring the progress of treatment . however , the use of such photographs for other purposes raises significant ethical questions,23 although we do not wish to obstruct the proper use of such images and strongly believe that the following steps should be undertaken to improve ethical practices and protect patients and clinicians : professional bodies should develop guidelines to assist in the acquisition and use of digital photographs for health education purposes.training programs should then be developed to raise awareness of these guidelines and local institutional policies to raise awareness and promote ethical conduct.appropriate consent is critical to the management of digital photographs . professional bodies should develop guidelines to assist in the acquisition and use of digital photographs for health education purposes . appropriately acquired consent will protect both patients and staff from ethical dilemmas surrounding the use of digital photography within the domain of teaching and learning . in order to comply with ethical standards in the face of ever - changing technology , the need for auditing of health care facilities on their practices around digital photography is critical and may shed light on the frequency and use of digital photography and the presence of policies and guidelines that reflect the current technological climate . this has become increasingly difficult as technology is far outpacing legislative and legal systems.6 as such , this has resulted in some health care institutions deeming smartphone cameras as a significant ethical dilemma for patient privacy , resulting in the development and implementation of smartphone policies.6 therefore , the development of policies and guidelines in accordance with the relevant legislation is warranted . similarly , the development of logic tables and a search strategy across five major relevant databases , including the educational resources information database ( educational resources information center ) , offer reassurance that the review is both rigorous and comprehensive . however , given the methodological complexities of evaluating issues as ethical implications , surveys can still offer useful and practical evidence that has the potential to guide policy and for guideline development . despite the fact that the database search was extensive and inclusive , it was limited to only english language publications over the past 10 years and we did not review the gray literature , and therefore , may have overlooked some relevant studies . this integrative review suggests that there is currently a deficit in knowledge pertaining to ethical considerations of clinicians in obtaining , storing , utilizing , and distributing digital photography within the clinical and teaching environment . these results have implications for informing the practices of those who utilize digital photography routinely in their practice , in particular , newly graduated health professionals , with the opportunity to lay the foundations and embed their future career in sound ethical practices that ensure the safety of their patients and themselves in a dynamic highly technological and litigious health care environment .

since their emergence a decade ago , ionic liquids ( il ) have had a constantly growing influence on organic , bio- and green chemistry , due to the unique physico - chemical properties manifested by their typical salt structure : a heterocyclic nitrogen - containing organic cation ( in general ) and an inorganic or organic anion , with melting points below 100 c and no vapor pressure . the latter property leads to the practical replacement of conventional volatile organic compounds ( vocs ) from the point of view of atmospheric emissions , though they do present the serious drawback that a small amount of il could enter the environment through groundwater . this risk makes it necessary to perform further eco - toxicological studies of il on various species , in order to improve the design rules for synthesized il with minimal toxicity to environment integrated organisms . ionic liquids display variable stability in terms of moisture and solubility in water , polar and nonpolar organic solvents . various values of ionic liquid hydrophobicity and polarity may be tailored with the help of nucleoside chemistry according to the main principles of green chemistry : the new chemicals must be designed to preserve effectiveness of function while reducing toxicity , and not persisting in the environment at the end of their usage , but breaking down into inoffensive degradation products . most of the ionic liquids with imidazolium , phosphonium , pyridinium and ammonium that were tested were resistant to ready biodegradation . their toxicity to microorganisms can limit biodegradation , while their toxicity to humans and others organisms is obviously significant . the examination regarding the biodegradation of surfactant compounds focuses on a close resemblance between many quaternary ammonium compounds as well as surfactants based around an imidazolium core . the factors that improved the biodegradation of surfactants have successfully been applied to ionic liquids . for instance , bis(trifluoromethylsulfonyl)imide ( tfmsi ) and pf6 ionic liquids containing an ester in the side chain exhibit the same hydrophobic character as 1-n - butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ( [ bmim][tfmsi ] ) and 1-n - butyl-3-methylimidazolium hexafluorophosphate ( [ bmim][pf6 ] ) . the enzymatic hydrolysis step , which initiates a pathway to further breakdown products , improves the biodegradation . therefore , compound stability and toxicity are the factors biodegradability depends on . the effect of the counter - ion was not noticeable in biodegradability even though modifications of the anion led to changes in physical and chemical properties . still , the introduction of an organic anion clearly improves the extent of ultimate biodegradation . from the point of view of reactivity , il generally do not coordinate to metal complexes , enzymes and different organic substrates ; however , they are usually the major component of the mixtures having pre - organized structures with the aid of many hydrogen bonds ( structural directionality ) in contrast to classical salts in which the compounds are mostly formed with the aid of ionic bonds ( charge - ordering structures ) . on the other way , the recycling ability of il , especially dialkyl - imidazolium based ionic liquids ( the most studied until now ) , is based on their lack of solubility in some key organic solvents ( e.g. diethyl ether ) and in water - for the special case of 1-n - butyl-3-methylimidazolium hexafluorophosphate ( [ bmim][pf6 ] ) . water / soluble ionic liquids are more difficult to recycle , since their water immiscible complements , the secondary products , can not be easily removed . the detailed examination of relative energies and structural interactions ( like ion position , h - bonding and anion conformational variability ) in gas - phase ion - pairs has emphasized the way these quantities can be used to build up a picture of the local structure and interactions occurring in ionic liquids . for instance , while 1-n - butyl-3-methylimidazolium chloride ( [ bmim][cl ] ) forms a highly connected liquid with relatively strong interactions at one extreme , and 1-n - butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ( [ bmim][tfmsi ] ) forms a low connectivity network of weakly linked ions at the other extreme , [ bmim][bf4 ] lying between these two extremes forms a weak but more regular network . melting points and viscosity are partly dependent on local interactions between an ion and other molecules in the first solvation shell . for imidazolium based cations , hunt et al proved that the hydrogen bond is primarily ionic with a moderate covalent character . the fact that the coulombic attraction is the dominant stabilization force was demonstrated by the analysis of the charge distribution , molecular orbitals and electron density of the dimer complex 1-n - butyl-3-methylimidazolium chloride ( [ bmim][cl ] ) : the interactions governing the top conformers are very different from those in which the cl anion remains in plane ( were cl anion interacts with the manifold of orbitals ) ; the ion - pair lumo is the cation anti - bonding lumo and thus electron acceptance is not favorable . the effect of the chloride anion on rotation of the butyl chain is investigated and found to lower some rotational barriers while enhancing others . since the coulombic forces are significant in an ionic liquid , charge distribution or point charges on the constituent ions are likely to be more significant than in liquids made up of neutral charge molecules and deserve a more detailed study . in this respect , the costs of all approaches for sustainable product design can be reduced using sar and qsar methods . it has already been proved that the anti - microbial activity of quaternary ammonium chlorides is lipophilicity - dependent . while the 1-octanol - water partition coefficient could be seen only as the first approximation for compound lipophylicity , bioaccumulation and toxicity in fish , as well as sorption to soil and sediments assumes that lipophylicity is the main factor of anti - microbial activity . nevertheless , aiming at a deeper understanding of the specific mechanistic description of il eco - toxicity , it is worth considering that the ionic liquid properties are more comprehensively quantified through lipophylicity , polarizability and total energy as a unitarily complex of factors in developing appropriate structure - activity relationship ( sar ) studies . however , the main problem in assessing the viable qsar studies to predict ionic liquid toxicities concerns the anionic - cationic interaction superimposed on the anionic and cationic subsystems containing ionic liquids . one may address the search of special rules for assessing the anionic - cationic structural separately from the individual anionic and cationic ones , and then generating the qsar models . yet , because the cationic and anionic effects on liquid toxicity are merely separately studied at the moment , the appropriate strategy would be to firstly derive the anionic and cationic qsars and only then to move on to a qsar of the ionic liquid viewed as an anionic - cationic interaction . the current paper shows , for the first time , how the latter procedure may be implemented by means of the vectorial approach of the spectral - sar analysis . the illustration of the s - sar - il model presented is performed by studying the aquatic bacteria vibrio fischeri toxicity against a list of twenty - two ionic liquids , appropriately chosen so that they should contain a wide variety of heads , side chains , and anions . this way , the present methodology and results may be extended over a wide range of organisms towards designing specific eco - toxicological ionic liquid batteries . although ionic liquid structure is currently defined through three types of substructures , the head group ( the positively charged moiety ) with the side chain , r1 , r2 , etc . , which are substituents on heads group , and the anion , the present approach views il as consisting mainly of its cationic ( head and side chain ) and anionic subsystems in mutual interaction . this way , the two sub - systems containing ionic liquid can be modeled through their vectorial activities that sum up into the predicted activity of the ionic liquid as a whole . this picture is further sustained by the possibility of employment of the spectral - sar method to the present purposes . without going into details , if one has to solve the correlation between a set of biological activities of n - compounds with the set of m - structural properties of each of them , a spectral algebraic algorithm can be applied based on the vectorial view of the descriptors and measured activity combined with the gram - schmidt orthogonalization recipe and on the scalar product rule giving out a real number from any two arbitrary n - dimensional vectors let s also note that when considering the n - biological activities as well as their respective predictor variables grouped into vectors , as displayed in table 1 , the unity vector x0 = 1 1 ... 1 was added to account for the free correlation term in ( 1 ) . actually , the s - sar equation is derived from the determinant : once it is expanded along its first column , and where with these ingredients the qsar equation ( 1 ) becomes operational , with all coefficients effectively worked out . however , besides the effectiveness of the s - sar methodology in reproducing the old - fashioned multi - linear qsar analysis , one of its advances concerns the possibility of introducing norms associated with either predicted ( computed ) or experimental ( measured ) activities , thus gaining the possibility of the unique assignment of a number to a specific type of correlation , i.e. performing a sort of final quantification of the models . nevertheless , the activity norm given in eq . ( 6 ) opens the possibility of replacing the classical statistical correlation factor with a new definition , introducing the so called algebraic s - sar correlation factor as the ratio of the spectral norm of the predicted activity versus that of the measured one : in fact this new correlation factor definition compares the vectorial lengths of the predicted activity against the measured one , thus being an indicator of the extent to which certain computed property or activity approaches the dimension of the observed quantity . however , it was also shown that practically the algebraic correlation factor ( 8) furnishes higher values than its statistical counterpart definition ( 7 ) , in a systematical manner , thus making it the ideal tool for the present attempt to consider the ionic liquid activity taken from its cationic and anionic sub - system ones . moreover , with the help of both spectral norms and correlation factors we can introduce the so called the least path principle in terms of paths between the tested models ( or endpoints ) : thus providing a practical tool for deciding the dominant hierarchies along the possible ones with the important consequence of picturing the mechanistic and almost temporal evolution of structural causes that trigger the observed effects . this methodology was successfully applied in ecotoxicology and for designing the behavior of the species interactions within a test battery , promising to furnish the frame also in ionic liquids analysis . basically , since we may consider the ionic liquid as composed by the anionic and cationic subsystems , we can employ the spectral vectorial space of anionic and cationic activities to form the ionic liquid one throughout the vectorial resultant as displayed also in figure 1 . the vectorial summation in ( 11 ) may also be seen as the interference of the anionic and cationic activities in the space of ionic liquid so that their mutual interaction is included . actually , searching for the predicted norm of the vectorial ionic liquid activity one can notice the interference effects between the anionic and cationic systems through the correlation angle between the associated vectors , cosac . nevertheless , its practical definition can be achieved , since the anionic - cationic norm ( 13 ) is rewritten employing the vectorial relation ( 11 ) to the scalar product between the anionic and cationic vectors now , from the two equivalent expressions ( 13 ) and ( 14 ) , the anionic - cationic correlation angle results in a numerical form from the vectorial components of the anionic and cationic activities basically , the degree of anionic - cationic activity interaction in ionic liquid is fixed by the value of the angle ( 15 ) for each envisaged qsar model or endpoint . finally , aiming to obtain the ionic liquid working qsar equation from the cationic and anionic counterparts one simply needs to expand the norm and the scalar product in ( 14 ) as the square sum of components , according to the norm and scalar product definitions ( 12 ) and ( 2 ) , respectively , with the intermediate result : hence , by the direct identification of right and left side terms the quested equation arises with these all the required concepts and analytical tools are given for that any ionic liquid anionic - cationic interaction and resulted activity analysis on certain species or organism be performed , while concrete example will in next be exposed . without going into details , if one has to solve the correlation between a set of biological activities of n - compounds with the set of m - structural properties of each of them , a spectral algebraic algorithm can be applied based on the vectorial view of the descriptors and measured activity combined with the gram - schmidt orthogonalization recipe and on the scalar product rule giving out a real number from any two arbitrary n - dimensional vectors let s also note that when considering the n - biological activities as well as their respective predictor variables grouped into vectors , as displayed in table 1 , the unity vector x0 = 1 1 ... 1 was added to account for the free correlation term in ( 1 ) . actually , the s - sar equation is derived from the determinant : once it is expanded along its first column , and where with these ingredients the qsar equation ( 1 ) becomes operational , with all coefficients effectively worked out . however , besides the effectiveness of the s - sar methodology in reproducing the old - fashioned multi - linear qsar analysis , one of its advances concerns the possibility of introducing norms associated with either predicted ( computed ) or experimental ( measured ) activities , thus gaining the possibility of the unique assignment of a number to a specific type of correlation , i.e. performing a sort of final quantification of the models . nevertheless , the activity norm given in eq . ( 6 ) opens the possibility of replacing the classical statistical correlation factor with a new definition , introducing the so called algebraic s - sar correlation factor as the ratio of the spectral norm of the predicted activity versus that of the measured one : in fact this new correlation factor definition compares the vectorial lengths of the predicted activity against the measured one , thus being an indicator of the extent to which certain computed property or activity approaches the dimension of the observed quantity . however , it was also shown that practically the algebraic correlation factor ( 8) furnishes higher values than its statistical counterpart definition ( 7 ) , in a systematical manner , thus making it the ideal tool for the present attempt to consider the ionic liquid activity taken from its cationic and anionic sub - system ones . moreover , with the help of both spectral norms and correlation factors we can introduce the so called the least path principle in terms of paths between the tested models ( or endpoints ) : thus providing a practical tool for deciding the dominant hierarchies along the possible ones with the important consequence of picturing the mechanistic and almost temporal evolution of structural causes that trigger the observed effects . this methodology was successfully applied in ecotoxicology and for designing the behavior of the species interactions within a test battery , promising to furnish the frame also in ionic liquids analysis . basically , since we may consider the ionic liquid as composed by the anionic and cationic subsystems , we can employ the spectral vectorial space of anionic and cationic activities to form the ionic liquid one throughout the vectorial resultant as displayed also in figure 1 . the vectorial summation in ( 11 ) may also be seen as the interference of the anionic and cationic activities in the space of ionic liquid so that their mutual interaction is included . actually , searching for the predicted norm of the vectorial ionic liquid activity one can notice the interference effects between the anionic and cationic systems through the correlation angle between the associated vectors , cosac . nevertheless , its practical definition can be achieved , since the anionic - cationic norm ( 13 ) is rewritten employing the vectorial relation ( 11 ) to the scalar product between the anionic and cationic vectors now , from the two equivalent expressions ( 13 ) and ( 14 ) , the anionic - cationic correlation angle results in a numerical form from the vectorial components of the anionic and cationic activities basically , the degree of anionic - cationic activity interaction in ionic liquid is fixed by the value of the angle ( 15 ) for each envisaged qsar model or endpoint . finally , aiming to obtain the ionic liquid working qsar equation from the cationic and anionic counterparts one simply needs to expand the norm and the scalar product in ( 14 ) as the square sum of components , according to the norm and scalar product definitions ( 12 ) and ( 2 ) , respectively , with the intermediate result : hence , by the direct identification of right and left side terms the quested equation arises with these all the required concepts and analytical tools are given for that any ionic liquid anionic - cationic interaction and resulted activity analysis on certain species or organism be performed , while concrete example will in next be exposed . usually , the classical assessment of new industrial chemicals is inflexible , being directed by regulations and standardized procedures , while biological test systems are very expensive and the costs increase with the number of new chemicals released . so , a flexible test strategy is needed in correlating the chemical compounds with the systems they act upon . although qualitative ( eco ) toxicological algorithms for selection of test systems have been proposed in terms of identification of individual effects of different head - groups ( with identical side chains and anions ) and different anions ( with identical cations ) , a quantitative theoretical prediction algorithm for tested compounds in biological systems is still need be proposed due the lack of readily accessible ecotoxicological data . the principles of green chemistry say that one has to consider the whole process ( life cycle analysis ) rather than individual components of reaction ( single issue sustainability ) , based on the fact that the acute toxicity measurements do not provide a complete characterization of the full impact of a substance release into environment , but are only part of the environmental impact assessment [ 6 , 8 ] . in this context , ionic liquids with cations like pyridinium , imidazolium and pyrrolidinium have already been nominated in the united states national toxicology program ( ntp ) for toxicological testing based upon their potential of new solvents but also due they ability to enter in aquatic system ; if an accidentally discharge of ionic liquids into water occur , many of them being water soluble , they may be an environmental risk to aquatic plants and animals . for this reason the current application will develop the s - sar complete algorithm up to the mechanistic prediction of the correlated structural causes and ecotoxicological effects for the ionic liquids of figure 2 , containing ammonium , pyridinium , phosphonium , choline , and imidazolium cations , on aquatic bacteria vibrio fischeri . whereas the vibrio fisheri species was previously found to be one of the most resistant species to ordinary phenol compounds toxicity , the present ionic liquids show quite a wide structural variety to furnish useful information of their environmental action . the cationic and anionic structural properties , the lipophylicity , the electronic polarizability and the total energy , where computed with the hyperchem computational environment and displayed in table 2 together with the reported measured activity of their containing ionic liquids , respectively . the data in table 2 are suitable arranged so that the s - sar analysis is performed successively at the cationic and anionic level and then at the ionic liquid levels based on equations ( 4)(10 ) and ( 15)(17 ) in a hansch type expansion : when accounting for different combinations between the lipophylicity ( hydrophobic character ) , polarizability ( electronic character ) and total energy ( steric character ) factors , respectively . the spectral hierarchy of the predicted activities with respect to different concerned endpoints would lead to the mechanistic scheme according with the cationic and anionic sides as well as the overall ionic liquids influences the environmental ( vibrio fischeri ) toxicity . as earlier mentioned , the first step in our analysis consists in deriving the cationic and anionic qsars that link the structural lipophilic - electronic - steric parameters of the ionic liquids of figure 2 and table 2 with the observed activities of the whole containing ionic liquids upon the vibrio fischeri species . the results are presented in tables 3 and 4 for the cationic and anionic subsystems for all main combinations , i.e. generating the uni - modes ia - to - ic when only one structural parameter is correlated , the two - modes iia - to - iic when two combined structural factors are taken into account and for the three - mode correlation iii with all structural factors involved , respectively . for each such mode of action , the associated endpoint norm , the statistic and algebraic correlation factors were reported , computed with the equations ( 6 ) , ( 7 ) , and ( 8) , respectively . as a general observation , in all cases there was recorded a systematic increase of the correlation factor when computed in spectral space , i.e. using the algebraic definition , as compared with the standard statistical values . we would like to take this opportunity to advocate the use of the algebraic definition instead of the statistical one since the first one has the physical meaning of the length of action respecting the old - fashioned dispersion analysis . nevertheless , dispersion being a consequence of the appropriateness of the fit , the vectorial norm or the length of action accounts merely for the degree with which a certain model approaches the observed , or measured or manifested ( chemical - biological ) interaction . in this respect , it is also worth noting that both cationic and anionic predicted activities poorly resemble the experimentally expected activities with a smooth increase on the anionic influence , for all computed models except two cases based on the lipophylicity correlation ( ia and iia ) . apparently , this behaviour disagrees with the previously reported studies in which the anionic effect was only marginal in cytotoxicity , but in some special cases of certain ionic liquids tests . this situation was based on the observation that , for instance , since imidazolium ring in cations is a delocalized aromatic system with high electron acceptor potential , the nitrogen atoms are not capable to form any hydrogen bonds and the result is a very rigid and sterically inflexible system , while the elongation of r2 residue in side chain leads to a continuous increase of flexibility implying more conformational freedom . the reason for this actions relies on the fragmental hydrophobicity of each carbon connected to a quaternary amine which , combining the geometric bond factor ( that applies to the neutral solute ) with a negative electronic bond factor , decreases its magnitude with the square of the distance from the central nitrogen atom . moreover , the systematic variation of r1 , r2 , etc . , at identical head groups and anions , in all published data , from the molecular to individual organism level , leads to the conclusion that the shorter the chain lengths of side chains the lower the cytotoxicity ( higher ec50 values ) . the electronic portion of the bond factor extends along a chain of no more than 5 alkane carbons causes a decrease in overall hydrophobicity so the chains longer than 56 atom carbons will have a greater permeability through the cell membrane ( see for example [ dmim][bf4 ] ) . this , probably because the chemical transformation of the side chains of ionic liquids may reduce toxicity as far as the metabolites are less toxic compared to their parent chemicals . next , aiming to combine the two somewhat separate effects of cations and anions in the ionic liquid activities , the spectral - sar results are given in the table 5 , showing the working ionic liquid qsar equation as well the actual vectorial norm , statistic and algebraic correlation factors for each mode of action envisaged so far . the data in table 5 clearly demonstrate that the ionic liquid s - sar models always predict higher norms in endpoint activities thus providing the considerable increase in the algebraic correlation factors with respect to the cationic and anionic subsystem effects . it is very interesting to see that the statistical values not only furnish lower values than the algebraic outputs for the ionic liquids , but often lie even below the corresponding statistical values of the cationic and anionic subsystems . this situation gives us a chance to establish the limits of using the dispersion based correlation factor definition since it does not properly reproduced the addition effect of the two interacting subsystems as the anionic - cationic interaction in the ionic liquid structures . on the other hand , the mixture effect of the cationic and anionic vectorial actions in ( eco)toxicological studies is well established as far as the single substances of a mixture acts in similar and close quantified manner in all considered modes . the fact that this is the present case can be firstly visualized by the close inspection of tables 3 and 4 where the spectral norms of the predicted activities feature close values in relatively narrow domain of actions . moreover , the reinforcement of this idea comes from the data of table 6 in which the angle of interactions between the cationic and anionic subsystems are computed , as given by the formula ( 15 ) , for all considered endpoints with almost constant values around the 0.600 cosines of the angle , while the only higher fluctuation deviation appears in the ia and iia cases the same previously evidenced for the cationic dominancy over the anion effects . overall , once the electronic and steric mechanisms have also been considered , the anion could play a central role as technicophore because it exhibits a high potential for change technological properties ( solubility , viscosity ) or due its peculiarity to partially decompose itself in the ion pair interaction , leading to its dominant effect in the chemical - biological engaged activity . with these consideration we can safely assume that the spectral - sar in general and the algebraic correlation factors in particular are especially suited for modelling the ( eco)toxicological activities of ionic liquids from its anionic and cationic component effects . the final part of discussion is devoted for picturing a mechanistically mode of action for cationic , anionic and of their summed effects in containing ionic liquids on the considered vibrio fischeri species . that is , the minimum path procedure among all possible ways connecting endpoints from each category of models ( i.e. with one , two or three factors dependency ) is to be considered . the path lengths were computed employing the equation ( 10 ) to all cationic , anionic and ionic liquids data of tables 3 , 4 , and 5 , and the results are displayed in the table 7 , respectively . however , in order to identify the shortest paths in each category of endpoint connections , the following rules are applied : the first choice is the overall minimum path in a certain column of table 7 ( i.e. a system with a specific way of correlation factor ) ; if the overall minimum belongs to many equivalent paths ( as is the case of cationic with algebraic factor column in table 7 , for instance ) the minimum path will be considered the one that links the starting endpoint with the closest endpoint in the sense of norms ( as is the norm of iia mode the closest to the norm of ia mode in cationic - algebraic column of table 7 , for example ) ; the overall minimum path will set the dominant hierarchy of the mechanistically mode of action towards the experimentally observed activity and will be called the alpha path ( ) ; once the alpha path has been set the next minimum path will be looked for such that the starting endpoint should be different from the one involved in the alpha path ( that is , if in the alpha path the starting end point was ia , the next path to be identified will begin either from the ib or the ic mode ) ; the next minimum paths are chosen on the same rules as before and will be called as beta and gamma paths , and , respectively . at the end of this procedure each mode of action is touched only once , except for the final endpoint , here iii ( as a computational substitute of exp ) , so that all the methodology being regarded as searching minimum path throughout variation of paths with the fixed final endpoint . now , the alpha , beta and gamma path can be easily identified in table 7 and there are accordingly marked . with the aim of giving the complete results of the analysis in a single shot , figure 3 is a representation of the data , with the norms for each cationic , anionic , and resulted cationic liquid being drawn , linked by the major hierarchical paths against the considered mode of actions toward the observed length ( norm ) of the toxicity activity of the vibrio fischeri species , for both statistical and algebraic correlation pictures . figure 7 clearly underlines the fact that while anionic and cationic activity tendencies are somewhat complementary , they do not cancel each other in the ionic liquid that contains them but add up to attain the overall observed toxicity , in the spectral norm besides this , some other useful information can be extracted from figure 7 concerning the major path of structural causes in manifested toxicological action as revealed bellow . while in cationic case the statistical and algebraic path does not coincide at all ( e.g. what the alpha ia - iic - iii path in statistic differs than the alpha ia - iia - iii path in algebraic views ) , in the anionic case they are identically predicted ( excepting the fact that in the algebraic case the shortened paths are registered ) , together providing a mixed behavior for the resulted ionic liquid ( only the beta path ic - iic - iii is overlapping between statistic and algebraic views ) . while in cationic and anionic subsystems the path hierarchies are reversed , as and , in the resulting ionic liquid again the mixture effect is recorded since the succession , against the successions of starting endpoints iaibic , respectively . it is worth pointing out here that , indeed , the cationic alpha path is started on the lipophylicity causes ( ia ) , the same as for the containing ionic liquid , a different situation arising for the alpha anionic path that is beginning with the steric influence ( ic ) . this way the previously noted dominance of cationic influence when correlated with lipophylicity as well the recorded anionic influence related with steric effects are in this picture theoretically confirmed . other interesting features about ecotoxicological paths rely on the fact that , while for cationic and anionic subsystems the algebraic paths are systematically lower that the corresponding statistical ones , in the ionic liquid case the situation is reversed . the interpretation of this fascinating result is that it also confirms that the chemical - biological ionic liquid dispersive ( not specific ) actions in environment are merely through its subsystem components than from itself as a whole . this result further motivates the design of ionic liquids by tailoring the properties of its containing anionic and cationic components for prevent their hazardous toxicity . finally , let us also note that the ecotoxicological paths in cationic and anionic subsystems are summed up in the paths of the corresponding ionic liquids in a not trivial ways : from these s - sar equations some conceptual , however computationally based , ecotoxicological path rules for ionic liquids can be concluded , namely : the anionic gamma path effect is marginal over the cationic alpha path equation ( 19 ) ; the cationic and anionic beta paths decay into the gamma ionic liquid path when met together so that recording a sort of reciprocal cancellation of their effects equation ( 20 ) ; the anionic alpha path effect is reinforcing over the cationic gamma path averaging both at the beta path level of the resulted ionic liquid equation ( 21 ) . this way , the s - sar model presented seems to provide a unitary picture of the anionic - cationic interaction in ionic liquids as conciliating the anionic and cationic effects observed so far . however , further studies on different species with diverse computation schemes and parameters are required in order to conceptually asses a definitely [ a definitive ] theory of ionic liquid inter- and intra- mode of action . usually , the classical assessment of new industrial chemicals is inflexible , being directed by regulations and standardized procedures , while biological test systems are very expensive and the costs increase with the number of new chemicals released . so , a flexible test strategy is needed in correlating the chemical compounds with the systems they act upon . although qualitative ( eco ) toxicological algorithms for selection of test systems have been proposed in terms of identification of individual effects of different head - groups ( with identical side chains and anions ) and different anions ( with identical cations ) , a quantitative theoretical prediction algorithm for tested compounds in biological systems is still need be proposed due the lack of readily accessible ecotoxicological data . the principles of green chemistry say that one has to consider the whole process ( life cycle analysis ) rather than individual components of reaction ( single issue sustainability ) , based on the fact that the acute toxicity measurements do not provide a complete characterization of the full impact of a substance release into environment , but are only part of the environmental impact assessment [ 6 , 8 ] . in this context , ionic liquids with cations like pyridinium , imidazolium and pyrrolidinium have already been nominated in the united states national toxicology program ( ntp ) for toxicological testing based upon their potential of new solvents but also due they ability to enter in aquatic system ; if an accidentally discharge of ionic liquids into water occur , many of them being water soluble , they may be an environmental risk to aquatic plants and animals . for this reason the current application will develop the s - sar complete algorithm up to the mechanistic prediction of the correlated structural causes and ecotoxicological effects for the ionic liquids of figure 2 , containing ammonium , pyridinium , phosphonium , choline , and imidazolium cations , on aquatic bacteria vibrio fischeri . whereas the vibrio fisheri species was previously found to be one of the most resistant species to ordinary phenol compounds toxicity , the present ionic liquids show quite a wide structural variety to furnish useful information of their environmental action . the cationic and anionic structural properties , the lipophylicity , the electronic polarizability and the total energy , where computed with the hyperchem computational environment and displayed in table 2 together with the reported measured activity of their containing ionic liquids , respectively . the data in table 2 are suitable arranged so that the s - sar analysis is performed successively at the cationic and anionic level and then at the ionic liquid levels based on equations ( 4)(10 ) and ( 15)(17 ) in a hansch type expansion : when accounting for different combinations between the lipophylicity ( hydrophobic character ) , polarizability ( electronic character ) and total energy ( steric character ) factors , respectively . the spectral hierarchy of the predicted activities with respect to different concerned endpoints would lead to the mechanistic scheme according with the cationic and anionic sides as well as the overall ionic liquids influences the environmental ( vibrio fischeri ) toxicity . as earlier mentioned , the first step in our analysis consists in deriving the cationic and anionic qsars that link the structural lipophilic - electronic - steric parameters of the ionic liquids of figure 2 and table 2 with the observed activities of the whole containing ionic liquids upon the vibrio fischeri species . the results are presented in tables 3 and 4 for the cationic and anionic subsystems for all main combinations , i.e. generating the uni - modes ia - to - ic when only one structural parameter is correlated , the two - modes iia - to - iic when two combined structural factors are taken into account and for the three - mode correlation iii with all structural factors involved , respectively . for each such mode of action , the associated endpoint norm , the statistic and algebraic correlation factors were reported , computed with the equations ( 6 ) , ( 7 ) , and ( 8) , respectively . as a general observation , in all cases there was recorded a systematic increase of the correlation factor when computed in spectral space , i.e. using the algebraic definition , as compared with the standard statistical values . we would like to take this opportunity to advocate the use of the algebraic definition instead of the statistical one since the first one has the physical meaning of the length of action respecting the old - fashioned dispersion analysis . nevertheless , dispersion being a consequence of the appropriateness of the fit , the vectorial norm or the length of action accounts merely for the degree with which a certain model approaches the observed , or measured or manifested ( chemical - biological ) interaction . in this respect , it is also worth noting that both cationic and anionic predicted activities poorly resemble the experimentally expected activities with a smooth increase on the anionic influence , for all computed models except two cases based on the lipophylicity correlation ( ia and iia ) . apparently , this behaviour disagrees with the previously reported studies in which the anionic effect was only marginal in cytotoxicity , but in some special cases of certain ionic liquids tests . this situation was based on the observation that , for instance , since imidazolium ring in cations is a delocalized aromatic system with high electron acceptor potential , the nitrogen atoms are not capable to form any hydrogen bonds and the result is a very rigid and sterically inflexible system , while the elongation of r2 residue in side chain leads to a continuous increase of flexibility implying more conformational freedom . the reason for this actions relies on the fragmental hydrophobicity of each carbon connected to a quaternary amine which , combining the geometric bond factor ( that applies to the neutral solute ) with a negative electronic bond factor , decreases its magnitude with the square of the distance from the central nitrogen atom . moreover , the systematic variation of r1 , r2 , etc . , at identical head groups and anions , in all published data , from the molecular to individual organism level , leads to the conclusion that the shorter the chain lengths of side chains the lower the cytotoxicity ( higher ec50 values ) . the electronic portion of the bond factor extends along a chain of no more than 5 alkane carbons causes a decrease in overall hydrophobicity so the chains longer than 56 atom carbons will have a greater permeability through the cell membrane ( see for example [ dmim][bf4 ] ) . this , probably because the chemical transformation of the side chains of ionic liquids may reduce toxicity as far as the metabolites are less toxic compared to their parent chemicals . next , aiming to combine the two somewhat separate effects of cations and anions in the ionic liquid activities , the spectral - sar results are given in the table 5 , showing the working ionic liquid qsar equation as well the actual vectorial norm , statistic and algebraic correlation factors for each mode of action envisaged so far . the data in table 5 clearly demonstrate that the ionic liquid s - sar models always predict higher norms in endpoint activities thus providing the considerable increase in the algebraic correlation factors with respect to the cationic and anionic subsystem effects . it is very interesting to see that the statistical values not only furnish lower values than the algebraic outputs for the ionic liquids , but often lie even below the corresponding statistical values of the cationic and anionic subsystems . this situation gives us a chance to establish the limits of using the dispersion based correlation factor definition since it does not properly reproduced the addition effect of the two interacting subsystems as the anionic - cationic interaction in the ionic liquid structures . on the other hand , the mixture effect of the cationic and anionic vectorial actions in ( eco)toxicological studies is well established as far as the single substances of a mixture acts in similar and close quantified manner in all considered modes the fact that this is the present case can be firstly visualized by the close inspection of tables 3 and 4 where the spectral norms of the predicted activities feature close values in relatively narrow domain of actions . moreover , the reinforcement of this idea comes from the data of table 6 in which the angle of interactions between the cationic and anionic subsystems are computed , as given by the formula ( 15 ) , for all considered endpoints with almost constant values around the 0.600 cosines of the angle , while the only higher fluctuation deviation appears in the ia and iia cases the same previously evidenced for the cationic dominancy over the anion effects . overall , once the electronic and steric mechanisms have also been considered , the anion could play a central role as technicophore because it exhibits a high potential for change technological properties ( solubility , viscosity ) or due its peculiarity to partially decompose itself in the ion pair interaction , leading to its dominant effect in the chemical - biological engaged activity . with these consideration we can safely assume that the spectral - sar in general and the algebraic correlation factors in particular are especially suited for modelling the ( eco)toxicological activities of ionic liquids from its anionic and cationic component effects . the final part of discussion is devoted for picturing a mechanistically mode of action for cationic , anionic and of their summed effects in containing ionic liquids on the considered vibrio fischeri species . that is , the minimum path procedure among all possible ways connecting endpoints from each category of models ( i.e. with one , two or three factors dependency ) is to be considered . the path lengths were computed employing the equation ( 10 ) to all cationic , anionic and ionic liquids data of tables 3 , 4 , and 5 , and the results are displayed in the table 7 , respectively . however , in order to identify the shortest paths in each category of endpoint connections , the following rules are applied : the first choice is the overall minimum path in a certain column of table 7 ( i.e. a system with a specific way of correlation factor ) ; if the overall minimum belongs to many equivalent paths ( as is the case of cationic with algebraic factor column in table 7 , for instance ) the minimum path will be considered the one that links the starting endpoint with the closest endpoint in the sense of norms ( as is the norm of iia mode the closest to the norm of ia mode in cationic - algebraic column of table 7 , for example ) ; the overall minimum path will set the dominant hierarchy of the mechanistically mode of action towards the experimentally observed activity and will be called the alpha path ( ) ; once the alpha path has been set the next minimum path will be looked for such that the starting endpoint should be different from the one involved in the alpha path ( that is , if in the alpha path the starting end point was ia , the next path to be identified will begin either from the ib or the ic mode ) ; the next minimum paths are chosen on the same rules as before and will be called as beta and gamma paths , and , respectively . at the end of this procedure each mode of action is touched only once , except for the final endpoint , here iii ( as a computational substitute of exp ) , so that all the methodology being regarded as searching minimum path throughout variation of paths with the fixed final endpoint . now , the alpha , beta and gamma path can be easily identified in table 7 and there are accordingly marked . with the aim of giving the complete results of the analysis in a single shot , figure 3 is a representation of the data , with the norms for each cationic , anionic , and resulted cationic liquid being drawn , linked by the major hierarchical paths against the considered mode of actions toward the observed length ( norm ) of the toxicity activity of the vibrio fischeri species , for both statistical and algebraic correlation pictures . figure 7 clearly underlines the fact that while anionic and cationic activity tendencies are somewhat complementary , they do not cancel each other in the ionic liquid that contains them but add up to attain the overall observed toxicity , in the spectral norm besides this , some other useful information can be extracted from figure 7 concerning the major path of structural causes in manifested toxicological action as revealed bellow . while in cationic case the statistical and algebraic path does not coincide at all ( e.g. what the alpha ia - iic - iii path in statistic differs than the alpha ia - iia - iii path in algebraic views ) , in the anionic case they are identically predicted ( excepting the fact that in the algebraic case the shortened paths are registered ) , together providing a mixed behavior for the resulted ionic liquid ( only the beta path ic - iic - iii is overlapping between statistic and algebraic views ) . while in cationic and anionic subsystems the path hierarchies are reversed , as and , in the resulting ionic liquid again the mixture effect is recorded since the succession , against the successions of starting endpoints iaibic , respectively . it is worth pointing out here that , indeed , the cationic alpha path is started on the lipophylicity causes ( ia ) , the same as for the containing ionic liquid , a different situation arising for the alpha anionic path that is beginning with the steric influence ( ic ) . this way the previously noted dominance of cationic influence when correlated with lipophylicity as well the recorded anionic influence related with steric effects are in this picture theoretically confirmed . other interesting features about ecotoxicological paths rely on the fact that , while for cationic and anionic subsystems the algebraic paths are systematically lower that the corresponding statistical ones , in the ionic liquid case the situation is reversed . the interpretation of this fascinating result is that it also confirms that the chemical - biological ionic liquid dispersive ( not specific ) actions in environment are merely through its subsystem components than from itself as a whole . this result further motivates the design of ionic liquids by tailoring the properties of its containing anionic and cationic components for prevent their hazardous toxicity . finally , let us also note that the ecotoxicological paths in cationic and anionic subsystems are summed up in the paths of the corresponding ionic liquids in a not trivial ways : from these s - sar equations some conceptual , however computationally based , ecotoxicological path rules for ionic liquids can be concluded , namely : the anionic gamma path effect is marginal over the cationic alpha path equation ( 19 ) ; the cationic and anionic beta paths decay into the gamma ionic liquid path when met together so that recording a sort of reciprocal cancellation of their effects equation ( 20 ) ; the anionic alpha path effect is reinforcing over the cationic gamma path averaging both at the beta path level of the resulted ionic liquid equation ( 21 ) . this way , the s - sar model presented seems to provide a unitary picture of the anionic - cationic interaction in ionic liquids as conciliating the anionic and cationic effects observed so far . however , further studies on different species with diverse computation schemes and parameters are required in order to conceptually asses a definitely [ a definitive ] theory of ionic liquid inter- and intra- mode of action . despite the promise to change the face of organic chemistry , the ionic liquids are becoming a central issue in green chemistry as well , due to their unique physico - chemical properties . for instance , their high viscosities compared with conventional molecular solvents have impact on lowering the reaction rates while through their higher degree of immersion into ground soil and sediments the ecotoxicity of various species and organisms are recorded . the latter effects are based on their common supposed mechanism through membrane disruption : having structural similarity with detergents , pesticides and antibiotics they induce polar narcosis due to their interfacial properties and may cause membrane - bond protein disruption . ionic liquids modes of action are based on the fact that the disrupt membranes and hydrophobic molecules have a greater ability to accumulate at this interface , while other mechanisms may arise from acetylcholinesterase inhibition , from common cellular structure or processes , from structural dna damage [ 23 , 32 ] . eventually , some bacteria could potentially break down imidazolium into different metabolites which follow further inhibition on themselves . in any case , low lipophylicity imidazolium based ionic liquids values indicate low permeability of these ionic liquids . in addition , to the certain role that the lipophylicity has in the assessment of ionic liquid toxicity on various species , before constructing kit- or battery tests involving chemical - biological level of organization , from enzyme to cellular to short and long lived organism [ 2,4,79,23,24,26,3144 ] , worth focusing on the quantitative algorithm with the help of which the experimental data will be interpret . this is because , criticized for its lacking in real ecological meaning , the dose - response approach for estimating the lethal effects of toxicants on organisms regulatory norms have been built around lc50 values that can be compared between different toxicants and organisms , while the statistical correlation factors of the proposed qsars so far are based on dispersion ( i.e. hazardous ) action and less accounting on the specific ways of ionic liquids action . in this regard , it is important to know if the tested chemicals could reach the target site into organism in a specifically manner first , so that an extrapolation from in vitro to in vivo is necessarily while other factors like biodegradation and bioaccumulation are necessarily be considered before conclusions are drawn . having to consider both specific and hazardous paths of action concerning the environmental risk posed by the newly released ionic liquids the recently introduced spectral - sar scheme of quantification of chemical - biological interaction is adapted here to the particular ionic liquid cationic - anionic structural interaction . the present method is based on the previous evaluation of qsars with specific norms and algebraic correlation factors for cationic and anionic influences followed by their vectorial interference in the spectral norm space . with a short generation lifetime , bacteria is an ideal starting point for ionic liquid structure - activity relationship investigation and serves as a basis for further toxicity tests to higher organisms and more complex systems . this way , the s - sar - il model adapted here was applied to vibrio fischeri species , this species being reported as the most resistant to other conventional environmental releasing chemical compounds . the results are promising , confirming at some level of analysis all previous certified facts , the cationic generally dominance , the special anionic effects , the ionic pair effect , etc . , adding in an elegant manner the least ( or minimum ) path rule in the spectral norm - correlation factor space as an effective tool in which the ecotoxicological rules of the ionic liquids can be derived . worth , however , noted that when choosing the hansch factors , although there was already pointed out that the use of free enthalpy instead of total energy provides better statistical correlation factors , in the present study the total energy was preferred due to its close connection with the sterical effects through the computational geometrical optimization procedure involved . this way , the minimal comprehensive set of qsar descriptors , i.e. hydrophobic , electronic , and steric , when predict the ecotoxicity endpoints was assured . in this respect , further studies may address the effect of the length of alkyl chain of the imidazolium - based ionic liquid on the activity . the present approach leaves room for further investigation at both the conceptual and computational levels of expertise when improving the specificity of analysis in terms of considered structural factors and of their sar combinations or when generalizing it to the eco - design of multiple species battery , respectively .

within the recently launched the spectral - structure activity relationship ( s - sar ) analysis , the vectorial anionic - cationic model of a generic ionic liquid is proposed , along with the associated algebraic correlation factor in terms of the measured and predicted activity norms . the reliability of the present scheme is tested by assessing the hansch factors , i.e. lipophylicity , polarizability and total energy , to predict the ecotoxicity endpoints of wide types of ionic liquids with ammonium , pyridinium , phosphonium , choline and imidazolium cations on the aquatic bacteria vibrio fischeri . the results , while confirming the cationic dominant influence when only lipophylicity is considered , demonstrate that the anionic effect dominates all other more specific interactions . it was also proved that the s - sar vectorial model predicts considerably higher activity for the ionic liquids than for its anionic and cationic subsystems separately , in all considered cases . moreover , through applying the least norm - correlation path principle , the complete toxicological hierarchies are presented , unfolding the ecological rules of combined cationic and anionic influences in ionic liquid toxicity .

1. Introduction 2. The Spectral-SAR Ionic Liquid (S-SAR-IL) Model 2.1. S-SAR Concepts 2.2. S-SAR for Ionic Liquids 3. Application to Ecotoxicology 3.1. The Working System 3.2. Results and Discussion 4. Conclusions

most of the ionic liquids with imidazolium , phosphonium , pyridinium and ammonium that were tested were resistant to ready biodegradation . nevertheless , aiming at a deeper understanding of the specific mechanistic description of il eco - toxicity , it is worth considering that the ionic liquid properties are more comprehensively quantified through lipophylicity , polarizability and total energy as a unitarily complex of factors in developing appropriate structure - activity relationship ( sar ) studies . however , the main problem in assessing the viable qsar studies to predict ionic liquid toxicities concerns the anionic - cationic interaction superimposed on the anionic and cationic subsystems containing ionic liquids . yet , because the cationic and anionic effects on liquid toxicity are merely separately studied at the moment , the appropriate strategy would be to firstly derive the anionic and cationic qsars and only then to move on to a qsar of the ionic liquid viewed as an anionic - cationic interaction . the illustration of the s - sar - il model presented is performed by studying the aquatic bacteria vibrio fischeri toxicity against a list of twenty - two ionic liquids , appropriately chosen so that they should contain a wide variety of heads , side chains , and anions . without going into details , if one has to solve the correlation between a set of biological activities of n - compounds with the set of m - structural properties of each of them , a spectral algebraic algorithm can be applied based on the vectorial view of the descriptors and measured activity combined with the gram - schmidt orthogonalization recipe and on the scalar product rule giving out a real number from any two arbitrary n - dimensional vectors let s also note that when considering the n - biological activities as well as their respective predictor variables grouped into vectors , as displayed in table 1 , the unity vector x0 = 1 1 ... 1 was added to account for the free correlation term in ( 1 ) . however , besides the effectiveness of the s - sar methodology in reproducing the old - fashioned multi - linear qsar analysis , one of its advances concerns the possibility of introducing norms associated with either predicted ( computed ) or experimental ( measured ) activities , thus gaining the possibility of the unique assignment of a number to a specific type of correlation , i.e. ( 6 ) opens the possibility of replacing the classical statistical correlation factor with a new definition , introducing the so called algebraic s - sar correlation factor as the ratio of the spectral norm of the predicted activity versus that of the measured one : in fact this new correlation factor definition compares the vectorial lengths of the predicted activity against the measured one , thus being an indicator of the extent to which certain computed property or activity approaches the dimension of the observed quantity . however , it was also shown that practically the algebraic correlation factor ( 8) furnishes higher values than its statistical counterpart definition ( 7 ) , in a systematical manner , thus making it the ideal tool for the present attempt to consider the ionic liquid activity taken from its cationic and anionic sub - system ones . basically , since we may consider the ionic liquid as composed by the anionic and cationic subsystems , we can employ the spectral vectorial space of anionic and cationic activities to form the ionic liquid one throughout the vectorial resultant as displayed also in figure 1 . the vectorial summation in ( 11 ) may also be seen as the interference of the anionic and cationic activities in the space of ionic liquid so that their mutual interaction is included . actually , searching for the predicted norm of the vectorial ionic liquid activity one can notice the interference effects between the anionic and cationic systems through the correlation angle between the associated vectors , cosac . nevertheless , its practical definition can be achieved , since the anionic - cationic norm ( 13 ) is rewritten employing the vectorial relation ( 11 ) to the scalar product between the anionic and cationic vectors now , from the two equivalent expressions ( 13 ) and ( 14 ) , the anionic - cationic correlation angle results in a numerical form from the vectorial components of the anionic and cationic activities basically , the degree of anionic - cationic activity interaction in ionic liquid is fixed by the value of the angle ( 15 ) for each envisaged qsar model or endpoint . finally , aiming to obtain the ionic liquid working qsar equation from the cationic and anionic counterparts one simply needs to expand the norm and the scalar product in ( 14 ) as the square sum of components , according to the norm and scalar product definitions ( 12 ) and ( 2 ) , respectively , with the intermediate result : hence , by the direct identification of right and left side terms the quested equation arises with these all the required concepts and analytical tools are given for that any ionic liquid anionic - cationic interaction and resulted activity analysis on certain species or organism be performed , while concrete example will in next be exposed . however , besides the effectiveness of the s - sar methodology in reproducing the old - fashioned multi - linear qsar analysis , one of its advances concerns the possibility of introducing norms associated with either predicted ( computed ) or experimental ( measured ) activities , thus gaining the possibility of the unique assignment of a number to a specific type of correlation , i.e. ( 6 ) opens the possibility of replacing the classical statistical correlation factor with a new definition , introducing the so called algebraic s - sar correlation factor as the ratio of the spectral norm of the predicted activity versus that of the measured one : in fact this new correlation factor definition compares the vectorial lengths of the predicted activity against the measured one , thus being an indicator of the extent to which certain computed property or activity approaches the dimension of the observed quantity . however , it was also shown that practically the algebraic correlation factor ( 8) furnishes higher values than its statistical counterpart definition ( 7 ) , in a systematical manner , thus making it the ideal tool for the present attempt to consider the ionic liquid activity taken from its cationic and anionic sub - system ones . basically , since we may consider the ionic liquid as composed by the anionic and cationic subsystems , we can employ the spectral vectorial space of anionic and cationic activities to form the ionic liquid one throughout the vectorial resultant as displayed also in figure 1 . the vectorial summation in ( 11 ) may also be seen as the interference of the anionic and cationic activities in the space of ionic liquid so that their mutual interaction is included . actually , searching for the predicted norm of the vectorial ionic liquid activity one can notice the interference effects between the anionic and cationic systems through the correlation angle between the associated vectors , cosac . nevertheless , its practical definition can be achieved , since the anionic - cationic norm ( 13 ) is rewritten employing the vectorial relation ( 11 ) to the scalar product between the anionic and cationic vectors now , from the two equivalent expressions ( 13 ) and ( 14 ) , the anionic - cationic correlation angle results in a numerical form from the vectorial components of the anionic and cationic activities basically , the degree of anionic - cationic activity interaction in ionic liquid is fixed by the value of the angle ( 15 ) for each envisaged qsar model or endpoint . finally , aiming to obtain the ionic liquid working qsar equation from the cationic and anionic counterparts one simply needs to expand the norm and the scalar product in ( 14 ) as the square sum of components , according to the norm and scalar product definitions ( 12 ) and ( 2 ) , respectively , with the intermediate result : hence , by the direct identification of right and left side terms the quested equation arises with these all the required concepts and analytical tools are given for that any ionic liquid anionic - cationic interaction and resulted activity analysis on certain species or organism be performed , while concrete example will in next be exposed . for this reason the current application will develop the s - sar complete algorithm up to the mechanistic prediction of the correlated structural causes and ecotoxicological effects for the ionic liquids of figure 2 , containing ammonium , pyridinium , phosphonium , choline , and imidazolium cations , on aquatic bacteria vibrio fischeri . the cationic and anionic structural properties , the lipophylicity , the electronic polarizability and the total energy , where computed with the hyperchem computational environment and displayed in table 2 together with the reported measured activity of their containing ionic liquids , respectively . the data in table 2 are suitable arranged so that the s - sar analysis is performed successively at the cationic and anionic level and then at the ionic liquid levels based on equations ( 4)(10 ) and ( 15)(17 ) in a hansch type expansion : when accounting for different combinations between the lipophylicity ( hydrophobic character ) , polarizability ( electronic character ) and total energy ( steric character ) factors , respectively . the spectral hierarchy of the predicted activities with respect to different concerned endpoints would lead to the mechanistic scheme according with the cationic and anionic sides as well as the overall ionic liquids influences the environmental ( vibrio fischeri ) toxicity . as earlier mentioned , the first step in our analysis consists in deriving the cationic and anionic qsars that link the structural lipophilic - electronic - steric parameters of the ionic liquids of figure 2 and table 2 with the observed activities of the whole containing ionic liquids upon the vibrio fischeri species . the results are presented in tables 3 and 4 for the cationic and anionic subsystems for all main combinations , i.e. next , aiming to combine the two somewhat separate effects of cations and anions in the ionic liquid activities , the spectral - sar results are given in the table 5 , showing the working ionic liquid qsar equation as well the actual vectorial norm , statistic and algebraic correlation factors for each mode of action envisaged so far . the data in table 5 clearly demonstrate that the ionic liquid s - sar models always predict higher norms in endpoint activities thus providing the considerable increase in the algebraic correlation factors with respect to the cationic and anionic subsystem effects . it is very interesting to see that the statistical values not only furnish lower values than the algebraic outputs for the ionic liquids , but often lie even below the corresponding statistical values of the cationic and anionic subsystems . this situation gives us a chance to establish the limits of using the dispersion based correlation factor definition since it does not properly reproduced the addition effect of the two interacting subsystems as the anionic - cationic interaction in the ionic liquid structures . on the other hand , the mixture effect of the cationic and anionic vectorial actions in ( eco)toxicological studies is well established as far as the single substances of a mixture acts in similar and close quantified manner in all considered modes . moreover , the reinforcement of this idea comes from the data of table 6 in which the angle of interactions between the cationic and anionic subsystems are computed , as given by the formula ( 15 ) , for all considered endpoints with almost constant values around the 0.600 cosines of the angle , while the only higher fluctuation deviation appears in the ia and iia cases the same previously evidenced for the cationic dominancy over the anion effects . with these consideration we can safely assume that the spectral - sar in general and the algebraic correlation factors in particular are especially suited for modelling the ( eco)toxicological activities of ionic liquids from its anionic and cationic component effects . it is worth pointing out here that , indeed , the cationic alpha path is started on the lipophylicity causes ( ia ) , the same as for the containing ionic liquid , a different situation arising for the alpha anionic path that is beginning with the steric influence ( ic ) . other interesting features about ecotoxicological paths rely on the fact that , while for cationic and anionic subsystems the algebraic paths are systematically lower that the corresponding statistical ones , in the ionic liquid case the situation is reversed . finally , let us also note that the ecotoxicological paths in cationic and anionic subsystems are summed up in the paths of the corresponding ionic liquids in a not trivial ways : from these s - sar equations some conceptual , however computationally based , ecotoxicological path rules for ionic liquids can be concluded , namely : the anionic gamma path effect is marginal over the cationic alpha path equation ( 19 ) ; the cationic and anionic beta paths decay into the gamma ionic liquid path when met together so that recording a sort of reciprocal cancellation of their effects equation ( 20 ) ; the anionic alpha path effect is reinforcing over the cationic gamma path averaging both at the beta path level of the resulted ionic liquid equation ( 21 ) . this way , the s - sar model presented seems to provide a unitary picture of the anionic - cationic interaction in ionic liquids as conciliating the anionic and cationic effects observed so far . for this reason the current application will develop the s - sar complete algorithm up to the mechanistic prediction of the correlated structural causes and ecotoxicological effects for the ionic liquids of figure 2 , containing ammonium , pyridinium , phosphonium , choline , and imidazolium cations , on aquatic bacteria vibrio fischeri . the cationic and anionic structural properties , the lipophylicity , the electronic polarizability and the total energy , where computed with the hyperchem computational environment and displayed in table 2 together with the reported measured activity of their containing ionic liquids , respectively . the data in table 2 are suitable arranged so that the s - sar analysis is performed successively at the cationic and anionic level and then at the ionic liquid levels based on equations ( 4)(10 ) and ( 15)(17 ) in a hansch type expansion : when accounting for different combinations between the lipophylicity ( hydrophobic character ) , polarizability ( electronic character ) and total energy ( steric character ) factors , respectively . the spectral hierarchy of the predicted activities with respect to different concerned endpoints would lead to the mechanistic scheme according with the cationic and anionic sides as well as the overall ionic liquids influences the environmental ( vibrio fischeri ) toxicity . as earlier mentioned , the first step in our analysis consists in deriving the cationic and anionic qsars that link the structural lipophilic - electronic - steric parameters of the ionic liquids of figure 2 and table 2 with the observed activities of the whole containing ionic liquids upon the vibrio fischeri species . the results are presented in tables 3 and 4 for the cationic and anionic subsystems for all main combinations , i.e. next , aiming to combine the two somewhat separate effects of cations and anions in the ionic liquid activities , the spectral - sar results are given in the table 5 , showing the working ionic liquid qsar equation as well the actual vectorial norm , statistic and algebraic correlation factors for each mode of action envisaged so far . the data in table 5 clearly demonstrate that the ionic liquid s - sar models always predict higher norms in endpoint activities thus providing the considerable increase in the algebraic correlation factors with respect to the cationic and anionic subsystem effects . it is very interesting to see that the statistical values not only furnish lower values than the algebraic outputs for the ionic liquids , but often lie even below the corresponding statistical values of the cationic and anionic subsystems . this situation gives us a chance to establish the limits of using the dispersion based correlation factor definition since it does not properly reproduced the addition effect of the two interacting subsystems as the anionic - cationic interaction in the ionic liquid structures . on the other hand , the mixture effect of the cationic and anionic vectorial actions in ( eco)toxicological studies is well established as far as the single substances of a mixture acts in similar and close quantified manner in all considered modes the fact that this is the present case can be firstly visualized by the close inspection of tables 3 and 4 where the spectral norms of the predicted activities feature close values in relatively narrow domain of actions . moreover , the reinforcement of this idea comes from the data of table 6 in which the angle of interactions between the cationic and anionic subsystems are computed , as given by the formula ( 15 ) , for all considered endpoints with almost constant values around the 0.600 cosines of the angle , while the only higher fluctuation deviation appears in the ia and iia cases the same previously evidenced for the cationic dominancy over the anion effects . with these consideration we can safely assume that the spectral - sar in general and the algebraic correlation factors in particular are especially suited for modelling the ( eco)toxicological activities of ionic liquids from its anionic and cationic component effects . other interesting features about ecotoxicological paths rely on the fact that , while for cationic and anionic subsystems the algebraic paths are systematically lower that the corresponding statistical ones , in the ionic liquid case the situation is reversed . finally , let us also note that the ecotoxicological paths in cationic and anionic subsystems are summed up in the paths of the corresponding ionic liquids in a not trivial ways : from these s - sar equations some conceptual , however computationally based , ecotoxicological path rules for ionic liquids can be concluded , namely : the anionic gamma path effect is marginal over the cationic alpha path equation ( 19 ) ; the cationic and anionic beta paths decay into the gamma ionic liquid path when met together so that recording a sort of reciprocal cancellation of their effects equation ( 20 ) ; the anionic alpha path effect is reinforcing over the cationic gamma path averaging both at the beta path level of the resulted ionic liquid equation ( 21 ) . this way , the s - sar model presented seems to provide a unitary picture of the anionic - cationic interaction in ionic liquids as conciliating the anionic and cationic effects observed so far . in addition , to the certain role that the lipophylicity has in the assessment of ionic liquid toxicity on various species , before constructing kit- or battery tests involving chemical - biological level of organization , from enzyme to cellular to short and long lived organism [ 2,4,79,23,24,26,3144 ] , worth focusing on the quantitative algorithm with the help of which the experimental data will be interpret . the present method is based on the previous evaluation of qsars with specific norms and algebraic correlation factors for cationic and anionic influences followed by their vectorial interference in the spectral norm space . , adding in an elegant manner the least ( or minimum ) path rule in the spectral norm - correlation factor space as an effective tool in which the ecotoxicological rules of the ionic liquids can be derived . worth , however , noted that when choosing the hansch factors , although there was already pointed out that the use of free enthalpy instead of total energy provides better statistical correlation factors , in the present study the total energy was preferred due to its close connection with the sterical effects through the computational geometrical optimization procedure involved .

it is mainly transmitted from person to person through direct contact with exudates from early skin lesions of infected people . yaws is considered a disease of poverty occurring in tropical regions throughout the world with heavy rainfall and high humidity . it is more common in rural and isolated populations where access to health care is often limited . crowded environments and poor hygiene are also considered as factors facilitating transmission [ 4 , 5 ] . the disease affects predominantly children younger than 15 years ( the peak incidence of clinical manifestations is 2 to 10 years ) , who serve as the primary reservoir of the disease . the current knowledge is that transmission is by direct contact with infected lesions and that flies , including nonbiting haematophagous ones , can infect skin breaches through their dejecta or regurgitation [ 6 , 7 ] . reported that a yaws - like treponema was identified in african monkeys and baboons , and more recently robed et al . mentioned that the genetic analysis of a strain collected from a guinean baboon demonstrated a close relation to the human strains of yaws . furthermore , yaws - like infections have been identified in nonhuman primates in africa , in particular in the republic of congo where 17% of a wild gorilla population have been found with typical yaw lesions leading the authors to speculate that yaws infections in gorillas and humans living in tropical rain forests might be due to the same bacterium treponema pertenue . considering that in humans and gorillas t. pertenue spreads by direct contact with infected lesions and that flies also play a role in transmission of the bacterium , a risk of contamination between humans and other primates might exist . these findings argue in favor of a potential role of yaws - infected nonhuman primates in humans ' infections . however , there is no evidence of such transmission in the literature , and the actual significance of these findings to human is not known [ 2 , 3 , 9 ] . this debate is interesting and certainly deserves more research to establish if pathogens cross - transmit between humans and primates populations . nevertheless , for the western pacific region , we did not find evidence of recent resurgence of yaws in countries where frequent encounters between humans and monkeys occur ( cambodia , malaysia , or vietnam , e.g. ) , but we found evidence of recent yaws reappearance in pacific countries where there is no population of nonhuman primates . furthermore , india which hosts a large population of monkeys with frequent encounters with humans managed to eliminate yaws . therefore , the provocative question of whether humans are in fact the reservoir of the disease to other primates might be raised . in the absence of nonhuman primates in the pacific , humans are the only known reservoir of the disease in this part of the region . it is estimated that there were 50 to 150 million cases of active yaws worldwide in the early 1950s . a global campaign to control endemic treponematoses , including yaws , was launched in 1952 targeting 46 countries . by the end of 1964 , the global campaign , supported by the world health organization and the united nations children 's fund , successfully reduced the prevalence of yaws by 95% to an estimated 2.5 million cases [ 4 , 1012 ] . yaws surveillance and control activities subsequently became integrated into the primary health care systems of individual countries , where remaining cases were to be identified and treated . yaws transmission persisted , however , although at low levels , and the passive approach for yaws control under primary health care systems was not efficient in detecting and treating cases remaining in remote and isolated areas of developing countries . in the 1970s , despite efforts to renew the commitment to yaws control and to reengage the international community ( e.g. , world health assembly resolution 31.58 of 1978 on yaws ; global and regional meetings in the early 1980s ) , yaws persisted in many parts of the world , with the largest number of cases found in west and central africa [ 13 , 14 ] . in the south pacific , records show that yaws was highly prevalent prior to mass treatment campaigns carried out in the late 1950s and early 1960s . following mass treatment campaigns , the number of reported cases dramatically declined , and yaws was considered eliminated in most areas of the south pacific [ 16 , 17 ] . since the late 1970s , however , suspected cases of yaws were reported in various areas of papua new guinea ( png ) [ 1823 ] , the solomon islands [ 16 , 2426 ] , and vanuatu [ 17 , 27 , 28 ] . while available records suggest that these countries remain endemic to this date , the extend of the current burden due to yaws is not well known . in our twelve - year experience as a field public health expert in the pacific , we had to deal with yaws in many instances . over the years , we spent time doing literature researches to address , when they aroused , specific issues linked to yaws control activities in the pacific , and we often felt that a single document providing all available information together with relevant references would be useful to clinicians and to health workers . in the past three decades , yaws control activities have been localized in a number of areas in the pacific , and there is an obvious disconnect between regular field observations of yaws coherent with verbal reports from front line health workers mentioning that yaws has never stopped or has reappeared in the past few decades and the patchy and scarce information found in the literature . there is no document providing an overall picture of the current yaws situation in the region , and the literature consulted over the years gave the impression that most activities have been conducted in silos , answering to a sudden and localized resurgence or interest on yaws . discussions with colleagues and health workers confirmed this overall sense of a heterogeneous approach in the region and the lack of a clear idea of the current situation in regards to a disease that has supposedly been eradicated more than half a century ago but that is regularly encountered in certain parts of the region . it then became clear that a review of all the available information and references for the region would be a useful tool not only to clinicians but also to public health decision makers . a thorough assessment of the yaws situation in the western pacific region ( wpr ) over the past 60 years and the potential challenges yaws eradication poses today could serve as a reminder for the public health community in the region about this neglected disease . it could stimulate a discussion among regional public health decision makers and pave the way for a coordinated approach in the areas still affected . with this in mind , we reviewed the literature currently available , and we provide in this paper a summary of epidemiological records on yaws in the wpr with a special focus on the pacific area since the 1980s . it also reviews past control efforts in the region with an emphasis on png , the solomon islands , and vanuatu . in addition to data provided by who member states , we searched on medline and wholis , the world health organization 's library database , for relevant articles using the term yaws . to identify references specific to the region ( primarily south pacific ) , the following terms were used : south pacific , pacific , and western pacific for countries outside the pacific , and individual country names . some articles from the 1950s to the 1970s in particular were excluded due to the contents being too similar to the ones already referenced or because they were quoted in more recent articles . the whole process identified 53 relevant documents , all of which are referenced in this paper . an additional search with endemic treponematosis and with endemic treponematoses was conducted which found 300 references . the search was then limited to western pacific , pacific , asia , and individual country names in the region together with the term endemic treponematosis or endemic treponematoses . only two additional relevant articles were found but were excluded for similar reasons to those explained above . yaws control during the 1950s and 1960s put a strong emphasis on assessing the population - level prevalence of active and infectious yaws cases . initial surveys often covered a large proportion of endemic populations and were conducted together with mass or selective treatment using procaine penicillin g with 2% aluminum monostearate ( pam ) . for example , in the cook islands , 99% of the population were screened in 1960 , and in vanuatu , an initial mass treatment survey conducted in 1958 covered 94% of the indigenous population . the results of these surveys were also utilized to determine appropriate control methods . in most cases , the national control programs were established with the assistance of who and unicef in most areas of the south pacific and received technical assistance from an interregional treponematosis advisory team consisting of a medical officer , a serologist , and a nurse / administrative officer based in the region , in particular for training of national staff members in conducting surveys and diagnostic techniques . surveys conducted in the 1960s and 1970s showed successful reduction of yaws below detectable levels in most areas of the south pacific . for example , geizer reported that only 6 cases were found in niue in 1957 , while the disease was widely spread earlier . according to geizer , only 3 cases were diagnosed between 1964 and 1984 in fiji , the last one being in 1983 , while initial surveys conducted in 1954 found 10% to 70% prevalence of reactive serology . in tonga , the last 7 cases were reported in 1976 while 7,452 cases were reported in 1962 when the eradication campaign was launched . several authors reported that yaws control activities were then gradually integrated with other communicable disease programs into basic health services without providing further details [ 3 , 9 , 30 ] . in 1985 , meheus and hopkins discussed integration of yaws control activities into primary health care ( phc ) interventions . meheus explains that control of yaws was then relying on several strategies combining population screening with mass treatment , and case finding / diagnosis at consultation with contact tracing / treatment . we did not find concrete examples on how these yaws control activities were introduced , implemented , and integrated in the south pacific . lack of funding associated with weak political commitment and fragile primary health care systems resulted in the continued occurrence of yaws and its poor reporting . by the 1970s and 1980s , sporadic outbreaks were reported in png , the solomon islands , and vanuatu , and in most cases , the immediate responses to these outbreaks were recorded and published . however , as geizer discussed in 1986 , information on yaws became scarce once the outbreaks were contained , and very limited information about surveillance activities is currently available for these countries . in 1984 , the who recommendations were published in response to resurgence in the 1970s . according to the recommendations , a treatment strategy for yaws control thus , the prevalence of active case needs to be assessed in a representative sample of a population of interest ( e.g. , district , province , or country ) in order to determine an appropriate treatment policy for the population . the recommendations also suggest that treatment of the entire population ( total mass treatment ) , treatment of all active cases and obvious contacts ( contacts being defined as those who have frequent , direct , person - to - person contact with a patient with active yaws lesions ) as well as all children under 15 years of age ( juvenile mass treatment ) , or treatment of all active cases and all household and other obvious contacts ( selective mass treatment ) should be adopted based on the level of prevalence of clinically active yaws in the community ( table 1 ) . in addition , it was indicated that in remote areas where access to health services is limited , total mass treatment may be a more appropriate option even when the population prevalence is less than 10% . benzathine benzylpenicillin was recommended in preference to the other forms of penicillin , penicillin aluminium monostearate ( pam ) in particular as the serum concentration of penicillin produced by benzathine benzylpenicillin persists above the treponemicidal level much longer than that produced by pam . the following dosages were recommended : 600.000 units for children below 6 years ; 1.2 million units for children between 6 and 14 years ; 2.4 million units for children above 14 years and adults . these recommendations are still applicable today for the control of yaws , and the same treatment using a single dose of benzathine penicillin is still considered the standard treatment for yaws . tetracycline , erythromycin , or doxycycline can be used for patients with penicillin hypersensitivity . to be successful mass treatments must reach a very high level of coverage . while no studies we reviewed specified the minimum level of coverage to be targeted , hudson et al . argued that the successful eradication campaigns in the 1950s and 1960s can be in part attributed to the fact that the treatment of the entire treponemal reservoir , including active and latent cases , was achieved . stated that all contacts of infectious cases must be treated if yaws is to be eliminated from the community and estimated that 1 to 3% of yaws - affected patients may not be cured by the recommended treatment due to quality issues with the penicillin used or the use of altered penicillin associated with improper storage , reconstitution , or expiration of drugs . while mass treatment is challenging in terms of logistics and requires high political commitment , the low cost of benzathin penicillin and its efficacy make it a valuable option for treating yaws in a population . de noray et al . estimated the cost of the 2001 mass treatment conducted in sanma province , vanuatu , to be usd 1.30 per person . treatment with penicillin always carries the risk of severe side effects such as anaphylactic shock , and this must be considered when planning such interventions [ 2 , 17 ] . based on the who recommendations , serological testing is necessary to identify latent or incubating cases . however , in some cases , serological testing during a prevalence survey may not be necessary , since most latent or incubating cases are found in clusters near an infectious case . these would receive treatment as household or obvious contacts ( even if they were not identified as cases ) . . indicated that the prevalence of active yaws observed during a survey could be used to estimate the prevalence of seroreactor defined as seroactive yaws cases . whether or not these estimations apply to the current epidemiology of yaws is not well understood ( table 2 ) . most yaws control activities implemented in the south pacific since the 1990s followed the above - recommended strategies and treatment regimen . however , in 2010 , fegan et al . raised a question on the use of benzathin penicillin , mainly because of its logistical constraints . the same authors suggested exploring the possibility of using an oral treatment such as azithromycin , a macrolide antibiotic with a known efficacy in t. pallidum infections and with a long half - life in tissue . compared to the challenges posed by treatments with benzathine penicillin ( logistics , possible allergy , and pain associated with the injection ) , azythromycin presents some true advantages . its oral presentation and its efficacy in single - dose administration for the treatment of syphilis in adults make it potentially a more accessible and more acceptable treatment at peripheral level . first , macrolide - resistant t. pallidum initially identified in groups with high - risk sexual behavior in the united states and ireland has now been isolated in other countries such as china where martin et al . report an apparent high prevalence of macrolide resistant t. pallidum in shanghai , the czech republic where the prevalence of the mutations responsible for macrolide - resistance is reported to be as high as 20% in clinical specimens or in canada where 29% of syphilis cases were found to be caused by a strain resistant to azythromycin . the recent identification of macrolide - resistant t. pallidum subspecies pallidum in several countries raises concerns on the possible independent emergence of multiple strains and on the role selective pressure and other conditions may have played . furthermore , it raises questions on the impact of these findings on other subspecies of treponema pallidum including pertenue . it also allows speculations that similar mutations leading to macrolide resistance might quickly appear with yaws bacterium . these reservations and the consistent success of penicillin in the treatment of syphilis and yaws for more than fifty years strongly argue in favor of benzathine penicillin as the treatment of choice for yaws . in 1998 , following a study in 39 children with clinical yaws in karkar island , png , backhouse et al . reported that while initial clinical and serologic responses to benzathine penicillin were satisfactory in more than 90% of the children , 11 ( 28% ) later showed clinical and/or serologic evidence of relapse or reinfection . these authors concluded in favor of treatment failures due to reduced susceptibility to penicillin because reinfection was unlikely in this particular community and under the circumstances of the study . to our knowledge , this possible reduction in susceptibility or tolerance to benzathine penicillin of t. pallidum subsp . pertenue has not been further documented , and additional data to support the finding could not be found in the south pacific . in most cases , diagnosis was based on clinical criteria during the 1950s and 1960s . with the disappearance of yaws in a number of countries and the progressive retirement of those who were involved in the eradication campaigns in the 1950s and 1960s , some authors suggested that most health workers have never seen the disease and may not be able to make a clinical diagnosis . however , our review shows that yaws has been most often detected clinically by primary health care workers , for example , in vanuatu in 2000 and png in 1995 , who then reported to their local authorities , leading to further investigations and control activities . it is also possible that some of those workers had been involved in the past or more recent control efforts and were capable of recognizing the symptoms . on another hand , a number of skin conditions common in the tropics may appear similar to yaws lesions , and thus , it is possible that some proportion of yaws reported in primary health care settings may in fact be false positive cases . in some instances , nevertheless , clinical diagnosis made in recent years may be less specific as the number of primary health care workers experienced in yaws diagnosis is declining , and a large number of active cases identified in recent years are reported as presenting milder symptoms compared to the 1950s [ 18 , 28 , 40 ] . one can speculate about the accuracy of this contention , especially as there is nearly no survey available comparing results of yaws clinical studies in the same area in the 1950s and nowadays . however , for the western pacific region , we found one survey where the authors compare clinical findings from two surveys conducted in 1953 and in 1987 in the same island of the solomon islands and found 10% of cases with secondary or tertiary yaws in 1987 compared to 30% in 1953 . the reasons for these attenuated clinical manifestations are unclear , but they are likely the consequence of a mix of different factors such as more accessible health systems possibly resulting in earlier diagnosis of infections and a wide use of antibiotics ( especially penicillin since the 1950s ) which might delay the occurrence of clinical lesions . common serological tests currently used in the south pacific are the venereal disease research laboratory ( vdrl ) test and the rapid plasma reagin ( rpr ) test . both are sensitive , but they are nontreponemal antigen tests and do not differentiate yaws from other treponemal infections such as syphilis [ 2 , 4 ] . however , it is important to acknowledge that treponemal tests such as the fluorescent treponemal antibody - absorption enzyme immunoassay ( fta - abs ) , the treponema pallidum particle agglutination ( tppa ) test , or the enzyme immunoassay ( eia ) can not make such a distinction either . this reality can be of importance as it represents a particular challenge in countries where both yaws and syphilis are endemic . for example , a question was raised regarding the specificity of the rpr test in vanuatu where surveys in 2000 found an unexpected syphilis prevalence of 2.4% in antenatal clinics women ( rpr confirmed by tpha ) . one of the authors ( c. capuano ) was involved in the discussion , which concluded that in the absence of clinical symptoms , it was not possible to differentiate among the two infections ( personal communications dr . c. capuano , 2001 ) . similarly , the inability to serologically differentiate yaws and syphilis can be an issue in countries where yaws was endemic in the past , and the prevalence of syphilis is known to be high . in 1992 , gershman et al . also raised the issue following a large increase of serologically reported cases of syphilis in the marshall islands , a previously yaws endemic country where it was believed to have been eradicated in the late 1940s and early 1950s . new techniques such as the molecular methods reported in 2006 by centurion - lara et al . to differentiate the three t. pallidum subspecies could be of great assistance in similar situations . however , such methods are expensive , complex , and they require more than the standard laboratory equipment found in the resources - limited countries still affected by yaws in the wpr . these constraints make such methods currently not affordable and of limited value for field use . however , although prevention of yaws and syphilis is very different , both infections warrant treatment regardless of the ability to differentiate them . thus , one can expect that in the wpr , diagnosis of yaws will continue to require the assessment of test results and clinical manifestations while carefully taking into account epidemiologic characteristics of yaws . a nationwide mass treatment targeting the entire population of png took place between 1953 and 1958 under the australian administration covering more than 90% of the population . many of the untreated individuals were residents of remote and isolated areas of the country where campaign reach was limited . the campaign was successful ; only 2,352 cases were reported in 1959 , and fewer than 500 cases were reported each year until 1973 . a slight increase in the number of cases was recorded between 1973 and 1978 . however , it did not exceed 1,000 cases per year , and most of them occurred in rural areas , in bougainville , and around rabaul in new britain . the disease was rare in the highland districts and reported to be nonexistent in the central province and port moresby . the clinical appearance of yaws observed during this time period was milder ( fewer lesions , plantar lesions , and bone involvement rarely observed ) than in the 1950s , and these milder cases were described as attenuated cases [ 18 , 40 ] . in 1964 - 65 , a serological survey for treponemal disease involving 844 sera was conducted in the eastern highlands of png . no clinical case of active yaws was found in the population , but some of the elderly were identified with clinical evidence of old yaws lesions . the seroprevalence reported by the authors varies from 3.9% to 79.2% . in 3 out of the 10 census units surveyed , the prevalence in children below 15 years ranged from 14.3% to 40% , and the authors concluded that the treponemal disease involved was yaws . in december 1978 , a mass campaign targeting the entire population was carried out on karkar island , madang province , after a rapid increase of yaws cases was recorded in the previous year . based on surveys carried out preceding the mass treatment , the estimated prevalence varied from 0% to 27% , and the prevalence of infectious cases ( examined using dark - field microscopy ) also varied from 2% to 22% on the island . although the mass treatment reported to have covered above 90% of the island population , the island continued to report cases of yaws . in 1981 , a more comprehensive survey reported 0.36% prevalence of infectious cases and 2% prevalence of noninfectious clinical cases in the surveyed villages . cases with attenuated manifestation were also recorded during this outbreak , and a local doctor also noted that the response to pam was poorer than in the 1950s . it is possible that penicillin was improperly stored and thus lost its potency . in the early 1980s , clusters of cases were reported in the provinces of east and west sepik , east and west new britain , new ireland , and milne bay . the outbreak and following mass treatments in yilui village , west sepik province , is well documented . in yilui village ( population 509 based on 1980 census ) , the initial mass treatments in early 1984 found 193 clinical cases ( more than 35% of the 1980 population ) . a follow - up treatment conducted six months later found 60 clinical cases , mainly children , including 49 clinical cases treated during the initial mass treatments . serological tests were performed on a sample of clinical and nonsymptomatic individuals . a large proportion of nonsymptomatic individuals presented reactive vdrl tests ( 12 out of 19 ) , suggesting that a large proportion of the village population had been exposed to the infection in the recent past , just prior to the mass treatments . cases with attenuated symptoms were also reported in yilui . in the kiriwina and trobriand islands of milne bay province , a small number of suspected cases among children aged 4 to 13 were reported in 1984 . , another outbreak was reported on karkar island in the village of takia ( population 1600 ) . among 632 children aged 0 to 15 years blood samples were repeatedly collected from the clinical cases over 22 months following the initial treatment , and the authors found that 13% of the cases remained or became serologically positive again at 22 months despite the fact that many of the initial cases were treated multiple times . clinical relapse among the initial clinical cases between april 2000 and september 2001 , the nine mile clinic in port moresby identified 494 cases confirmed by serological tests ( tpha and vdrl ) through clinic - based case detection . the clinic serves approximately 20,000 individuals in the periurban population of northeast port moresby , where many live in squatter settlements with poor access to water and electricity . yaws cases were seen at the clinic as early as 1995 , and a yaws register was created in april 2000 after an increase in numbers was observed in 1999 . the clinic also conducted a prevalence survey in 2001 at settlements near the clinic where prevalence was expected to be high . among 227 children under the age of 17 examined , 33 had active yaws lesions as diagnosed by experienced medical staff . yaws cases had been rare in port moresby , and thus , this finding suggests that the cases observed at the clinic may have contracted yaws outside port moresby or from persons with yaws who migrated from other parts of the country . the population in port moresby is young and rapidly growing with a continuous migration of people throughout the country . with poor hygiene , crowded environments , and a large number of people previously unexposed to yaws records on yaws in most areas of png are not available since the late 1980s . however , as found in port moresby in 2000 and 2001 , cases exist and transmission is still likely to be active . however , it is important to note that no late - stage cases have been seen since the mass treatment in 1950s and many of the more recent cases presented with a small number of lesions . table 3 summarizes yaws reported cases and prevalence surveys in png from 1959 to 2001 . a nationwide mass treatment campaign was carried out in the british solomon islands protectorate between 1956 and 1958 , covering all main islands and most of the other inhabited islands . an initial survey prior to the treatment campaign found the prevalence of active yaws cases above 14.5% . the campaign was successful , and only a few cases were reported ( from malaita ) following the campaign . no cases were documented from 1970 until 1981 when an outbreak of cases was reported in the western province [ 16 , 24 , 44 ] . the cases were initially misdiagnosed as tropical ulcers until suspected cases presenting large leg ulcers were serologically confirmed as yaws in 1984 ( serology done in australia ) . following the confirmation , a mass treatment of the entire population was carried out in the islands of gizo , vella la vella , ranonga , simbo , new georgia , kolombangara , and north choiseul . by the end of the campaign , 3,994 out of 29,235 persons examined ( 13.7% ) the disease was more prevalent among children under the age of 15 ; 28% of children under 15 examined were diagnosed to have yaws . yaws was diagnosed clinically in the field by medical staff involved in the previous mass campaign in the 1950s . a higher prevalence was reported in the islands of vella la vella , ranonga , and simbo . no case had been reported in the shorthand islands , despite its proximity to png . in 1986 , monthly reports from rural health clinics indicated a recurrence of yaws in areas of western province treated during the 1984 mass campaign . a sample survey was conducted in areas where a large number of cases were reported . 83 definite cases ( 10% , papillomatous or ulceropapillomatous ) and 68 suspected cases ( 8.2% , scanty macules and maculopapulomatous ) were identified among 833 examined . based on this finding , a treatment campaign was carried out in 1987 on the islands of vella la vella , ranonga , simbo , kolombangara , new georgia , and gizo . among the total of 24,216 blood was collected from a random sample of the population , and 11% of 453 serum samples showed positive vdrl tests . in simbo island , where 47% of treated individuals ( 580 of 1,220 ) were clinically diagnosed to have yaws , 57% of cases presented primary lesions ( a single raised lesion , often ulcerated ) , while 43% had secondary lesions ( multiple ulcers and small papillomas ) . investigators also noted that cases in 1984 and 1987 presented milder attenuated symptoms ( a few scanty lesions ) than cases in 1950s who often presented with abundant large elevated papillomas . while tertiary yaws was common in the 1950s , it was not observed during the campaign in 1987 . the annual health report of 2007 compiled by the ministry of health provides some evidence of persistent foci existing in the country for the last decade . between 1998 and 2007 , all 10 provinces reported cases of yaws , detected among patients attending primary health care facilities . no survey or mass treatment were reported to have been carried out since 1987 . according to the report , the national incidence rate of yaws in 2007 estimated using the 2007 projected population of the solomon islands the incidence rate in 2007 was high in makira ( 70.4 per 1000 ) and guadalcanal ( 60.3 per 1000 ) , and low in isabel ( 19 per 1000 ) and choiseul ( 17 per 1000 ) . based on the reported incidence rates and the 2007 projected provincial population , the absolute number of cases in 2007 was estimated to exceed 4,000 in guadalcanal and malaita . however , the rates have fluctuated in the past , and the observed change in the rates may be due to changes in diagnosis and recording practices over the 10 year period . in 2007 , a who consultant noted that for the period from 1997 to 2006 the rate of clinical yaws reported was constantly highest in children aged between one and four years , and that in 2003 , the rate increased with a marked rise in children and people aged five years and over . since 2003 , the rate has shown a downward trend , reaching its lowest point in 2006 . in addition , data gathered since 1998 only include cases diagnosed in primary health care facilities . the figures may be overestimated if cases are misdiagnosed or overdiagnosed , or underestimated if many yaws cases do not seek care from the health system . in 1984 and 1986 , a woman in australia who migrated from the solomon islands in 1975 was treated for tertiary stage yaws . she visited the solomon islands in 1981 and suffered from likely secondary - stage yaws upon her visit . the authors speculated that she may have been reinfected during the visit leading to the destructive bony lesions consistent with a tertiary stage of yaws observed in 1984 and 1986 . there has been no other report of imported yaws from the south pacific in australia or new zealand . as indicated by the 2007 national health report , it is likely that yaws is still found in most of the provinces in the solomon islands . however , more information on reported clinical cases , such as detailed descriptions of symptoms and serological status , is necessary to confirm whether the reported cases are true cases of yaws , and to identify the most affected areas . table 4 summarizes yaws reported cases and prevalence surveys in the solomon islands from 1956 to 2007 . in vanuatu , an initial survey treated approximately 94% of the resident population , and a followup was estimated to have covered 92% of the population . about two - thirds of tanna island in the province of tafea was not treated during the initial treatment surveys due to refusal for political reasons . the estimated prevalence after the mass campaigns was reduced to 0.5 per 1000 . throughout the 1970s , further investigation of these cases revealed active yaws transmission in the island . in 1981 and 1985 , mass treatments were carried out in several villages in northwestern tanna where clinical cases had been reported . despite these efforts , yaws continued to reappear in tanna , and a large - scale treatment campaign was planned in 1989 . a survey conducted prior to the mass treatment found 116 ( 16.5% ) clinically suspected cases out of 704 treatment participants in 13 villages and 1 school in tanna . among the 97 clinically suspected cases , 34 showed vdrl titer above 1 : 4 . based on this finding , the mass treatment was planned to cover the entire population of tanna , about 20,700 people . approximately 90% of the population were treated , although children under the age of 3 months were excluded from examination and treatment . during the mass treatment , a large proportion of clinical cases ( 79.3% ) were under the age of 15 , and 32% of blood samples from 189 suspected cases had positive vdrl results . following the campaign , only a few cases were reported in 1990 . however , by 1992 , a number of cases were reported from villages that previously had not participated in the treatment campaign . the laboratory reports between 1995 and 1998 also indicated that yaws was still not eliminated from the island . in 2000 , patients with suspicious lesions were reported on the island of santo , sanma province , where laboratory records showed no confirmed cases between 1995 and 1998 . a preliminary investigation found 21 clinical cases in central and south santo including 20 cases serologically confirmed as yaws . from may to july 2001 , a sample survey representative of the entire island was conducted . among 273 individuals , 57 ( 20.9% ) were serologically positive . the majority ( 70% ) of seropositives were males , and 40% were under 15 years of age . following the survey , a mass treatment of the entire sanma province was carried out . the treatment coverage was estimated to be 92.4% based on the campaign registers and exceeded 100% based on the census population of 1999 . during the treatment campaign , 251 clinically suspected cases were found and treated in 82 villages . a large proportion of clinical cases ( 78% ) were under 15 . among suspected clinical cases who were later serologically tested , 40% ( 96 out of 230 ) had positive vdrl results . it was also noted that cases were concentrated in central and southern parts of the province . the total estimated cost of the 2001 eradication campaign in sanma province reached usd 41,700 for a population of 36,414 or an estimated usd 1.15 per person . in 2007 , 789 cases were reported on a routine health report from the island of tanna . a serological and clinical survey using a cluster sampling method was conducted in 2008 to assess the endemicity of yaws on this island . among 306 individuals from whom blood samples were taken , 95 ( 31% ) were positives for rapid plasma reagin ( rpr ) and/or rapid diagnostic test for syphilis ( rdt ) . it is not stated whether the seropositive individuals showed clinical manifestations of yaws , had been treated for the disease in the recent past , or are active cases of yaws . out of 473 individuals enrolled in the clinical survey , 55 ( 11% ) had typical yaws lesions . sixty six ( 14% ) had skin lesions which may possibly have been yaws , and most of them ( 94% ) were below 15 years of age . during this survey , only one tertiary case of yaws was seen in an adult , presenting with a mild saber tibia . all cases reported since the 1970s were found in clusters on certain islands of tafea and sanma provinces . active case detection and appropriate treatment may be necessary in some communities where a large number of cases were previously reported . yaws has not been officially reported in the other provinces since the mass treatment campaigns in the late 1950s . table 5 summarizes yaws reported cases and prevalence surveys in vanuatu from 1958 to 2008 . with the technical assistance of who and unicef , national yaws control programs were established in fiji and western samoa ( samoa ) in 1955 , gilbert and ellice islands ( kiribati and tuvalu ) in 1957 , and tonga in 1962 . initial surveys conducted by these national programs showed a varying degree of yaws endemicity in the region with the highest prevalence found in fiji ( 28.81% ) . based on the results of these surveys , selective or mass treatment with pam was implemented , and the disease was nearly eliminated in these countries by the 1960s . passive surveillance continued in these areas , and a very small number of cases were reported over the next few decades . between 1969 and 1984 , fiji reported three cases , thought to be the very last in that country . the last reports of yaws in tonga were 7 cases in 1976 . in the cook islands , a nationwide eradication campaign in 1960 covering approximately 99% of the population found 17 cases of infectious yaws and 14 cases of noninfectious yaws . no case has been documented in these areas of the south pacific in recent years . data on past yaws control efforts in remaining areas of the south pacific are very limited . the national control programs were established in cambodia , laos , malaysia , and the philippines in the 1950s . a significant reduction of prevalence to near elimination however , in 1985 , lo reviewed yaws control activities since the 1950s in malaysia and concluded that the program has been a success and that yaws disappearance from malaysia could be expected . in the following years , only a small outbreak consisting of 10 active cases was reported in baling , malaysia in 1989 , and 6 imported cases of yaws were found in a family in johor , malaysia , in 1988 . in australia , the most recent information we found is from garner et al . who reported in 1972 results from a serological survey in the aboriginal population of the northern territory . they concluded that while no case of active treponemal infection was found , the prevalence of treponemal infection varied from 3.4% to 58% indicating that yaws , endemic syphilis , and probably venereal syphilis were present in the aboriginal population . figures 1 , 2 , 3 , and 4 summarize the prevalence of yaws for the wpr from the 1950s to the present time . nearly 98% of cases reported in 2003 were from the provinces of east nusa tenggara , south east sulewasi , papua , and maluku . in indonesia , yaws control became integrated with sexually transmitted disease control in 1980 , further with the leprosy program in 2000 , and then with the water and sanitation program in 2003 . in the early 2000s , the reported annual incidence ranged from 2,000 to 4,500 cases . in timor - leste , the disease has been reported from at least 6 of 13 districts in recent years . yaws control is currently part of an infectious disease control program targeting leprosy , lymphatic filariasis , and soil - transmitted helminthiasis . the estimated incidence of yaws in timor - leste ranges from 500 to 1,000 cases per year . in both indonesia and timor - leste , the assessment of incidence relies on reports from primary health facilities ; therefore , the reported incidences may not be accurate due to underreporting or overdiagnosis . obtaining greater political commitment and sufficient resources has been a key challenge for strengthening the existing yaws control programs in these countries . india declared yaws elimination in 2006 , after an absence of yaws cases since 2004 . the yaws eradication programme in india commenced in 1996 as a pilot program following the development of an eradication strategy by the national institute of communicable disease and was expanded to all endemic areas of the country in 1999 . the strategy consisted of two core activities : ( 1 ) active case detection and treatment of cases and contacts with long - acting penicillin and ( 2 ) community mobilization to raise awareness through information education communication programs . the program was phased into three stages ( attack , maintenance , and elimination / eradication ) and had well - defined criteria for elimination ( nil reporting of new infectious cases ) and eradication ( noseroactivity to rpr or vdrl in children below 5 years of age after achieving nil reporting for three years ) as well as certification processes . four appraisals were conducted in 2000 , 2002 , 2004 , and 2006 , each conducted by an independent team consisting of a public health expert , a dermatologist , and a microbiologist . in addition to careful and detailed planning of program activities , factors thought to have contributed to the success include high - level political commitment maintained throughout the campaign , advocacy for sustained commitment , and the availability of resources . rigorous active case detection , where all suspected cases and contacts were treated , was conducted biannually . supportive monitoring and supervision of peripheral health workers also assured the high quality of case detection and helped to maintain their motivation throughout the campaign . following the declaration of elimination in india , the who south - east asia region has set 2012 as a target of yaws elimination from indonesia and timor - leste . after the successful eradication campaigns of the 1960s , the primary health care systems were supposed to give the last push towards eradication of yaws . however , a combination of various factors including poor political commitment and limited funding resulted in a missed opportunity and disappointed the hopes raised half a century ago . yaws presents new challenges such as poor awareness and knowledge among health care workers , unknown epidemiological situation , and attenuated clinical forms of the disease . these challenges , combined with the current competing priorities in the public health arena , require new , innovative , and country - tailored approaches . recent experience in india as well as past experiences in the south pacific clearly demonstrate that with a well - designed elimination strategy based on reliable epidemiological data and sustained high - level political commitment , it is possible to eliminate yaws . the increased attention required to address the resurgence of yaws in the affected countries does not necessarily mean increased resources . in view of the many health priorities and the human and financial resources constraints , utilization of existing programs with similar primary target population ( i.e. , children under 15 years of age ) in implementing yaws control activities may provide a cost - effective option . for example , yaws screening and detection could be included in school - based health promotion programs such as a deworming program for soil - transmitted helminthiasis or dental surveys . cost - effective strategies in identifying cases , in providing treatment , and in tracing all contacts need to be identified at country level in order to take into account logistical constraints , resources available locally , and other health priorities in the affected countries . many opportunities to revitalize yaws control activities already exist at country level : maternal and child health clinics and their related activities , water and sanitation programmes , or school - based programmes are such examples . one way to approach yaws control might be to address it in the broader context of the primary health care ( phc ) where lessons learnt from past failures would be used to avoid repeating the same mistakes . yaws control activities could be powerful tools for revitalizing and/or strengthening phc in endemic countries , especially if they use the momentum usually created by mass campaigns , which are still necessary to bring down the level of transmission in highly endemic areas . such an approach may mean that yaws eradication will take longer than with repeated mass campaigns , but considering the competing health priorities and other challenges faced by the affected countries , it might be a more sustainable and realistic strategy . it also means that before a mass campaign is considered due consideration must be given to the role of the phc system before , during , and after the campaign is carried out . however , at this stage it is of tremendous importance to get a better understanding of the current epidemiological situation through a strong and systematic assessment of yaws in the pacific in general and in png , vanuatu , and solomon in particular . only then , sound strategies towards elimination of yaws can be developed , and the work started more than half a century ago can be completed .

until the middle of the 20th century , yaws was highly endemic and considered a serious public health problem in the western pacific region ( wpr ) , leading to intensive control efforts in the 1950s1960s . since then , little attention has been paid to its reemergence . its current burden is unknown . this paper presents the results of an extensive literature review , focusing on yaws in the south pacific . available records suggest that the region remains largely free of yaws except for papua new guinea , solomon islands , and vanuatu . many clinical cases reported recently were described as attenuated ; advanced stages are rare . a single intramuscular injection of benzathine penicillin is still effective in curing yaws . in the pacific , yaws may be amenable to elimination if adequate resources are provided and political commitment revived . a mapping of yaws prevalence in png , solomon , and vanuatu is needed before comprehensive country - tailored strategies towards yaws elimination can be developed .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusions

mentioned that the genetic analysis of a strain collected from a guinean baboon demonstrated a close relation to the human strains of yaws . however , there is no evidence of such transmission in the literature , and the actual significance of these findings to human is not known [ 2 , 3 , 9 ] . nevertheless , for the western pacific region , we did not find evidence of recent resurgence of yaws in countries where frequent encounters between humans and monkeys occur ( cambodia , malaysia , or vietnam , e.g. ) in the absence of nonhuman primates in the pacific , humans are the only known reservoir of the disease in this part of the region . , world health assembly resolution 31.58 of 1978 on yaws ; global and regional meetings in the early 1980s ) , yaws persisted in many parts of the world , with the largest number of cases found in west and central africa [ 13 , 14 ] . in the south pacific , records show that yaws was highly prevalent prior to mass treatment campaigns carried out in the late 1950s and early 1960s . following mass treatment campaigns , the number of reported cases dramatically declined , and yaws was considered eliminated in most areas of the south pacific [ 16 , 17 ] . since the late 1970s , however , suspected cases of yaws were reported in various areas of papua new guinea ( png ) [ 1823 ] , the solomon islands [ 16 , 2426 ] , and vanuatu [ 17 , 27 , 28 ] . while available records suggest that these countries remain endemic to this date , the extend of the current burden due to yaws is not well known . in our twelve - year experience as a field public health expert in the pacific , we had to deal with yaws in many instances . over the years , we spent time doing literature researches to address , when they aroused , specific issues linked to yaws control activities in the pacific , and we often felt that a single document providing all available information together with relevant references would be useful to clinicians and to health workers . in the past three decades , yaws control activities have been localized in a number of areas in the pacific , and there is an obvious disconnect between regular field observations of yaws coherent with verbal reports from front line health workers mentioning that yaws has never stopped or has reappeared in the past few decades and the patchy and scarce information found in the literature . there is no document providing an overall picture of the current yaws situation in the region , and the literature consulted over the years gave the impression that most activities have been conducted in silos , answering to a sudden and localized resurgence or interest on yaws . discussions with colleagues and health workers confirmed this overall sense of a heterogeneous approach in the region and the lack of a clear idea of the current situation in regards to a disease that has supposedly been eradicated more than half a century ago but that is regularly encountered in certain parts of the region . a thorough assessment of the yaws situation in the western pacific region ( wpr ) over the past 60 years and the potential challenges yaws eradication poses today could serve as a reminder for the public health community in the region about this neglected disease . it could stimulate a discussion among regional public health decision makers and pave the way for a coordinated approach in the areas still affected . with this in mind , we reviewed the literature currently available , and we provide in this paper a summary of epidemiological records on yaws in the wpr with a special focus on the pacific area since the 1980s . it also reviews past control efforts in the region with an emphasis on png , the solomon islands , and vanuatu . to identify references specific to the region ( primarily south pacific ) , the following terms were used : south pacific , pacific , and western pacific for countries outside the pacific , and individual country names . the search was then limited to western pacific , pacific , asia , and individual country names in the region together with the term endemic treponematosis or endemic treponematoses . for example , in the cook islands , 99% of the population were screened in 1960 , and in vanuatu , an initial mass treatment survey conducted in 1958 covered 94% of the indigenous population . in most cases , the national control programs were established with the assistance of who and unicef in most areas of the south pacific and received technical assistance from an interregional treponematosis advisory team consisting of a medical officer , a serologist , and a nurse / administrative officer based in the region , in particular for training of national staff members in conducting surveys and diagnostic techniques . surveys conducted in the 1960s and 1970s showed successful reduction of yaws below detectable levels in most areas of the south pacific . meheus explains that control of yaws was then relying on several strategies combining population screening with mass treatment , and case finding / diagnosis at consultation with contact tracing / treatment . we did not find concrete examples on how these yaws control activities were introduced , implemented , and integrated in the south pacific . lack of funding associated with weak political commitment and fragile primary health care systems resulted in the continued occurrence of yaws and its poor reporting . by the 1970s and 1980s , sporadic outbreaks were reported in png , the solomon islands , and vanuatu , and in most cases , the immediate responses to these outbreaks were recorded and published . however , as geizer discussed in 1986 , information on yaws became scarce once the outbreaks were contained , and very limited information about surveillance activities is currently available for these countries . the recommendations also suggest that treatment of the entire population ( total mass treatment ) , treatment of all active cases and obvious contacts ( contacts being defined as those who have frequent , direct , person - to - person contact with a patient with active yaws lesions ) as well as all children under 15 years of age ( juvenile mass treatment ) , or treatment of all active cases and all household and other obvious contacts ( selective mass treatment ) should be adopted based on the level of prevalence of clinically active yaws in the community ( table 1 ) . these recommendations are still applicable today for the control of yaws , and the same treatment using a single dose of benzathine penicillin is still considered the standard treatment for yaws . argued that the successful eradication campaigns in the 1950s and 1960s can be in part attributed to the fact that the treatment of the entire treponemal reservoir , including active and latent cases , was achieved . while mass treatment is challenging in terms of logistics and requires high political commitment , the low cost of benzathin penicillin and its efficacy make it a valuable option for treating yaws in a population . most yaws control activities implemented in the south pacific since the 1990s followed the above - recommended strategies and treatment regimen . compared to the challenges posed by treatments with benzathine penicillin ( logistics , possible allergy , and pain associated with the injection ) , azythromycin presents some true advantages . these reservations and the consistent success of penicillin in the treatment of syphilis and yaws for more than fifty years strongly argue in favor of benzathine penicillin as the treatment of choice for yaws . in 1998 , following a study in 39 children with clinical yaws in karkar island , png , backhouse et al . reported that while initial clinical and serologic responses to benzathine penicillin were satisfactory in more than 90% of the children , 11 ( 28% ) later showed clinical and/or serologic evidence of relapse or reinfection . pertenue has not been further documented , and additional data to support the finding could not be found in the south pacific . with the disappearance of yaws in a number of countries and the progressive retirement of those who were involved in the eradication campaigns in the 1950s and 1960s , some authors suggested that most health workers have never seen the disease and may not be able to make a clinical diagnosis . however , our review shows that yaws has been most often detected clinically by primary health care workers , for example , in vanuatu in 2000 and png in 1995 , who then reported to their local authorities , leading to further investigations and control activities . it is also possible that some of those workers had been involved in the past or more recent control efforts and were capable of recognizing the symptoms . on another hand , a number of skin conditions common in the tropics may appear similar to yaws lesions , and thus , it is possible that some proportion of yaws reported in primary health care settings may in fact be false positive cases . in some instances , nevertheless , clinical diagnosis made in recent years may be less specific as the number of primary health care workers experienced in yaws diagnosis is declining , and a large number of active cases identified in recent years are reported as presenting milder symptoms compared to the 1950s [ 18 , 28 , 40 ] . one can speculate about the accuracy of this contention , especially as there is nearly no survey available comparing results of yaws clinical studies in the same area in the 1950s and nowadays . however , for the western pacific region , we found one survey where the authors compare clinical findings from two surveys conducted in 1953 and in 1987 in the same island of the solomon islands and found 10% of cases with secondary or tertiary yaws in 1987 compared to 30% in 1953 . common serological tests currently used in the south pacific are the venereal disease research laboratory ( vdrl ) test and the rapid plasma reagin ( rpr ) test . similarly , the inability to serologically differentiate yaws and syphilis can be an issue in countries where yaws was endemic in the past , and the prevalence of syphilis is known to be high . also raised the issue following a large increase of serologically reported cases of syphilis in the marshall islands , a previously yaws endemic country where it was believed to have been eradicated in the late 1940s and early 1950s . however , such methods are expensive , complex , and they require more than the standard laboratory equipment found in the resources - limited countries still affected by yaws in the wpr . however , although prevention of yaws and syphilis is very different , both infections warrant treatment regardless of the ability to differentiate them . thus , one can expect that in the wpr , diagnosis of yaws will continue to require the assessment of test results and clinical manifestations while carefully taking into account epidemiologic characteristics of yaws . the clinical appearance of yaws observed during this time period was milder ( fewer lesions , plantar lesions , and bone involvement rarely observed ) than in the 1950s , and these milder cases were described as attenuated cases [ 18 , 40 ] . no clinical case of active yaws was found in the population , but some of the elderly were identified with clinical evidence of old yaws lesions . in 3 out of the 10 census units surveyed , the prevalence in children below 15 years ranged from 14.3% to 40% , and the authors concluded that the treponemal disease involved was yaws . in december 1978 , a mass campaign targeting the entire population was carried out on karkar island , madang province , after a rapid increase of yaws cases was recorded in the previous year . although the mass treatment reported to have covered above 90% of the island population , the island continued to report cases of yaws . in 1981 , a more comprehensive survey reported 0.36% prevalence of infectious cases and 2% prevalence of noninfectious clinical cases in the surveyed villages . cases with attenuated manifestation were also recorded during this outbreak , and a local doctor also noted that the response to pam was poorer than in the 1950s . in the early 1980s , clusters of cases were reported in the provinces of east and west sepik , east and west new britain , new ireland , and milne bay . in yilui village ( population 509 based on 1980 census ) , the initial mass treatments in early 1984 found 193 clinical cases ( more than 35% of the 1980 population ) . a large proportion of nonsymptomatic individuals presented reactive vdrl tests ( 12 out of 19 ) , suggesting that a large proportion of the village population had been exposed to the infection in the recent past , just prior to the mass treatments . among 632 children aged 0 to 15 years blood samples were repeatedly collected from the clinical cases over 22 months following the initial treatment , and the authors found that 13% of the cases remained or became serologically positive again at 22 months despite the fact that many of the initial cases were treated multiple times . yaws cases had been rare in port moresby , and thus , this finding suggests that the cases observed at the clinic may have contracted yaws outside port moresby or from persons with yaws who migrated from other parts of the country . with poor hygiene , crowded environments , and a large number of people previously unexposed to yaws records on yaws in most areas of png are not available since the late 1980s . a nationwide mass treatment campaign was carried out in the british solomon islands protectorate between 1956 and 1958 , covering all main islands and most of the other inhabited islands . following the confirmation , a mass treatment of the entire population was carried out in the islands of gizo , vella la vella , ranonga , simbo , new georgia , kolombangara , and north choiseul . yaws was diagnosed clinically in the field by medical staff involved in the previous mass campaign in the 1950s . a higher prevalence was reported in the islands of vella la vella , ranonga , and simbo . no case had been reported in the shorthand islands , despite its proximity to png . while tertiary yaws was common in the 1950s , it was not observed during the campaign in 1987 . according to the report , the national incidence rate of yaws in 2007 estimated using the 2007 projected population of the solomon islands the incidence rate in 2007 was high in makira ( 70.4 per 1000 ) and guadalcanal ( 60.3 per 1000 ) , and low in isabel ( 19 per 1000 ) and choiseul ( 17 per 1000 ) . however , the rates have fluctuated in the past , and the observed change in the rates may be due to changes in diagnosis and recording practices over the 10 year period . the authors speculated that she may have been reinfected during the visit leading to the destructive bony lesions consistent with a tertiary stage of yaws observed in 1984 and 1986 . there has been no other report of imported yaws from the south pacific in australia or new zealand . as indicated by the 2007 national health report , it is likely that yaws is still found in most of the provinces in the solomon islands . however , more information on reported clinical cases , such as detailed descriptions of symptoms and serological status , is necessary to confirm whether the reported cases are true cases of yaws , and to identify the most affected areas . despite these efforts , yaws continued to reappear in tanna , and a large - scale treatment campaign was planned in 1989 . during this survey , only one tertiary case of yaws was seen in an adult , presenting with a mild saber tibia . initial surveys conducted by these national programs showed a varying degree of yaws endemicity in the region with the highest prevalence found in fiji ( 28.81% ) . based on the results of these surveys , selective or mass treatment with pam was implemented , and the disease was nearly eliminated in these countries by the 1960s . the last reports of yaws in tonga were 7 cases in 1976 . in the cook islands , a nationwide eradication campaign in 1960 covering approximately 99% of the population found 17 cases of infectious yaws and 14 cases of noninfectious yaws . no case has been documented in these areas of the south pacific in recent years . data on past yaws control efforts in remaining areas of the south pacific are very limited . in the following years , only a small outbreak consisting of 10 active cases was reported in baling , malaysia in 1989 , and 6 imported cases of yaws were found in a family in johor , malaysia , in 1988 . who reported in 1972 results from a serological survey in the aboriginal population of the northern territory . they concluded that while no case of active treponemal infection was found , the prevalence of treponemal infection varied from 3.4% to 58% indicating that yaws , endemic syphilis , and probably venereal syphilis were present in the aboriginal population . nearly 98% of cases reported in 2003 were from the provinces of east nusa tenggara , south east sulewasi , papua , and maluku . in indonesia , yaws control became integrated with sexually transmitted disease control in 1980 , further with the leprosy program in 2000 , and then with the water and sanitation program in 2003 . yaws control is currently part of an infectious disease control program targeting leprosy , lymphatic filariasis , and soil - transmitted helminthiasis . the estimated incidence of yaws in timor - leste ranges from 500 to 1,000 cases per year . obtaining greater political commitment and sufficient resources has been a key challenge for strengthening the existing yaws control programs in these countries . india declared yaws elimination in 2006 , after an absence of yaws cases since 2004 . the yaws eradication programme in india commenced in 1996 as a pilot program following the development of an eradication strategy by the national institute of communicable disease and was expanded to all endemic areas of the country in 1999 . four appraisals were conducted in 2000 , 2002 , 2004 , and 2006 , each conducted by an independent team consisting of a public health expert , a dermatologist , and a microbiologist . in addition to careful and detailed planning of program activities , factors thought to have contributed to the success include high - level political commitment maintained throughout the campaign , advocacy for sustained commitment , and the availability of resources . after the successful eradication campaigns of the 1960s , the primary health care systems were supposed to give the last push towards eradication of yaws . yaws presents new challenges such as poor awareness and knowledge among health care workers , unknown epidemiological situation , and attenuated clinical forms of the disease . these challenges , combined with the current competing priorities in the public health arena , require new , innovative , and country - tailored approaches . recent experience in india as well as past experiences in the south pacific clearly demonstrate that with a well - designed elimination strategy based on reliable epidemiological data and sustained high - level political commitment , it is possible to eliminate yaws . the increased attention required to address the resurgence of yaws in the affected countries does not necessarily mean increased resources . cost - effective strategies in identifying cases , in providing treatment , and in tracing all contacts need to be identified at country level in order to take into account logistical constraints , resources available locally , and other health priorities in the affected countries . one way to approach yaws control might be to address it in the broader context of the primary health care ( phc ) where lessons learnt from past failures would be used to avoid repeating the same mistakes . however , at this stage it is of tremendous importance to get a better understanding of the current epidemiological situation through a strong and systematic assessment of yaws in the pacific in general and in png , vanuatu , and solomon in particular . only then , sound strategies towards elimination of yaws can be developed , and the work started more than half a century ago can be completed .

the pittsburgh epidemiology of diabetes complications ( edc ) study is a historical , prospective investigation of incident cases of childhood - onset type 1 diabetes diagnosed , or seen within 1 year of diagnosis , at the children s hospital of pittsburgh between 1950 and 1980 . a total of 658 patients who were eligible and willing to participate in the study had an initial clinical assessment between 1986 and 1988 ( mean age 28 years ; mean duration of diabetes 19 years ) and have been subsequently reexamined or surveyed biennially for up to 20 years . for the analyses in this study , edc study participants who were free of cad at study entry and had three stored samples ( blood and either 24-h or overnight urine ) available before the development of cad ( for incident cases ) or the end of follow - up were selected for study ( n = 356 ) . given availability , samples were chosen to reflect an early , midpoint , and late assessment during a participant s follow - up . thus , biologic determinations were approximately 6 years apart for noncases , whereas , given the shorter available follow - up for those developing cad , biomarker measurements were approximately 4 years apart for incident cases . demographic , health care , diabetes self - care , and medical history information were ascertained via self - administered questionnaires before each clinic visit . blood pressure was measured with a random zero sphygmomanometer after a 5-min rest ( 7 ) , and hypertension was defined as blood pressure 140/90 mmhg or use of antihypertensive medications . although the definition of hypertension has been altered in recent years , given that the majority of the follow - up within the edc study , which was initiated in 1986 , falls within the time period during which hypertension was still defined as 140/90 mmhg , we felt that it would not be appropriate to reclassify the cohort retrospectively . nevertheless , statistical analyses were repeated using 130/80 mmhg as the cutoff points for the definition of hypertension . stable glycosylated hemoglobin ( hba1 ) was measured by ion exchange chromatography ( isolab , akron , oh ) for the first 18 months and by automated high - performance liquid chromatography ( diamat , biorad , hercules , ca ) for the subsequent 10 years ; the two assays were highly correlated ( r = 0.95 ) . for follow - up beyond 10 years , hba1c was measured with the dca 2000 analyzer ( bayer , tarrytown , ny ) . the dca 2000 and diamat assays also were highly correlated ( r = 0.95 ) . original hba1 ( 19861998 ) and later hba1c values ( 19982004 ) were converted to standard hba1c values aligned with the diabetes control and complications trial ( dcct ) using regression formulae derived from duplicate assays [ dcct hba1c = ( 0.83 diamat hba1 ) + 0.14 and dcct hba1c = ( dca hba1c 1.13 ) / 0.81 ] . hdl cholesterol was determined enzymatically after precipitation with heparin and manganese chloride , using a modified version of the lipid research clinics method ( 8) . cholesterol and triglycerides were measured enzymatically ( 9,10 ) . urinary albumin was measured by immunonephelometry ( 11 ) , and creatinine was assayed using an ectachem 400 analyzer ( eastman kodak co. , rochester , ny ) . glomerular filtration rate was estimated using the equation used in the chronic kidney disease epidemiology collaboration study ( 12 ) . cad was defined as myocardial infarction confirmed by q - waves on electrocardiogram ( minnesota code 1.1 , 1.2 ) or hospital records , angiographic stenosis 50% , revascularization , or death due to cad . plasma levels of - and -tocopherol as well as urinary 15-isoprostane f2 t ( isop ) were measured from samples collected at the clinical visit at three time points during the 20-year follow - up of the edc study and stored at 70c until the present analyses . the concentrations of - and -tocopherol in plasma samples anticoagulated with edta were measured under subdued lighting , as previously reported ( 13,14 ) . the coefficient of variation between runs is 6.0 and 2.8% for -tocopherol and -tocopherol , respectively . the effect of prolonged storage on serum antioxidant samples was addressed within the multiple risk factor intervention trial ( mrfit ) ; no appreciable degradation was seen in samples stored at 50 to 70c ( 15 ) . urine samples were obtained in 24-h ( 58% ) or overnight ( 42% ) collections . the same type of timed sample was used at all time points for a given individual . levels of isop were measured using a competitive elisa procedure developed by oxford biomedical research ( oxford , mi ) , which has a correlation ( r ) of > 0.8 with gas chromatography mass spectrometry procedures . because there are no published data on the potential effects of prolonged storage on the measurement of isop , we simultaneously performed the assay on stored ( 20- and 10-year ) samples and fresh samples from 6 controls and 11 individuals with type 1 diabetes without renal failure . isop levels were similar and within the normal range at all time points ( controls : 0.96 , 1.6 , and 1.01 ng / mg creatinine , respectively ; individuals with type 1 diabetes : 1.3 , 1.2 , and 0.98 ng / mg creatinine , respectively ) , suggesting stability of the assay . all statistical analyses were conducted using sas version 9.3 ( sas institute , cary , nc ) . given the dependence of plasma tocopherol concentration on lipid levels , - and -tocopherol were reported per unit cholesterol ( 16 ) . urinary isop ( in nanograms per milliliter ) were normalized for urinary creatinine concentration and expressed as nanograms per milligram of creatinine . univariate associations between measurements at the first time point ( baseline ) and subsequent cad status were determined using the student t test for normally distributed continuous variables or the wilcoxon two - sample rank sum test for nonnormally distributed continuous variables . the or fisher exact test were used as appropriate for univariate analysis of categorical variables . since -tocopherol has been shown to act as a pro - oxidant under conditions of low reactive oxidant fluxes ( 17 ) , spearman rank correlations were used at each time point for the entire cohort , and separately for incident cases and noncases , to investigate the association between -tocopherol and urinary isop according to high versus low / normal hba1c . mixed models were used to graph trajectories of the main independent variables over time by subsequent cad status . trajectories were plotted as a function of time : time before cad incidence in cases and time before the end of follow - up in noncases ; cubic splines with continuous second derivatives were used to smooth lines ( 18 ) . the genmod procedure in sas was used to assess the association between the repeated measurements of the main independent variables and cad incidence after adjustment for duration of diabetes and significant univariate risk factors . nonnormally distributed variables were logarithmically transformed to more closely satisfy the assumption of normality for entry into multivariable models ( as noted in tables 13 and fig . participant characteristics at the first time point ( baseline ) by subsequent cad status multivariable mixed models for the subsequent development of cad number of cases and noncases at each time point -tocopherol and urinary isop trajectories ( a ) and -tocopherol - to - urinary isop ratio trajectories ( b ) ( adjusted for duration of diabetes , race , sex , bmi , having ever smoked , hba1c , hypertension , hdl and non - hdl cholesterol , albumin excretion rate ( log ) , estimated glomerular filtration rate , and white blood cell count ) before a cad event or the end of follow - up . all statistical analyses were conducted using sas version 9.3 ( sas institute , cary , nc ) . given the dependence of plasma tocopherol concentration on lipid levels , - and -tocopherol were reported per unit cholesterol ( 16 ) . urinary isop ( in nanograms per milliliter ) were normalized for urinary creatinine concentration and expressed as nanograms per milligram of creatinine . univariate associations between measurements at the first time point ( baseline ) and subsequent cad status were determined using the student t test for normally distributed continuous variables or the wilcoxon two - sample rank sum test for nonnormally distributed continuous variables . the or fisher exact test were used as appropriate for univariate analysis of categorical variables . since -tocopherol has been shown to act as a pro - oxidant under conditions of low reactive oxidant fluxes ( 17 ) , spearman rank correlations were used at each time point for the entire cohort , and separately for incident cases and noncases , to investigate the association between -tocopherol and urinary isop according to high versus low / normal hba1c . mixed models were used to graph trajectories of the main independent variables over time by subsequent cad status . trajectories were plotted as a function of time : time before cad incidence in cases and time before the end of follow - up in noncases ; cubic splines with continuous second derivatives were used to smooth lines ( 18 ) . the genmod procedure in sas was used to assess the association between the repeated measurements of the main independent variables and cad incidence after adjustment for duration of diabetes and significant univariate risk factors . nonnormally distributed variables were logarithmically transformed to more closely satisfy the assumption of normality for entry into multivariable models ( as noted in tables 13 and fig . participant characteristics at the first time point ( baseline ) by subsequent cad status multivariable mixed models for the subsequent development of cad number of cases and noncases at each time point -tocopherol and urinary isop trajectories ( a ) and -tocopherol - to - urinary isop ratio trajectories ( b ) ( adjusted for duration of diabetes , race , sex , bmi , having ever smoked , hba1c , hypertension , hdl and non - hdl cholesterol , albumin excretion rate ( log ) , estimated glomerular filtration rate , and white blood cell count ) before a cad event or the end of follow - up . the concentrations of measured biomarkers of interest ( i.e. , urinary isop and - and -tocopherol ) over time in the entire study group are given in supplementary table 1 . the concentration of -tocopherol increased over time , paralleling the increase in the use of vitamin supplements in the entire edc population ( data not shown ) . no statistically significant changes , however , were observed overall in urinary isop or -tocopherol . among individuals free of cad at study entry and with stored samples available at three times during the 20-year follow - up ( n = 356 ) , 88 ( 24.7% ) developed a cad event . participant characteristics at the first of three time points ( baseline ) by subsequent cad status are shown in table 1 . as expected , individuals who subsequently developed cad were more likely to be older , with a longer duration of diabetes and higher blood pressure , lipid levels , albumin excretion rate , and white blood cell count compared with those who did not develop an event . incident cases were also more likely to have smoked , have micro- or macroalbuminuria , and lower estimated glomerular filtration rate . although no significant differences in urinary isop , -tocopherol per unit cholesterol , or the ratio of -tocopherol to urinary isop were observed between subsequent cases and noncases , it is surprising that -tocopherol ( not adjusting for cholesterol ) was elevated among subsequent cases , whereas -tocopherol levels per unit cholesterol were elevated in individuals without cad compared with subsequent cases . in investigating the strength and direction of a potential association between urinary isop and -tocopherol concentrations , generally inverse associations were observed regardless of the level of glycemic control ( < 7.5 or > 7.5% ) or subsequent cad status , although the strength of the correlation differed by time point ( data not shown ) . separate mixed models were constructed for each main independent variable [ i.e. , urinary isops ( per milligram of urinary creatinine ) , plasma - and -tocopherol ( per unit cholesterol ) , and the -tocopherol - to - isop ratio ] to assess the independent association between repeated measurements of each variable and the subsequent development of cad ( table 2 ) . in multivariable mixed models , a borderline significant direct association was observed between urinary isop concentrations and cad ( p = 0.06 ) , whereas a significant inverse association was observed between plasma -tocopherol concentration and cad ( p = 0.02 ) . repeated measurements of -tocopherol concentration did not seem to independently relate to subsequent cad status ( p = 0.54 ) . interestingly , the -tocopherol - to - urinary isop ratio was strongly inversely associated with cad ( p = 0.003 ) . similar results of an inverse association between the -tocopherol - to - urinary isop ratio and cad incidence ( p = 0.04 ) were observed when including in the data from analyses of an additional 93 individuals with isop measured only in morning 4-hour clinic urine samples . 1b ) from the fully adjusted models ( table 2 ) before the occurrence of a cad event or the end of follow - up ( table 3 gives the number of cases and noncases at each point during the follow - up period ) . although differences did not appear to be of a great magnitude throughout the follow - up period , -tocopherol and the -tocopherol - to - urinary isop ratio were higher , especially during the 7 years before a cad event or the end of follow - up , whereas urinary isop concentrations were lower among individuals who remained free of a cad event . these data suggest that among individuals with type 1 diabetes , the profile of oxs ( i.e. , urinary isoprostanes ) and its potential antioxidant defense ( i.e. , plasma -tocopherol ) differ over time in those who develop incident cad compared with those who do not . this difference is particularly apparent in the 7 years before the incident event and is well characterized by the ratio of -tocopherol to urinary isop . no association was observed between repeated measures of plasma -tocopherol and subsequent development of cad . thus , a direct association of urinary isop concentrations after adjustment for other covariates was observed , whereas plasma -tocopherol and the -tocopherol - to - urinary isop ratio were inversely related to subsequent cad status . clinical trials of the effect of the use of antioxidant vitamin supplements in both the general population as well as individuals with diabetes have generally suggested no benefit of supplementation for the primary prevention of cardiovascular disease and even an increased mortality risk ( 5 ) . indeed , a meta - analysis of six randomized clinical trials comparing cardiovascular death risk with vitamin e versus placebo reported no benefit of supplementation , whereas similar meta - analyses for the effect of -carotene supplementation suggested an increased risk of both all - cause and cardiovascular disease mortality ( 19 ) . interestingly , however , secondary prevention trials have provided somewhat more encouraging results for vitamin e supplementation , especially for myocardial infarction ( 2022 ) , although not all studies agree ( 23 ) . we also have previously shown that a protective effect of -tocopherol against cad seemed to be confined to persons with type 1 diabetes and kidney disease in a small case - control study ( 6 ) . thus , an issue raised is whether antioxidants may provide protection against cardiovascular disease only in susceptible population subgroups , such as those with a prior diagnosis ( secondary prevention ) or who are genetically susceptible . indeed , in three clinical trials a reduced risk of cardiovascular disease with vitamin e therapy was apparent only among individuals with both type 2 diabetes and the haptoglobin 22 genotype ( 2426 ) . we also have previously raised the premise that an adequate response ( e.g. , antioxidant intake ) to a specific insult ( e.g. , oxs ) may reduce or delay the development of pathologic conditions associated with the insult ( 27 ) . thus , despite difficulties in recognizing markers representing protection or resistance in response to a specific stress , the concurrent evaluation of markers representing insult against those representing protection from or resistance to insults has great implications . in the current study , we aimed to assess whether an adequate antioxidative response to oxs could delay or prevent the development of cad in a cohort of individuals with long - standing type 1 diabetes . given that the majority of clinical trials of antioxidant supplementation in relation to cardiovascular disease focused on vitamin e , we measured the plasma concentration of - and -tocopherol . the former has been reported to have greater biologic activity and to be the only form maintained in human plasma ( 28 ) , whereas the latter is the form most commonly found in the american diet ( 4 ) . we further assessed levels of urinary isops , prostaglandin - like compounds derived primarily from nonenzymatic , free radical - induced peroxidation of arachidonic acid ( 29 ) . contrary to reactions catalyzed by enzymes , it is reasonable to assume that the attack by reactive oxygen species on arachidonic acid yields equimolar levels of all f - series isoprostanes . f2-isoprostanes , the first isoprostane class discovered , are detectable in their esterified form in all normal biological tissues and in their free form in all normal biological fluids , including plasma and urine ( 3,30 ) . importantly , their concentration in urine is stable and not affected by arachidonic acid auto - oxidation or daily variation ( 29,31 ) , contributing to their establishment as the gold standard biomarker of oxidant stress . the framingham heart study reported that urinary isoprostane concentrations were significantly associated with smoking , diabetes , and bmi , suggesting a role for systemic oxs in cardiovascular disease ( 32 ) . a small case - control study from germany also noted that urinary isoprostane excretion increased with an increasing number of traditional coronary heart disease risk factors ( 33 ) . indeed , several case - control studies of humans suggested the presence of elevated isoprostane concentrations in either plasma or urine among individuals with cardiovascular disease ( 3337 ) . thus , plasma isoprostane levels were shown to be higher in individuals with atherosclerosis of the carotid or iliofemoral arteries ( 34 ) , as well as among those with angiographic evidence of cad ( 35 ) , compared with controls . urinary isoprostane levels also were reported to be increased with both subclinical atherosclerosis ( 36 ) and manifest cad ( 33,36,37 ) . an important question that remained unanswered , however , was , is oxs the initiator of processes leading to the development of cad ? similarly to previous reports in the general population , we also observed a direct association between urinary concentrations of isoprostane and subsequent cad in individuals with type 1 diabetes , although the association was not very strong , perhaps suggesting that the level of oxs per se is not the sole critical factor in disease pathogenesis , at least in this population . however , simultaneous evaluation of urinary isop and plasma -tocopherol resulted in significant effects for both biomarkers . the -tocopherol - to - urinary isop ratio over time was even more significant for subsequent cad , consistent with the hypothesis that an adequate response to an insult may reduce risk associated with that insult . thus , although the edc study is not a clinical trial , our findings point to the value of concurrent evaluation of insult and response to that insult to identify risk and potentially lead to novel approaches to preventing the progression of insults to full disease ( e.g. , by selective administration of antioxidant therapy to those with a low -tocopherol - to - isop ratio ) , an approach that may merit further evaluation . nonetheless , whether findings from the current study in type 1 diabetes could be directly applied to the general population is unclear . indeed , it has been shown that urinary isoprostane levels are increased when type 1 diabetes is diagnosed , and although they were shown to decline after 16 weeks of insulin treatment , isoprostane concentrations seem to remain elevated in persons with type 1 diabetes compared with controls ( 38 ) . it is , then , possible that a relationship with cad may be more difficult to observe in this compared with the general population if increased levels of oxs are present in type 1 diabetes before the initiation of atherosclerosis . pro - oxidant properties of -tocopherol have previously been described , especially at low oxidant flukes and in the absence of co - antioxidants ( 17 ) . thus , a direct association between isop and -tocopherol may have been expected at lower levels of glycemic control ( hba1c < 7.5% ) , contrary to our observations in this study . these findings , however , may suggest that the distribution of hba1c , even at lower levels , did not achieve the level of radical flux , leading to tocopherol - mediated peroxidation . strengths of the current study comprise the large cohort of individuals with type 1 diabetes , the long ( 20-year ) prospective follow - up , and the biennial assessment of both risk factors and the outcome . in addition , the measurement of plasma levels of the antioxidants - and -tocopherol at three time points is also a strength because , contrary to dietary intake or supplement use data , plasma concentrations reflect biologically relevant levels of the vitamins . finally , the measurement of urinary isop concentration , currently considered the gold standard biomarker of oxs , at three time points during the follow - up period is a further asset of this study . as with any research study , conclusions based on this investigation are limited by several weaknesses , imposed by our choice of study design and the assumptions we made . thus , although measuring biomarkers and assessing risk factors at multiple time points would have improved the precision of measurements , individuals with fewer than three stored samples had to be excluded from these analyses . this practice would have affected study participants with inadequate stored samples ( whether due to failure to collect samples , inadequate sample collection , or use of samples for previous analyses ) , especially those exhibiting cardiovascular and/or fatal events earlier during the follow - up period . moreover , despite improved precision , biomarker assessment at three time points over a 20-year follow - up may not be an adequate representation of an individual s oxs status . we further restricted the assessment of oxs and antioxidative response to measurements of urinary isop and plasma tocopherol concentrations , respectively , among an array of currently known oxidative / antioxidative biomarkers . whether other combinations of markers of stress and response or whether the total antioxidative potential / reserve of an individual may be more relevant to the subsequent risk for cad can not be derived from these data . nevertheless , previous published reports of an association between total antioxidant status and cardiovascular outcomes have produced conflicting results ( 6,3942 ) . finally , we can not exclude the presence of subclinical cad at study entry , a common limitation of both observational studies and clinical trials . in conclusion , lower -tocopherol and the -tocopherol - to - urinary isop ratio were inversely related to cad ; to a lesser extent , higher urinary isop concentrations were directly related to cad . these data thus provide some support for the hypothesis that a greater capability ( -tocopherol ) to respond to oxs ( isoprostanes ) relates to cad incidence .

objectivealthough oxidative stress ( oxs ) is thought to contribute to atherosclerosis and coronary artery disease ( cad ) , little is known about the variability in an individual s ability to respond to oxs . therefore , we assessed potential indices of response to oxs and evaluated whether they modify the association between oxs and cad.research design and methodswe evaluated plasma - and -tocopherol per unit cholesterol ( potential response markers ) ; urinary 15-isoprostane f2 t per milligram creatinine ( isoprostane [ isop ] , a potential stress marker ) ; and the -tocopherol - to - isop ratio ( as a measure of response to stress ) , measured three times during 20 years of follow - up , in relation to cad incidence in a cohort with childhood - onset type 1 diabetes ( n = 658 ; mean age at baseline , 28 years ; duration of diabetes , 19 years ) . participants with three samples ( blood and either 24-h or overnight urine ) available before the onset of cad or the end of follow - up ( n = 356 ) were selected for study.resultsin multivariable mixed models , -tocopherol over time was inversely associated with cad ( = 0.27 ; p = 0.02 ) , whereas a direct association was observed for isop ( = 0.0008 ; p = 0.06 ) . moreover , the -tocopherol - to - isop ratio was strongly and inversely related to cad incidence ( = 0.72 ; p = 0.003 ) , whereas in a separate model including -tocopherol and isop , both biomarkers maintained statistical significance . no association was observed for -tocopherol ( = 0.22 ; p = 0.54).conclusionsthese data suggest that a greater potential capability ( -tocopherol ) to respond to oxs ( urinary isop ) relates to cad incidence .

RESEARCH DESIGN AND METHODS Statistical analyses RESULTS CONCLUSIONS Supplementary Material

the pittsburgh epidemiology of diabetes complications ( edc ) study is a historical , prospective investigation of incident cases of childhood - onset type 1 diabetes diagnosed , or seen within 1 year of diagnosis , at the children s hospital of pittsburgh between 1950 and 1980 . a total of 658 patients who were eligible and willing to participate in the study had an initial clinical assessment between 1986 and 1988 ( mean age 28 years ; mean duration of diabetes 19 years ) and have been subsequently reexamined or surveyed biennially for up to 20 years . for the analyses in this study , edc study participants who were free of cad at study entry and had three stored samples ( blood and either 24-h or overnight urine ) available before the development of cad ( for incident cases ) or the end of follow - up were selected for study ( n = 356 ) . given availability , samples were chosen to reflect an early , midpoint , and late assessment during a participant s follow - up . thus , biologic determinations were approximately 6 years apart for noncases , whereas , given the shorter available follow - up for those developing cad , biomarker measurements were approximately 4 years apart for incident cases . although the definition of hypertension has been altered in recent years , given that the majority of the follow - up within the edc study , which was initiated in 1986 , falls within the time period during which hypertension was still defined as 140/90 mmhg , we felt that it would not be appropriate to reclassify the cohort retrospectively . stable glycosylated hemoglobin ( hba1 ) was measured by ion exchange chromatography ( isolab , akron , oh ) for the first 18 months and by automated high - performance liquid chromatography ( diamat , biorad , hercules , ca ) for the subsequent 10 years ; the two assays were highly correlated ( r = 0.95 ) . for follow - up beyond 10 years , hba1c was measured with the dca 2000 analyzer ( bayer , tarrytown , ny ) . the dca 2000 and diamat assays also were highly correlated ( r = 0.95 ) . original hba1 ( 19861998 ) and later hba1c values ( 19982004 ) were converted to standard hba1c values aligned with the diabetes control and complications trial ( dcct ) using regression formulae derived from duplicate assays [ dcct hba1c = ( 0.83 diamat hba1 ) + 0.14 and dcct hba1c = ( dca hba1c 1.13 ) / 0.81 ] . urinary albumin was measured by immunonephelometry ( 11 ) , and creatinine was assayed using an ectachem 400 analyzer ( eastman kodak co. , rochester , ny ) . cad was defined as myocardial infarction confirmed by q - waves on electrocardiogram ( minnesota code 1.1 , 1.2 ) or hospital records , angiographic stenosis 50% , revascularization , or death due to cad . plasma levels of - and -tocopherol as well as urinary 15-isoprostane f2 t ( isop ) were measured from samples collected at the clinical visit at three time points during the 20-year follow - up of the edc study and stored at 70c until the present analyses . the concentrations of - and -tocopherol in plasma samples anticoagulated with edta were measured under subdued lighting , as previously reported ( 13,14 ) . the coefficient of variation between runs is 6.0 and 2.8% for -tocopherol and -tocopherol , respectively . the effect of prolonged storage on serum antioxidant samples was addressed within the multiple risk factor intervention trial ( mrfit ) ; no appreciable degradation was seen in samples stored at 50 to 70c ( 15 ) . urine samples were obtained in 24-h ( 58% ) or overnight ( 42% ) collections . levels of isop were measured using a competitive elisa procedure developed by oxford biomedical research ( oxford , mi ) , which has a correlation ( r ) of > 0.8 with gas chromatography mass spectrometry procedures . because there are no published data on the potential effects of prolonged storage on the measurement of isop , we simultaneously performed the assay on stored ( 20- and 10-year ) samples and fresh samples from 6 controls and 11 individuals with type 1 diabetes without renal failure . isop levels were similar and within the normal range at all time points ( controls : 0.96 , 1.6 , and 1.01 ng / mg creatinine , respectively ; individuals with type 1 diabetes : 1.3 , 1.2 , and 0.98 ng / mg creatinine , respectively ) , suggesting stability of the assay . given the dependence of plasma tocopherol concentration on lipid levels , - and -tocopherol were reported per unit cholesterol ( 16 ) . urinary isop ( in nanograms per milliliter ) were normalized for urinary creatinine concentration and expressed as nanograms per milligram of creatinine . since -tocopherol has been shown to act as a pro - oxidant under conditions of low reactive oxidant fluxes ( 17 ) , spearman rank correlations were used at each time point for the entire cohort , and separately for incident cases and noncases , to investigate the association between -tocopherol and urinary isop according to high versus low / normal hba1c . mixed models were used to graph trajectories of the main independent variables over time by subsequent cad status . trajectories were plotted as a function of time : time before cad incidence in cases and time before the end of follow - up in noncases ; cubic splines with continuous second derivatives were used to smooth lines ( 18 ) . the genmod procedure in sas was used to assess the association between the repeated measurements of the main independent variables and cad incidence after adjustment for duration of diabetes and significant univariate risk factors . nonnormally distributed variables were logarithmically transformed to more closely satisfy the assumption of normality for entry into multivariable models ( as noted in tables 13 and fig . participant characteristics at the first time point ( baseline ) by subsequent cad status multivariable mixed models for the subsequent development of cad number of cases and noncases at each time point -tocopherol and urinary isop trajectories ( a ) and -tocopherol - to - urinary isop ratio trajectories ( b ) ( adjusted for duration of diabetes , race , sex , bmi , having ever smoked , hba1c , hypertension , hdl and non - hdl cholesterol , albumin excretion rate ( log ) , estimated glomerular filtration rate , and white blood cell count ) before a cad event or the end of follow - up . given the dependence of plasma tocopherol concentration on lipid levels , - and -tocopherol were reported per unit cholesterol ( 16 ) . urinary isop ( in nanograms per milliliter ) were normalized for urinary creatinine concentration and expressed as nanograms per milligram of creatinine . univariate associations between measurements at the first time point ( baseline ) and subsequent cad status were determined using the student t test for normally distributed continuous variables or the wilcoxon two - sample rank sum test for nonnormally distributed continuous variables . since -tocopherol has been shown to act as a pro - oxidant under conditions of low reactive oxidant fluxes ( 17 ) , spearman rank correlations were used at each time point for the entire cohort , and separately for incident cases and noncases , to investigate the association between -tocopherol and urinary isop according to high versus low / normal hba1c . mixed models were used to graph trajectories of the main independent variables over time by subsequent cad status . trajectories were plotted as a function of time : time before cad incidence in cases and time before the end of follow - up in noncases ; cubic splines with continuous second derivatives were used to smooth lines ( 18 ) . the genmod procedure in sas was used to assess the association between the repeated measurements of the main independent variables and cad incidence after adjustment for duration of diabetes and significant univariate risk factors . nonnormally distributed variables were logarithmically transformed to more closely satisfy the assumption of normality for entry into multivariable models ( as noted in tables 13 and fig . participant characteristics at the first time point ( baseline ) by subsequent cad status multivariable mixed models for the subsequent development of cad number of cases and noncases at each time point -tocopherol and urinary isop trajectories ( a ) and -tocopherol - to - urinary isop ratio trajectories ( b ) ( adjusted for duration of diabetes , race , sex , bmi , having ever smoked , hba1c , hypertension , hdl and non - hdl cholesterol , albumin excretion rate ( log ) , estimated glomerular filtration rate , and white blood cell count ) before a cad event or the end of follow - up . , urinary isop and - and -tocopherol ) over time in the entire study group are given in supplementary table 1 . no statistically significant changes , however , were observed overall in urinary isop or -tocopherol . among individuals free of cad at study entry and with stored samples available at three times during the 20-year follow - up ( n = 356 ) , 88 ( 24.7% ) developed a cad event . as expected , individuals who subsequently developed cad were more likely to be older , with a longer duration of diabetes and higher blood pressure , lipid levels , albumin excretion rate , and white blood cell count compared with those who did not develop an event . although no significant differences in urinary isop , -tocopherol per unit cholesterol , or the ratio of -tocopherol to urinary isop were observed between subsequent cases and noncases , it is surprising that -tocopherol ( not adjusting for cholesterol ) was elevated among subsequent cases , whereas -tocopherol levels per unit cholesterol were elevated in individuals without cad compared with subsequent cases . in investigating the strength and direction of a potential association between urinary isop and -tocopherol concentrations , generally inverse associations were observed regardless of the level of glycemic control ( < 7.5 or > 7.5% ) or subsequent cad status , although the strength of the correlation differed by time point ( data not shown ) . , urinary isops ( per milligram of urinary creatinine ) , plasma - and -tocopherol ( per unit cholesterol ) , and the -tocopherol - to - isop ratio ] to assess the independent association between repeated measurements of each variable and the subsequent development of cad ( table 2 ) . in multivariable mixed models , a borderline significant direct association was observed between urinary isop concentrations and cad ( p = 0.06 ) , whereas a significant inverse association was observed between plasma -tocopherol concentration and cad ( p = 0.02 ) . repeated measurements of -tocopherol concentration did not seem to independently relate to subsequent cad status ( p = 0.54 ) . interestingly , the -tocopherol - to - urinary isop ratio was strongly inversely associated with cad ( p = 0.003 ) . similar results of an inverse association between the -tocopherol - to - urinary isop ratio and cad incidence ( p = 0.04 ) were observed when including in the data from analyses of an additional 93 individuals with isop measured only in morning 4-hour clinic urine samples . 1b ) from the fully adjusted models ( table 2 ) before the occurrence of a cad event or the end of follow - up ( table 3 gives the number of cases and noncases at each point during the follow - up period ) . although differences did not appear to be of a great magnitude throughout the follow - up period , -tocopherol and the -tocopherol - to - urinary isop ratio were higher , especially during the 7 years before a cad event or the end of follow - up , whereas urinary isop concentrations were lower among individuals who remained free of a cad event . these data suggest that among individuals with type 1 diabetes , the profile of oxs ( i.e. , plasma -tocopherol ) differ over time in those who develop incident cad compared with those who do not . this difference is particularly apparent in the 7 years before the incident event and is well characterized by the ratio of -tocopherol to urinary isop . no association was observed between repeated measures of plasma -tocopherol and subsequent development of cad . thus , a direct association of urinary isop concentrations after adjustment for other covariates was observed , whereas plasma -tocopherol and the -tocopherol - to - urinary isop ratio were inversely related to subsequent cad status . indeed , a meta - analysis of six randomized clinical trials comparing cardiovascular death risk with vitamin e versus placebo reported no benefit of supplementation , whereas similar meta - analyses for the effect of -carotene supplementation suggested an increased risk of both all - cause and cardiovascular disease mortality ( 19 ) . interestingly , however , secondary prevention trials have provided somewhat more encouraging results for vitamin e supplementation , especially for myocardial infarction ( 2022 ) , although not all studies agree ( 23 ) . we also have previously shown that a protective effect of -tocopherol against cad seemed to be confined to persons with type 1 diabetes and kidney disease in a small case - control study ( 6 ) . indeed , in three clinical trials a reduced risk of cardiovascular disease with vitamin e therapy was apparent only among individuals with both type 2 diabetes and the haptoglobin 22 genotype ( 2426 ) . , antioxidant intake ) to a specific insult ( e.g. , oxs ) may reduce or delay the development of pathologic conditions associated with the insult ( 27 ) . thus , despite difficulties in recognizing markers representing protection or resistance in response to a specific stress , the concurrent evaluation of markers representing insult against those representing protection from or resistance to insults has great implications . in the current study , we aimed to assess whether an adequate antioxidative response to oxs could delay or prevent the development of cad in a cohort of individuals with long - standing type 1 diabetes . given that the majority of clinical trials of antioxidant supplementation in relation to cardiovascular disease focused on vitamin e , we measured the plasma concentration of - and -tocopherol . the former has been reported to have greater biologic activity and to be the only form maintained in human plasma ( 28 ) , whereas the latter is the form most commonly found in the american diet ( 4 ) . f2-isoprostanes , the first isoprostane class discovered , are detectable in their esterified form in all normal biological tissues and in their free form in all normal biological fluids , including plasma and urine ( 3,30 ) . importantly , their concentration in urine is stable and not affected by arachidonic acid auto - oxidation or daily variation ( 29,31 ) , contributing to their establishment as the gold standard biomarker of oxidant stress . the framingham heart study reported that urinary isoprostane concentrations were significantly associated with smoking , diabetes , and bmi , suggesting a role for systemic oxs in cardiovascular disease ( 32 ) . indeed , several case - control studies of humans suggested the presence of elevated isoprostane concentrations in either plasma or urine among individuals with cardiovascular disease ( 3337 ) . thus , plasma isoprostane levels were shown to be higher in individuals with atherosclerosis of the carotid or iliofemoral arteries ( 34 ) , as well as among those with angiographic evidence of cad ( 35 ) , compared with controls . urinary isoprostane levels also were reported to be increased with both subclinical atherosclerosis ( 36 ) and manifest cad ( 33,36,37 ) . similarly to previous reports in the general population , we also observed a direct association between urinary concentrations of isoprostane and subsequent cad in individuals with type 1 diabetes , although the association was not very strong , perhaps suggesting that the level of oxs per se is not the sole critical factor in disease pathogenesis , at least in this population . however , simultaneous evaluation of urinary isop and plasma -tocopherol resulted in significant effects for both biomarkers . the -tocopherol - to - urinary isop ratio over time was even more significant for subsequent cad , consistent with the hypothesis that an adequate response to an insult may reduce risk associated with that insult . thus , although the edc study is not a clinical trial , our findings point to the value of concurrent evaluation of insult and response to that insult to identify risk and potentially lead to novel approaches to preventing the progression of insults to full disease ( e.g. , by selective administration of antioxidant therapy to those with a low -tocopherol - to - isop ratio ) , an approach that may merit further evaluation . nonetheless , whether findings from the current study in type 1 diabetes could be directly applied to the general population is unclear . indeed , it has been shown that urinary isoprostane levels are increased when type 1 diabetes is diagnosed , and although they were shown to decline after 16 weeks of insulin treatment , isoprostane concentrations seem to remain elevated in persons with type 1 diabetes compared with controls ( 38 ) . it is , then , possible that a relationship with cad may be more difficult to observe in this compared with the general population if increased levels of oxs are present in type 1 diabetes before the initiation of atherosclerosis . thus , a direct association between isop and -tocopherol may have been expected at lower levels of glycemic control ( hba1c < 7.5% ) , contrary to our observations in this study . these findings , however , may suggest that the distribution of hba1c , even at lower levels , did not achieve the level of radical flux , leading to tocopherol - mediated peroxidation . strengths of the current study comprise the large cohort of individuals with type 1 diabetes , the long ( 20-year ) prospective follow - up , and the biennial assessment of both risk factors and the outcome . in addition , the measurement of plasma levels of the antioxidants - and -tocopherol at three time points is also a strength because , contrary to dietary intake or supplement use data , plasma concentrations reflect biologically relevant levels of the vitamins . finally , the measurement of urinary isop concentration , currently considered the gold standard biomarker of oxs , at three time points during the follow - up period is a further asset of this study . as with any research study , conclusions based on this investigation are limited by several weaknesses , imposed by our choice of study design and the assumptions we made . this practice would have affected study participants with inadequate stored samples ( whether due to failure to collect samples , inadequate sample collection , or use of samples for previous analyses ) , especially those exhibiting cardiovascular and/or fatal events earlier during the follow - up period . moreover , despite improved precision , biomarker assessment at three time points over a 20-year follow - up may not be an adequate representation of an individual s oxs status . we further restricted the assessment of oxs and antioxidative response to measurements of urinary isop and plasma tocopherol concentrations , respectively , among an array of currently known oxidative / antioxidative biomarkers . finally , we can not exclude the presence of subclinical cad at study entry , a common limitation of both observational studies and clinical trials . in conclusion , lower -tocopherol and the -tocopherol - to - urinary isop ratio were inversely related to cad ; to a lesser extent , higher urinary isop concentrations were directly related to cad . these data thus provide some support for the hypothesis that a greater capability ( -tocopherol ) to respond to oxs ( isoprostanes ) relates to cad incidence .

foodborne trematode ( fbt ) infections are an important public health concern in various asian countries , including lao pdr , vietnam , cambodia , thailand , the philippines , taiwan , china , and the republic of korea . human fbt infections are caused by habitual consumption of raw fish containing infective larvae ( metacercariae ) [ 1 - 3 ] . studies on fbt metacercarial infections have revealed that some species of freshwater and brackish water fish play important roles as the source of human infections in endemic areas [ 4 - 7 ] . it is known that fbt are not only pathogenic to the human host provoking remarkable morbidities but also harmful to fish hosts causing a serious economic loss in the aquaculture fish industry . surveys on metacercarial infection in second intermediate hosts , in combination with surveys on adult fluke infections in humans , are essential to understand the epidemiology and host - parasite relationships of fbt infections in particular geographical areas . however , fecal examinations are not usually suitable to determine the exact infection status in humans , since the specific identification of eggs is very difficult in cases of mixed infections with small - sized trematode eggs , in particular , the liver fluke , heterophyids , gymnophallids , and lecithodendriid flukes [ 8 - 10 ] . therefore , investigation of metacercarial infections in second intermediate hosts available in the area around can provide valuble information on the trematode epidemiology . hanoi city and nam dinh province are located in the northern part of vietnam . through adult fluke recovery by dung et al . , it has been known that many residents in nam dinh province are infected with fbt , such as clonorchis sinensis , heterophyids ( haplorchis pumilio , haplorchis taichui , haplorchis yokogawai , and stellantchasmus falcatus ) , and fasciolopsis buski . after that survey , metacercatrial infections were investigated in fish intermediate hosts caught in several local areas in vietnam [ 12 - 17 ] . in particular , phan et al . surveyed on fbt metacercarial infection in freshwater fish from small - scale farms ( family - based household fish farm ) in nam dinh province . however , those studies did not provide quantitative details of fbt metacercarial infections in fish hosts , mainly focusing on qualitative aspects of commercially important cultured fish species . therefore , in the present study , we intended to reveal the infection status ( both qualitative and quantitative aspects ) of fbt metacercariae in wild fish caught from 2 localities of northern vietnam ( hanoi and nam dinh province ) . in addition , we described the morphologic characteristics of fbt metacercariae and their adults obtained from experimentally infected hamsters . 1 ) , 76 and 79 fish ( 9 species each ) , respectively , were collected from local markets in november 2004 to april 2005 . all collected fish were transferred with ice to the laboratory of department of parasitology , gyeongsang national university school of medicine , jinju , korea . their length and weight were measured and the species were identified with the aid of fishbase site in internet ( table 1 ) . individual fish was ground with a mortar with pestle , or a grinder , and the ground fish meat was mixed with artificial gastric juice . the mixture was incubated at 36 for 2 hr . the digested material was filtered through a 11 mm mesh , and washed with 0.85% saline untill the supernatant became clear . the metacercariae were classified by their general features and were identified at the species level based on detailed morphologies and demensions using a light microscope . identified metacercariae were experimentally infected to hamsters to obtain adult worms . at day 8 after infection , the hamsters were killed by cervical dislocation , and their small intestines were isolated and longitudinally opened with a scissors in a beaker containing 0.85% saline . adult flukes were recovered from the sediment of the intestinal contents which were diluted and washed with 0.85% saline . recovered worms were fixed with 10% formalin under a cover glass pressure , stained with semichon 's acetocarmine , and observed using a light microscope equipped with a micrometer . in fish from hanoi , 3 species of metacercariae , h. pumilio , centrocestus formosanus , and procerovum varium were recovered . h. pumilio metacercariae were detected in 7 of 9 fish species examined . among them , ctenopharyngodon idella was most heavily infected ( table 2 ) . c. formosanus metacercariae were found in 4 fish species , and anabas testudineus revealed a relatively high infection rate and high metacercarial density ( table 3 ) . the infection rate of this fish with p. varium metacercariae was 100% , and the average metacercarial density was 466 per fish ( table 4 ) . in fish from nam dinh province , 6 species of metacercariae , h. taichui , h. pumilio , c. formosanus , p. varium , s. falcatus , and heterophyopsis continua , were recovered . taichui metacercariae ( 6 in total number ) were detected in 3 ( 60% ) of 5 rasbora aurotaenia fish . h. pumilio metacercariae were detected in 8 of 9 fish species examined . among them , a. testudineus revealed relatively higher prevalence ( table 2 ) . c. formosanus metacercariae were recovered in 5 fish species , and their infection rate and densities were relatively low ( table 3 ) . varium metacercariae were detected in 2 fish species ( climbing perch a. testudineus and striped mullet mugil cephalus ) . s. falcatus metacercariae were detected in all 10 m. cephalus , and the average metacercarial density was 84.4 . only 2 h. continua metacercariae were recovered in a lindman 's grenadier anchovy , coilia lindmani . 205)160 - 190 ( 175 ) in size , had a baseball glove - shaped ventrogenital sac with 11 - 18 rodlets and an o - shaped excretory bladder occupying the large portion of the posterior body . . h. pumilio metacercariae ( n=20 ) were elliptical , 160 - 195 ( 179)145 - 175 ( 159 ) in size , had 36 - 42 deer horn - like minute spines arranged in 1 - 2 rows around the ventrogenital complex , and an o - shaped excretory bladder occupying the large portion of the posterior body . c. formosanus metacercariae ( n=20 ) were elliptical , 155 - 190 ( 169)123 - 150 ( 138 ) in size , had 32 circumoral spines around the oral sucker arranged in 2 rows , and a x - shaped excretory bladder occupying the greater portion of the posterior body . p. varium metacercariae ( n=20 ) were elliptical , 165 - 208 ( 187)115 - 163 ( 147 ) in size , had yellowish - brown pigment granules scattering in the area near the intestinal bifurcation , a pair of eyespots lateral to the pharynx , a submedian ventral sucker , a thick - walled bulb - like expulser , and a d - shaped ( half moon - shaped ) excretory bladder with grouped granules ( fig . 2a ) . s. falcatus metacercariae ( n=10 ) were elliptical , 255 - 330 ( 297)225 - 250 ( 232 ) in size , had yellowish - brown pigment granules scattered all over the body , a submedian ventral sucker , a thick - walled bulb - like expulser , and a v - shaped excretory bladder ( fig . 2b ) . h. continua metacercariae ( n=2 ) were nearly round with relatively thick cyst wall , 440 - 475 ( 458)435 - 470 ( 453 ) in size , had brownish pigment scattered all over the body , a ventral sucker located median , a genital sucker located just behind the ventral sucker , and a y - shaped excretory bladder ( fig . 2c ) . 3a ) : body small , pear - shaped , 580 - 730 ( av . 655 ) long and 300 - 310 ( 305 ) wide , with the greatest width at middle , the ovarian level . pharynx subglobular or elliptical , 35 - 38 ( 36 ) by 28 - 30 ( 29 ) . ventrogenital sac small with 11 - 18 rodlets , baseball glove - shaped , 88 - 100 ( 94 ) by 70 - 80 ( 75 ) . seminal vesicle saccular , bipartite , 113 - 125 ( 119 ) by 73 - 75 ( 74 ) . ovary spherical or subspherical , 85 - 88 ( 86 ) by 71 - 74 ( 73 ) , dextral to midline . seminal receptacle ellipsoidal , 80 - 88 ( 84 ) by 50 - 53 ( 51 ) , lying at the right side of the ovary . testis single , globular or subglobular , 160 - 193 ( 176 ) by 120 - 165 ( 143 ) , lying in the posterior 1/4 of the body . uterus with eggs occupying from the anterior 1/3 to the posterior end ( most of the hind - body ) . eggs small , yellow , and 24 - 27 ( 26 ) by 12 - 14 ( 13 ) . 3b ) : body small , pear - shaped , 415 - 550 ( 496 ) long and 195 - 245 ( 217 ) wide , with the greatest width at middle , the ovarian level . oral sucker subterminal , 43 - 50 ( 46 ) by 50 - 58 ( 54 ) . pharynx subglobular or elliptical , 25 - 33 ( 29 ) by 20 - 30 ( 25 ) . ventrogenital sac small with 36 - 42 deer horn - like minute spines , 65 - 80 ( 74 ) by 50 - 75 ( 57 ) . seminal vesicle saccular , 28 - 100 ( 60 ) by 18 - 75 ( 45 ) . ovary spherical or subspherical , 55 - 70 ( 62 ) by 30 - 68 ( 52 ) , slightly dextral to midline . seminal receptacle elliptical , 38 - 85 ( 51 ) by 25 - 58 ( 38 ) , lying at the right side of the ovary . testis single , globular or subglobular , 65 - 103 ( 82 ) by 75 - 105 ( 85 ) , lying at the posterior 1/4 of the body . uterus with eggs occupying from the anterior 1/3 to the posterior end ( most of the hind - body ) . eggs small , yellow , and 28 - 31 ( 30 ) by 15 - 18 ( 16 ) . 3c ) : body small , pear - shaped , 355 - 475 ( 434 ) long and 190 - 250 ( 223 ) wide , with the greatest width at posterior 1/3 of the body . oral sucker subterminal , 35 - 43 ( 38 ) by 38 - 48 ( 42 ) . pharynx subglobular or elliptical , 23 - 30 ( 27 ) by 18 - 23 ( 20 ) . ventral sucker very small , 15 - 30 ( 25 ) by 18 - 33 ( 27 ) , embedded in the ventrogenital sac . expulsor long and thick - walled , 88 - 138 ( 115 ) by 25 - 35 ( 29 ) . ovary spherical or subspherical , 45 - 75 ( 59 ) by 38 - 70 ( 48 ) , slightly dextral to midline . seminal receptacle saccular , 45 - 83 ( 63 ) by 33 - 55 ( 44 ) , lying at the right side of the testis . testis single , globular or subglobular , 105 - 150 ( 127 ) by 100 - 180 ( 127 ) , situated at the middle of the hind - body . uterus with eggs occupying from the anterior 1/3 to the posterior end ( most of the hind - body ) . vitellaria follicular , distributing from the posterior border of the ovary to the posterior extremity . eggs small , yellow , and 25 - 28 ( 26 ) by 11 - 15 ( 13 ) . 3d ) : body small , 400 - 650 ( 481 ) long and 200 - 300 ( 239 ) wide . oral sucker subterminal , 30 - 43 ( 38 ) by 45 - 55 ( 49 ) . pharynx subglobular , 23 - 30 ( 27 ) by 20 - 38 ( 24 ) . ventral sucker small , 25 - 30 ( 27 ) by 28 - 30 ( 29 ) . expulsor long and thick - walled , 75 - 100 ( 87 ) by 30 - 38 ( 33 ) . seminal vesicle saccate , 63 - 70 ( 66 ) by 33 - 40 ( 37 ) . ovary spherical , 63 - 100 ( 79 ) by 43 - 75 ( 62 ) . testes paired , ovoid or globular , slightly oblique and widely separated ; right 83 - 180 ( 111 ) by 45 - 93 ( 62 ) ; left 83 - 188 ( 108 ) by 30 - 88 ( 55 ) . uterus with eggs occupying from the anterior 1/3 to the posterior end ( most of the hind - body ) . eggs small , yellow , and 20 - 23 ( 21 ) by 11 - 13 ( 12 ) . c. formosanus ( n=15 , fig . 3e ) : body very small , 320 - 470 ( 367 ) long and 170 - 235 ( 207 ) wide . oral sucker subterminal , 35 - 43 ( 40 ) by 38 - 50 ( 42 ) , armed with about 32 circumoral spines . pharynx globular , 30 - 43 ( 35 ) by 20 - 28 ( 23 ) . ventral sucker round or elliptical , 33 - 43 ( 37 ) by 45 - 53 ( 51 ) . ovary elliptical , 50 - 75 ( 59 ) by 30 - 53 ( 39 ) , dextral to midline . seminal receptacle large and saccular , 50 - 75 ( 63 ) by 40 - 55 ( 49 ) . two testes ellipsoidal , side by side near the posterior end ; right 60 - 88 ( 72 ) by 33 - 50 ( 41 ) , left 55 - 88 ( 66 ) by 30 - 43 ( 36 ) . vitellaria follicular , distributing along the extracecal margins from the pharyngeal level to the posterior end . eggs small , yellow , and 29 - 33 ( 30 ) by 15 - 18 ( 17 ) . in fish from hanoi , 3 species of metacercariae , h. pumilio , centrocestus formosanus , and procerovum varium were recovered . h. pumilio metacercariae were detected in 7 of 9 fish species examined . among them , ctenopharyngodon idella was most heavily infected ( table 2 ) . c. formosanus metacercariae were found in 4 fish species , and anabas testudineus revealed a relatively high infection rate and high metacercarial density ( table 3 ) . the infection rate of this fish with p. varium metacercariae was 100% , and the average metacercarial density was 466 per fish ( table 4 ) . in fish from nam dinh province , 6 species of metacercariae , h. taichui , h. pumilio , c. formosanus , p. varium , s. falcatus , and heterophyopsis continua , were recovered . taichui metacercariae ( 6 in total number ) were detected in 3 ( 60% ) of 5 rasbora aurotaenia fish . h. pumilio metacercariae were detected in 8 of 9 fish species examined . among them , a. testudineus revealed relatively higher prevalence ( table 2 ) . c. formosanus metacercariae were recovered in 5 fish species , and their infection rate and densities were relatively low ( table 3 ) . varium metacercariae were detected in 2 fish species ( climbing perch a. testudineus and striped mullet mugil cephalus ) . s. falcatus metacercariae were detected in all 10 m. cephalus , and the average metacercarial density was 84.4 . only 2 h. continua metacercariae were recovered in a lindman 's grenadier anchovy , coilia lindmani . 205)160 - 190 ( 175 ) in size , had a baseball glove - shaped ventrogenital sac with 11 - 18 rodlets and an o - shaped excretory bladder occupying the large portion of the posterior body . . h. pumilio metacercariae ( n=20 ) were elliptical , 160 - 195 ( 179)145 - 175 ( 159 ) in size , had 36 - 42 deer horn - like minute spines arranged in 1 - 2 rows around the ventrogenital complex , and an o - shaped excretory bladder occupying the large portion of the posterior body . c. formosanus metacercariae ( n=20 ) were elliptical , 155 - 190 ( 169)123 - 150 ( 138 ) in size , had 32 circumoral spines around the oral sucker arranged in 2 rows , and a x - shaped excretory bladder occupying the greater portion of the posterior body . p. varium metacercariae ( n=20 ) were elliptical , 165 - 208 ( 187)115 - 163 ( 147 ) in size , had yellowish - brown pigment granules scattering in the area near the intestinal bifurcation , a pair of eyespots lateral to the pharynx , a submedian ventral sucker , a thick - walled bulb - like expulser , and a d - shaped ( half moon - shaped ) excretory bladder with grouped granules ( fig . 2a ) . s. falcatus metacercariae ( n=10 ) were elliptical , 255 - 330 ( 297)225 - 250 ( 232 ) in size , had yellowish - brown pigment granules scattered all over the body , a submedian ventral sucker , a thick - walled bulb - like expulser , and a v - shaped excretory bladder ( fig . 2b ) . h. continua metacercariae ( n=2 ) were nearly round with relatively thick cyst wall , 440 - 475 ( 458)435 - 470 ( 453 ) in size , had brownish pigment scattered all over the body , a ventral sucker located median , a genital sucker located just behind the ventral sucker , and a y - shaped excretory bladder ( fig . 2c ) . 3a ) : body small , pear - shaped , 580 - 730 ( av . 655 ) long and 300 - 310 ( 305 ) wide , with the greatest width at middle , the ovarian level . pharynx subglobular or elliptical , 35 - 38 ( 36 ) by 28 - 30 ( 29 ) . ventrogenital sac small with 11 - 18 rodlets , baseball glove - shaped , 88 - 100 ( 94 ) by 70 - 80 ( 75 ) . seminal vesicle saccular , bipartite , 113 - 125 ( 119 ) by 73 - 75 ( 74 ) . ovary spherical or subspherical , 85 - 88 ( 86 ) by 71 - 74 ( 73 ) , dextral to midline . seminal receptacle ellipsoidal , 80 - 88 ( 84 ) by 50 - 53 ( 51 ) , lying at the right side of the ovary . testis single , globular or subglobular , 160 - 193 ( 176 ) by 120 - 165 ( 143 ) , lying in the posterior 1/4 of the body . uterus with eggs occupying from the anterior 1/3 to the posterior end ( most of the hind - body ) . eggs small , yellow , and 24 - 27 ( 26 ) by 12 - 14 ( 13 ) . 3b ) : body small , pear - shaped , 415 - 550 ( 496 ) long and 195 - 245 ( 217 ) wide , with the greatest width at middle , the ovarian level . oral sucker subterminal , 43 - 50 ( 46 ) by 50 - 58 ( 54 ) . pharynx subglobular or elliptical , 25 - 33 ( 29 ) by 20 - 30 ( 25 ) . ventrogenital sac small with 36 - 42 deer horn - like minute spines , 65 - 80 ( 74 ) by 50 - 75 ( 57 ) . seminal vesicle saccular , 28 - 100 ( 60 ) by 18 - 75 ( 45 ) . ovary spherical or subspherical , 55 - 70 ( 62 ) by 30 - 68 ( 52 ) , slightly dextral to midline . seminal receptacle elliptical , 38 - 85 ( 51 ) by 25 - 58 ( 38 ) , lying at the right side of the ovary . testis single , globular or subglobular , 65 - 103 ( 82 ) by 75 - 105 ( 85 ) , lying at the posterior 1/4 of the body . uterus with eggs occupying from the anterior 1/3 to the posterior end ( most of the hind - body ) . eggs small , yellow , and 28 - 31 ( 30 ) by 15 - 18 ( 16 ) . 3c ) : body small , pear - shaped , 355 - 475 ( 434 ) long and 190 - 250 ( 223 ) wide , with the greatest width at posterior 1/3 of the body . oral sucker subterminal , 35 - 43 ( 38 ) by 38 - 48 ( 42 ) . pharynx subglobular or elliptical , 23 - 30 ( 27 ) by 18 - 23 ( 20 ) . ventral sucker very small , 15 - 30 ( 25 ) by 18 - 33 ( 27 ) , embedded in the ventrogenital sac . expulsor long and thick - walled , 88 - 138 ( 115 ) by 25 - 35 ( 29 ) . ovary spherical or subspherical , 45 - 75 ( 59 ) by 38 - 70 ( 48 ) , slightly dextral to midline . seminal receptacle saccular , 45 - 83 ( 63 ) by 33 - 55 ( 44 ) , lying at the right side of the testis . testis single , globular or subglobular , 105 - 150 ( 127 ) by 100 - 180 ( 127 ) , situated at the middle of the hind - body . uterus with eggs occupying from the anterior 1/3 to the posterior end ( most of the hind - body ) . vitellaria follicular , distributing from the posterior border of the ovary to the posterior extremity . eggs small , yellow , and 25 - 28 ( 26 ) by 11 - 15 ( 13 ) . 3d ) : body small , 400 - 650 ( 481 ) long and 200 - 300 ( 239 ) wide . oral sucker subterminal , 30 - 43 ( 38 ) by 45 - 55 ( 49 ) . pharynx subglobular , 23 - 30 ( 27 ) by 20 - 38 ( 24 ) . ventral sucker small , 25 - 30 ( 27 ) by 28 - 30 ( 29 ) . expulsor long and thick - walled , 75 - 100 ( 87 ) by 30 - 38 ( 33 ) . seminal vesicle saccate , 63 - 70 ( 66 ) by 33 - 40 ( 37 ) . ovary spherical , 63 - 100 ( 79 ) by 43 - 75 ( 62 ) . testes paired , ovoid or globular , slightly oblique and widely separated ; right 83 - 180 ( 111 ) by 45 - 93 ( 62 ) ; left 83 - 188 ( 108 ) by 30 - 88 ( 55 ) . uterus with eggs occupying from the anterior 1/3 to the posterior end ( most of the hind - body ) . eggs small , yellow , and 20 - 23 ( 21 ) by 11 - 13 ( 12 ) . c. formosanus ( n=15 , fig . 3e ) : body very small , 320 - 470 ( 367 ) long and 170 - 235 ( 207 ) wide . oral sucker subterminal , 35 - 43 ( 40 ) by 38 - 50 ( 42 ) , armed with about 32 circumoral spines . pharynx globular , 30 - 43 ( 35 ) by 20 - 28 ( 23 ) . ventral sucker round or elliptical , 33 - 43 ( 37 ) by 45 - 53 ( 51 ) . ovary elliptical , 50 - 75 ( 59 ) by 30 - 53 ( 39 ) , dextral to midline . seminal receptacle large and saccular , 50 - 75 ( 63 ) by 40 - 55 ( 49 ) . two testes ellipsoidal , side by side near the posterior end ; right 60 - 88 ( 72 ) by 33 - 50 ( 41 ) , left 55 - 88 ( 66 ) by 30 - 43 ( 36 ) . vitellaria follicular , distributing along the extracecal margins from the pharyngeal level to the posterior end . eggs small , yellow , and 29 - 33 ( 30 ) by 15 - 18 ( 17 ) . the 6 species of fbt metacercariae ( h. taichui , h. pumilio , c. formosanus , p. varium , s. falcatus , and h. continua ) detected in this study were all minute intestinal flukes and members of the heterophyidae . among them , h. pumilio was the dominant species . . reported a high prevalence ( 64.9% ) of small trematode eggs and also high prevalence for soil - transmitted helminths among residents of 2 communes in nam dinh province , northern vietnam , in april 2005 . they recovered adult flukes of 6 trematode species ( clonorchis sinensis , h. pumilio , h. taichui , h. yokogawai , s. falcatus , and fasciolopsis buski ) from 33 peoples who revealed over 1,000 eggs per gram of feces ( epg ) for small trematode eggs after praziquantel treatment and purgation . among the 6 trematode species , h. pumilio was recovered in all 33 ( 100% ) residents and the worm load averaged 416 per person . accordingly , it is presumed that more than 8 species of fbt , including h. pumilio , may be distributed in the northern part of vietnam , although we could not find c. sinensis and h. yokogawai metacercariae . thu et al . surveyed on metacercarial infections in cultured catfish and snakeheads , and also in several wild fish species from an giang province , a major fish production area in the mekong delta of vietnam in 2005 - 2006 . thien et al . examined 13 major cultured fish species from tien giang province and can tho city , located in central areas of the mekong delta of vietnam , in 2005 - 2006 . chi et al . performed a cross - sectional survey of fbt metacercariae in farmed fish in nghe an province , about 300 km south of hanoi , in 2005 . vo et al . surveyed 2 species of groupers ( epinephelus coioides and epinepheles bleekeri ) , and mullet ( m. cephalus ) in a central coastal area of vietnam ( khanh hoa province ) in 2008 . surveyed on the infection status of fbt metacercariae in freshwater fish from small - scale farms ( family - based household fish farm ) in nam dinh province in 2010 . these studies focused mainly on metacercarial infections in commercially important fish species and in qualitative aspects . studies on quantitative aspects of fbt metacercarial infections in each species of fish and thereby the suitability and susceptability of fish hosts for each fbt species have not been available . in the present study , h. pumilio metacercariae were detected in 104 ( 80.0% ) of 130 fish examined , and the metacercarial density was 64.2 per fish infected . 129 metacercariae ) than those from nam dinh province ( av . 15 metacercariae ) . the most heavily infected fish with h. pumilio metacercariae was the grass carp ( ctenopharyngodon idella ) caught from hanoi . the high susceptibility of this fish species was also shown in the study of phan et al . which was performed in nam dinh province . a similar finding was shown in guangxi zhuang autonomous region located in the southern part of china , slightly north of our surveyed area . in this area of china , 18 fish species ( carassius auratus , acheilognathus tonkinensis , hemibarbus maculatus , hypophthalmichthys molitrix , hemiculter leucisculus , c. idella , toxabramis houdemeri , microphysogobio fukiensis , pseudohemiculter dispar , opsariichthys bidens , squalidus argentatus , metzia lineata , cyprinus carpio , puntius semifasciolatus , saurogobio dabryi , culter recurviceps , chanodichthys dabryi , and pseudorasbora parva ) were listed as hosts for h. pumilio . on the other hand , in nam dinh province of vietnam , phan et al . listed 17 fish species ( labeo rohita , h. molitrix , cirrhinus mrigala , c. idella , c. auratus , squaliobarbus curriculus , piaractus brachypomum , c. carpio , cirrhinus molitorella , hypophthalmichthys nobilis , barbonymus gonionotus , h. leucisculus , anabas testudineus , oreochromis niloticus , clarias batrachus , channa orientalis , and notopterus notopterus ) for the host for h. pumilio . in the present study , 7 more fish species ( acheilognathus barbatulus , albulichthys albuloides , barbodes balleroides , coilia lindmani , parabramis pekinensis , rasbora aurotaenia , and trichogaster trichopterus ) have been newly added as the host for h. pumilio . c. formosanus metacercariae were detected in 8 fish species ( a. barbatulus , a. testudineus , b. balleroides , c. auratus , c. molitorella , c. idella , p. pekinensis , and p. semifasciolatus ) in the present study . detected the same species of metacercariae in 10 fish species ( l. rohita , h. molitrix , c. mrigala , c. idella , s. curriculus , p. brachypomum , c. carpio , c. batrachus , c. orientalis , and a. testudineus ) in nam dinh province . in china , sohn et al . reported 10 fish species ( m. fukiensis , a. tonkinensis , s. argentatus , c. carpio , h. molitrix , a. rivularis , h. leucisculus , m. lineata , s. dabryi and p. parva ) as the fish hosts for c. formosanus . therefore , it appears that more than 24 fish species play the role of second intermediate hosts for c. formosanus in vietnam and china . it has been known that 2 species of liver flukes ( clonorchis sinensis and opisthorchis viverrini ) distribute in vietnam . c. sinensis is distributed in the northern part , whereas o. viverrini is found in the southern part . however , the metacercariae of these liver flukes were only rarely detected in fish from vietnam . in an giang province , a southern part , only 19 metacercariae of o. viverrini thien et al . could not find any liver fluke metacercariae in all 13 important cultured fish species from tien giang province and can tho city , in the mekong delta of vietnam . no liver fluke metacercariae were detected in a cross - sectional survey of fbt metacercariae in farmed fish from nghe an province . meanwhile , only a small number of c. sinensis metacercariae were found in 1 of 1,185 silver carps ( h. molitrix ) examined in small - scale farms in nam dinh province . in the present study , we could not find any c. sinensis metacercariae in fish from the same study area . by contrast , dung et al . reported a high positive rate ( 51.5% ) of c. sinensis adult worms ( their worm load was not so high ; 1 - 18 per individual ) from 33 residents with over 1,000 epg for small trematode eggs in nam dinh province . this discrepancy between the metacercarial infection in fish hosts and the adult fluke infections in humans should be clarified in the near future . in the present study , p. varium metacercariae were detected in 2 fish species ( a. testudineus and m. cephalus ) , although these metacercariae were previously recorded in 9 fish species ( l. rohita , h. molitrix , c. mrigala , c. idella , s. curriculus , p. brachypomum , c. batrachus , b. gonionotus , and a. testudineus ) in nam dinh province . varium metacercariae together with 2 other heterophyid metacercariae ( h. continua and p. summa ) in 2 species of groupers and mullet from khanh hoa province in vietnam . on the other hand , s. falcatus and h. continua metacercariae were found in m. cephalus and c. lindmani , respectively , in the present study . although morphologic characteristics of fbt metacercariae had previously been reported , we redescribed some of their characteristic features to provide a useful aid for epidemiologic studies in southeast asian countries . among the fbt metacercariae detected in our study , 3 species ( h. taichui , h. pumilio , and c. formosanus ) were morphologically identical with those from china and lao pdr . on the other hand , the metacercariae of p. varium ( 187147 m ) were smaller than those ( 210180 ) reported in a freshwater fish ( oryzias melastigma ) from visakhapatnam , india . however , our p. varium metacercariae were almost identical with those found in groupers from nha trang district in khanh hoa province , vietnam . with regard to s. falcatus metacercariae , they were previously detected in cultured giant gouramy ( osphronemus gourami ) from tien giang province and can tho city , and also in the common carp ( c. carpio ) and grass carp ( c. idella ) from nghe an province , vietnam . however , no morphologic descriptions were available on s. falcatus metacercariae in vietnam . h. continua metacercariae ( 458453 m ) detected in a c. lindmani fish in the present study were similar in shape with but larger than those ( 380 in diameter ) recovered in mullets from khanh hoa province . in the republic of korea , it is agreed that h. continua metacercariae detected in various fish species ( laterolabrax japonicus , acanthogobius flavimanus , clupanodon punctatus , plecoglossus altivelis , conger myriaster , boleophthalmus pectinirostris , and scartelaos sp . ) show wide size ranges by fish hosts examined . among the fbt species distributed in vietnam , 2 species of liver flukes ( c. sinensis and o. viverrini ) are highly important in clinical and pathological aspects , being similar in general shape , the liver fluke metacercariae can be distinguished from those of heterophyid flukes by the presence of a large well - developed ventral sucker which is nearly equal in size with the oral sucker . the ventral suckers of h. taichui , h. pumilio , c. formosanus , p. varium , and s. falcatus are characteristically smaller than their oral suckers . nevertheless , when these metacercariae were mixed together , it is not easy to distinguish them unless each metacercaria is subjected to a detailed observation under a light microscope . the experimentally obtained adults of the 5 species of fbt were morphologically compatible with those previously reported . only briefly mentioned on the adult worm morphologies of h. pumilio , h. taichui , h. yokogawai , and s. falcatus after recovery in residents of nam dinh province , vietnam . the size of h. taichui ( 756421 ) and h. pumilio ( 632291 ) adults obtained from residents were slightly larger than those from our study , whereas the adult of s. falcatus ( 468298 ) from residents was smaller in size than our specimens .

the prevalence of foodborne trematode ( fbt ) metacercariae was investigated in fish from 2 localities of northern vietnam in 2004 - 2005 . freshwater fish ( 9 species ) were collected from local markets in hanoi city ( n=76 ) and nam dinh province ( n=79 ) , and were examined for fbt metacercariae using the artificial digestion technique . adult flukes were obtained from hamsters experimentally infected with the metacercariae at day 8 post - infection . three ( haplorchis pumilio , centrocestus formosanus , and procerovum varium ) and 6 ( haplorchis taichui , h. pumilio , c. formosanus , p. varium , stellantchasmus falcatus , and heterophyopsis continua ) species of fbt metacercariae were detected in the 2 regions , respectively . overall , among the positive fish species , h. pumilio metacercariae were detected in 104 ( 80.0% ) of 130 fish examined ( metacercarial density per infected fish ; 64.2 ) . c. formosanus metacercariae were found in 37 ( 40.2% ) of 92 fish ( metacercarial density ; 14.7 ) . p. varium metacercariae were detected in 19 ( 63.3% ) of 30 fish ( anabas testudineus and mugil cephalus ) ( metacercarial density ; 247.7 ) . s. falcatus metacercariae were found in all 10 m. cephalus examined ( metacercarial density ; 84.4 ) . h. continua metacercariae ( 2 in number ) were detected in 1 fish of coilia lindmani . morphologic characteristics of the fbt metacercariae and their experimentally obtained adults were described . the results have demonstrated that various fbt species are prevalent in northen parts of vietnam .

INTRODUCTION MATERIALS AND METHODS RESULTS FBT metacercariae in fish from Hanoi FBT metacercariae in fish from Nam Dinh Province Morphology of metacercariae (All measurement unit is m) Morphology of adult flukes DISCUSSION

foodborne trematode ( fbt ) infections are an important public health concern in various asian countries , including lao pdr , vietnam , cambodia , thailand , the philippines , taiwan , china , and the republic of korea . hanoi city and nam dinh province are located in the northern part of vietnam . , it has been known that many residents in nam dinh province are infected with fbt , such as clonorchis sinensis , heterophyids ( haplorchis pumilio , haplorchis taichui , haplorchis yokogawai , and stellantchasmus falcatus ) , and fasciolopsis buski . surveyed on fbt metacercarial infection in freshwater fish from small - scale farms ( family - based household fish farm ) in nam dinh province . therefore , in the present study , we intended to reveal the infection status ( both qualitative and quantitative aspects ) of fbt metacercariae in wild fish caught from 2 localities of northern vietnam ( hanoi and nam dinh province ) . in addition , we described the morphologic characteristics of fbt metacercariae and their adults obtained from experimentally infected hamsters . 1 ) , 76 and 79 fish ( 9 species each ) , respectively , were collected from local markets in november 2004 to april 2005 . at day 8 after infection , the hamsters were killed by cervical dislocation , and their small intestines were isolated and longitudinally opened with a scissors in a beaker containing 0.85% saline . in fish from hanoi , 3 species of metacercariae , h. pumilio , centrocestus formosanus , and procerovum varium were recovered . h. pumilio metacercariae were detected in 7 of 9 fish species examined . c. formosanus metacercariae were found in 4 fish species , and anabas testudineus revealed a relatively high infection rate and high metacercarial density ( table 3 ) . the infection rate of this fish with p. varium metacercariae was 100% , and the average metacercarial density was 466 per fish ( table 4 ) . in fish from nam dinh province , 6 species of metacercariae , h. taichui , h. pumilio , c. formosanus , p. varium , s. falcatus , and heterophyopsis continua , were recovered . taichui metacercariae ( 6 in total number ) were detected in 3 ( 60% ) of 5 rasbora aurotaenia fish . h. pumilio metacercariae were detected in 8 of 9 fish species examined . c. formosanus metacercariae were recovered in 5 fish species , and their infection rate and densities were relatively low ( table 3 ) . varium metacercariae were detected in 2 fish species ( climbing perch a. testudineus and striped mullet mugil cephalus ) . s. falcatus metacercariae were detected in all 10 m. cephalus , and the average metacercarial density was 84.4 . only 2 h. continua metacercariae were recovered in a lindman 's grenadier anchovy , coilia lindmani . h. pumilio metacercariae ( n=20 ) were elliptical , 160 - 195 ( 179)145 - 175 ( 159 ) in size , had 36 - 42 deer horn - like minute spines arranged in 1 - 2 rows around the ventrogenital complex , and an o - shaped excretory bladder occupying the large portion of the posterior body . c. formosanus metacercariae ( n=20 ) were elliptical , 155 - 190 ( 169)123 - 150 ( 138 ) in size , had 32 circumoral spines around the oral sucker arranged in 2 rows , and a x - shaped excretory bladder occupying the greater portion of the posterior body . p. varium metacercariae ( n=20 ) were elliptical , 165 - 208 ( 187)115 - 163 ( 147 ) in size , had yellowish - brown pigment granules scattering in the area near the intestinal bifurcation , a pair of eyespots lateral to the pharynx , a submedian ventral sucker , a thick - walled bulb - like expulser , and a d - shaped ( half moon - shaped ) excretory bladder with grouped granules ( fig . s. falcatus metacercariae ( n=10 ) were elliptical , 255 - 330 ( 297)225 - 250 ( 232 ) in size , had yellowish - brown pigment granules scattered all over the body , a submedian ventral sucker , a thick - walled bulb - like expulser , and a v - shaped excretory bladder ( fig . h. continua metacercariae ( n=2 ) were nearly round with relatively thick cyst wall , 440 - 475 ( 458)435 - 470 ( 453 ) in size , had brownish pigment scattered all over the body , a ventral sucker located median , a genital sucker located just behind the ventral sucker , and a y - shaped excretory bladder ( fig . in fish from hanoi , 3 species of metacercariae , h. pumilio , centrocestus formosanus , and procerovum varium were recovered . h. pumilio metacercariae were detected in 7 of 9 fish species examined . c. formosanus metacercariae were found in 4 fish species , and anabas testudineus revealed a relatively high infection rate and high metacercarial density ( table 3 ) . the infection rate of this fish with p. varium metacercariae was 100% , and the average metacercarial density was 466 per fish ( table 4 ) . in fish from nam dinh province , 6 species of metacercariae , h. taichui , h. pumilio , c. formosanus , p. varium , s. falcatus , and heterophyopsis continua , were recovered . taichui metacercariae ( 6 in total number ) were detected in 3 ( 60% ) of 5 rasbora aurotaenia fish . h. pumilio metacercariae were detected in 8 of 9 fish species examined . c. formosanus metacercariae were recovered in 5 fish species , and their infection rate and densities were relatively low ( table 3 ) . varium metacercariae were detected in 2 fish species ( climbing perch a. testudineus and striped mullet mugil cephalus ) . s. falcatus metacercariae were detected in all 10 m. cephalus , and the average metacercarial density was 84.4 . only 2 h. continua metacercariae were recovered in a lindman 's grenadier anchovy , coilia lindmani . h. pumilio metacercariae ( n=20 ) were elliptical , 160 - 195 ( 179)145 - 175 ( 159 ) in size , had 36 - 42 deer horn - like minute spines arranged in 1 - 2 rows around the ventrogenital complex , and an o - shaped excretory bladder occupying the large portion of the posterior body . c. formosanus metacercariae ( n=20 ) were elliptical , 155 - 190 ( 169)123 - 150 ( 138 ) in size , had 32 circumoral spines around the oral sucker arranged in 2 rows , and a x - shaped excretory bladder occupying the greater portion of the posterior body . p. varium metacercariae ( n=20 ) were elliptical , 165 - 208 ( 187)115 - 163 ( 147 ) in size , had yellowish - brown pigment granules scattering in the area near the intestinal bifurcation , a pair of eyespots lateral to the pharynx , a submedian ventral sucker , a thick - walled bulb - like expulser , and a d - shaped ( half moon - shaped ) excretory bladder with grouped granules ( fig . s. falcatus metacercariae ( n=10 ) were elliptical , 255 - 330 ( 297)225 - 250 ( 232 ) in size , had yellowish - brown pigment granules scattered all over the body , a submedian ventral sucker , a thick - walled bulb - like expulser , and a v - shaped excretory bladder ( fig . h. continua metacercariae ( n=2 ) were nearly round with relatively thick cyst wall , 440 - 475 ( 458)435 - 470 ( 453 ) in size , had brownish pigment scattered all over the body , a ventral sucker located median , a genital sucker located just behind the ventral sucker , and a y - shaped excretory bladder ( fig . the 6 species of fbt metacercariae ( h. taichui , h. pumilio , c. formosanus , p. varium , s. falcatus , and h. continua ) detected in this study were all minute intestinal flukes and members of the heterophyidae . reported a high prevalence ( 64.9% ) of small trematode eggs and also high prevalence for soil - transmitted helminths among residents of 2 communes in nam dinh province , northern vietnam , in april 2005 . they recovered adult flukes of 6 trematode species ( clonorchis sinensis , h. pumilio , h. taichui , h. yokogawai , s. falcatus , and fasciolopsis buski ) from 33 peoples who revealed over 1,000 eggs per gram of feces ( epg ) for small trematode eggs after praziquantel treatment and purgation . among the 6 trematode species , h. pumilio was recovered in all 33 ( 100% ) residents and the worm load averaged 416 per person . accordingly , it is presumed that more than 8 species of fbt , including h. pumilio , may be distributed in the northern part of vietnam , although we could not find c. sinensis and h. yokogawai metacercariae . surveyed on metacercarial infections in cultured catfish and snakeheads , and also in several wild fish species from an giang province , a major fish production area in the mekong delta of vietnam in 2005 - 2006 . surveyed 2 species of groupers ( epinephelus coioides and epinepheles bleekeri ) , and mullet ( m. cephalus ) in a central coastal area of vietnam ( khanh hoa province ) in 2008 . surveyed on the infection status of fbt metacercariae in freshwater fish from small - scale farms ( family - based household fish farm ) in nam dinh province in 2010 . in the present study , h. pumilio metacercariae were detected in 104 ( 80.0% ) of 130 fish examined , and the metacercarial density was 64.2 per fish infected . the most heavily infected fish with h. pumilio metacercariae was the grass carp ( ctenopharyngodon idella ) caught from hanoi . in this area of china , 18 fish species ( carassius auratus , acheilognathus tonkinensis , hemibarbus maculatus , hypophthalmichthys molitrix , hemiculter leucisculus , c. idella , toxabramis houdemeri , microphysogobio fukiensis , pseudohemiculter dispar , opsariichthys bidens , squalidus argentatus , metzia lineata , cyprinus carpio , puntius semifasciolatus , saurogobio dabryi , culter recurviceps , chanodichthys dabryi , and pseudorasbora parva ) were listed as hosts for h. pumilio . listed 17 fish species ( labeo rohita , h. molitrix , cirrhinus mrigala , c. idella , c. auratus , squaliobarbus curriculus , piaractus brachypomum , c. carpio , cirrhinus molitorella , hypophthalmichthys nobilis , barbonymus gonionotus , h. leucisculus , anabas testudineus , oreochromis niloticus , clarias batrachus , channa orientalis , and notopterus notopterus ) for the host for h. pumilio . in the present study , 7 more fish species ( acheilognathus barbatulus , albulichthys albuloides , barbodes balleroides , coilia lindmani , parabramis pekinensis , rasbora aurotaenia , and trichogaster trichopterus ) have been newly added as the host for h. pumilio . c. formosanus metacercariae were detected in 8 fish species ( a. barbatulus , a. testudineus , b. balleroides , c. auratus , c. molitorella , c. idella , p. pekinensis , and p. semifasciolatus ) in the present study . detected the same species of metacercariae in 10 fish species ( l. rohita , h. molitrix , c. mrigala , c. idella , s. curriculus , p. brachypomum , c. carpio , c. batrachus , c. orientalis , and a. testudineus ) in nam dinh province . reported 10 fish species ( m. fukiensis , a. tonkinensis , s. argentatus , c. carpio , h. molitrix , a. rivularis , h. leucisculus , m. lineata , s. dabryi and p. parva ) as the fish hosts for c. formosanus . however , the metacercariae of these liver flukes were only rarely detected in fish from vietnam . could not find any liver fluke metacercariae in all 13 important cultured fish species from tien giang province and can tho city , in the mekong delta of vietnam . no liver fluke metacercariae were detected in a cross - sectional survey of fbt metacercariae in farmed fish from nghe an province . meanwhile , only a small number of c. sinensis metacercariae were found in 1 of 1,185 silver carps ( h. molitrix ) examined in small - scale farms in nam dinh province . in the present study , p. varium metacercariae were detected in 2 fish species ( a. testudineus and m. cephalus ) , although these metacercariae were previously recorded in 9 fish species ( l. rohita , h. molitrix , c. mrigala , c. idella , s. curriculus , p. brachypomum , c. batrachus , b. gonionotus , and a. testudineus ) in nam dinh province . varium metacercariae together with 2 other heterophyid metacercariae ( h. continua and p. summa ) in 2 species of groupers and mullet from khanh hoa province in vietnam . on the other hand , s. falcatus and h. continua metacercariae were found in m. cephalus and c. lindmani , respectively , in the present study . although morphologic characteristics of fbt metacercariae had previously been reported , we redescribed some of their characteristic features to provide a useful aid for epidemiologic studies in southeast asian countries . among the fbt metacercariae detected in our study , 3 species ( h. taichui , h. pumilio , and c. formosanus ) were morphologically identical with those from china and lao pdr . on the other hand , the metacercariae of p. varium ( 187147 m ) were smaller than those ( 210180 ) reported in a freshwater fish ( oryzias melastigma ) from visakhapatnam , india . with regard to s. falcatus metacercariae , they were previously detected in cultured giant gouramy ( osphronemus gourami ) from tien giang province and can tho city , and also in the common carp ( c. carpio ) and grass carp ( c. idella ) from nghe an province , vietnam . h. continua metacercariae ( 458453 m ) detected in a c. lindmani fish in the present study were similar in shape with but larger than those ( 380 in diameter ) recovered in mullets from khanh hoa province . in the republic of korea , it is agreed that h. continua metacercariae detected in various fish species ( laterolabrax japonicus , acanthogobius flavimanus , clupanodon punctatus , plecoglossus altivelis , conger myriaster , boleophthalmus pectinirostris , and scartelaos sp . ) among the fbt species distributed in vietnam , 2 species of liver flukes ( c. sinensis and o. viverrini ) are highly important in clinical and pathological aspects , being similar in general shape , the liver fluke metacercariae can be distinguished from those of heterophyid flukes by the presence of a large well - developed ventral sucker which is nearly equal in size with the oral sucker . the ventral suckers of h. taichui , h. pumilio , c. formosanus , p. varium , and s. falcatus are characteristically smaller than their oral suckers . the experimentally obtained adults of the 5 species of fbt were morphologically compatible with those previously reported . only briefly mentioned on the adult worm morphologies of h. pumilio , h. taichui , h. yokogawai , and s. falcatus after recovery in residents of nam dinh province , vietnam . the size of h. taichui ( 756421 ) and h. pumilio ( 632291 ) adults obtained from residents were slightly larger than those from our study , whereas the adult of s. falcatus ( 468298 ) from residents was smaller in size than our specimens .

the mitochondrion is a membrane - enclosed cytoplasmic organelle , which has evolved from a primitive aerobic bacteria by means of a symbiotic relationship that started 1.5 billions years ago . these organelles range from 0.5 to 10 micrometers ( m ) in diameter and are composed of compartments devoted to specific functions . these regions include the outer membrane , the intermembrane space , the inner membrane , the cristae , and the matrix . cellular power plants because they produce most of the cell 's supply of adenosine triphosphate ( atp ) , by means of the oxidative phosphorylation ( oxphos ) machinery , which comprises electron transport chain ( etc ) and atp synthase ( complex v ) . the etc provides the cell with the most efficient energetic outcome in terms of atp production . it consists of four multimeric protein complexes ( complex i to iv ) located in the inner mitochondrial membrane together with complex v . etc dysfunction leads to reduced atp production , impaired calcium buffering , and increased generation of reactive oxygen species ( ros ) . mitochondria have their own dna , the mitochondrial dna ( mtdna ) , represented by a circular molecule of 16.5 kb without introns , constituting of a heavy chain ( h ) and a light chain ( l ) [ 3 , 5 ] . mtdna carries 37 genes : 22 encoding for mitochondrial transfer rnas ( trnas ) ( for the 20 standard amino acids , plus an extra gene for leucine and serine ) , 2 for ribosomal rnas ( rrnas ) and 13 encoding for polypeptides subunits of complexes of the respiratory chain system , 7 of them belonging to complex i or nadh dehydrogenase ( nd1 , nd2 , nd3 , nd4 , nd4l , nd5 , nd6 ) , 1 to complex iii or cytochrome c reductase , 3 to complex iv or cytochrome c oxidase ( cox i , cox ii and cox iii ) , and 2 to complex v or atp synthase ( atpase6 and atpase8 ) . the remaining mitochondrial proteins , including all the complex ii subunits , are encoded by nuclear dna . mitochondrial genetics differs from mendelian genetics in three major aspects : maternal inheritance , heteroplasmy , and mitotic segregation . mitochondria are inherited in humans via the female line [ 3 , 5 ] , transmitted as a nonrecombining unit by maternal inheritance . furthermore , human mtdna is characterized by a much greater evolutionary rate than that of the average of nuclear genes . thus , its sequence variation has been generated by the sequential accumulation of new mutations along radiating maternal lineages . ancestor mother , the mitochondrial eve , by identifying common polymorphisms that have been accumulated with time . these common polymorphisms describe classes of continent - specific genotypes , the haplogroups , evolved from the same ancestor , which can be detected by restriction fragment length polymorphism ( rflp ) analysis . probably lived in africa about 200,000 years ago and phylogeographic studies allowed to identify the mtdna haplogroups tree and the mtdna migration route ( see figure 1 ) . because the process of molecular differentiation is relatively fast and occurred mainly during and after the recent process of dispersal into different parts of the world , the basal branching structure of mtdna variation in most parts of the world is now well understood . the major branches of the tree were usually restricted geographically , some to sub - saharan africans , others to east asians and yet others to europeans and near easterners . african haplogroups fall into seven major families ( l0 , l1 , l2 , l3 , l4 , l5 , l6 ) . about 85000 years ago , probably in the horn of africa , changes in climate , the glacial interstadial phase 21 , triggered the rising of many descendant haplogroups from the root of haplogroup l3 , the first multifurcation node , probably because of some colonization event or local population growth [ 6 , 9 , 10 ] . non - african mtdna ( excluding migrations from africa within the past few thousand years ) descend from l3 and belong either to the m or n superclades . haplogroup n soon gave rise to haplogroup r. in the indian subcontinent and in southeast asia there is the richest basal variation in the three founder haplogroups m , n , and r , and this suggests a rapid colonization along the southern coast of asia , about 60000 years ago . over 30 subclades of the haplogroup m haplogroups a , b , c , d , and x have been found in the americas , coming mainly from asia . the expansions northwards occurred later , about 45000 years ago when technology and climatic conditions enabled the exploration of the interior of eurasia . one of the more marginal extensions eventually led to the peopling of europe . in europeans and near easterners ( who share a rather recent common ancestor ) , nine different mitochondrial haplogroups have been identified ( h , i , j , k , t , u , v , w , x ) . european mtdna variation is surprisingly impoverished in the number of independent basal lineages , compared with the south asian mtdna variation , maybe for the peripheral role that the pioneer migration into the near east and subsequently europe must have had in the broader out - of - africa scenario . complete mtdna sequencing and the increasing number of samples analyzed allowed subdividing haplogroups in smaller groups identifying younger branches on the mtdna evolution tree . it has been supposed that genetic polymorphisms within the mitochondrial genome might lead to impaired energy generation and to increased amount of ros , causing subtle differences in the encoded proteins and , thus , minimum changes in mitochondrial respiratory chain oxphos activity and free radical overproduction . increased production of ros damages cell membranes and further accelerates the high mutation rate of mtdna . the mtdna is particularly susceptible to oxidative damage . because of its vicinity with ros source ( etc ) and because it is not protected by histones and it is inefficiently repaired , mtdna shows a high mutation rate . accumulation of mtdna mutations enhances normal ageing , leads to oxidative damage , and causes energy failure and increased production of ros , in a vicious cycle . this could predispose to diseases or modify longevity in individual or in a population sharing the same mtdna genotype . maybe the opposite could be also true for different polymorphism(s ) , which could be protective . several studies correlate mitochondrial haplogroups with disease 's susceptibility or confer them protective roles . in a study of 2008 , conducted on 114 healthy spanish males , marcuello et al . found that haplogroups j presents with lower vo(2max ) ( oxygen consumption ) than non j variants . j has been related with a lower efficiency of etc , diminished atp , and ros production . furthermore , the lower ros production associated to j could also account for the accrual of this variant in elderly people consequent to a decreased oxidative damage . the h haplogroup seemed to be responsible for the difference between j and non - j , and this group also had significantly higher mitochondrial oxidative damage than the j haplogroup , suggesting that ros production is responsible for the higher vo(2max ) found in this variant . in agreement with these results , vo(2max ) and mitochondrial oxidative damage supporting the hypothesis that j haplogroup may impair the oxphos coupling , pierron et al . demonstrated that j haplogroup was markedly underrepresented among the a3243 g mutation carriers . this mutation on the trnaleu gene ( uur ) is one of the most common mtdna mutation . the phenotypic expression of this mutation is quite variable , ranging from mild to severe clinical phenotypes ( melas ) . the authors , in order to explain the epidemiology of haplogroup j and a3243 g mutation speculated that this association is lifethreatening , and therefore lethal to the embryo or germ line . one of the most common manifestation of some mitochondrial disease is the presence of retinal pigmentary changes , which are similar to those observed in age - related maculopathy ( arm ) . jones et al . demonstrated an association between haplogroup h and a reduced prevalence of this disease , after adjusting for known arm risk factors . haplogroup h1 may be protective for ischemic stroke ; in fact , it was found to be significantly less frequent in stroke patients than in controls , when comparing each clade against all other haplogroups pooled together , in a paper of rosa et al . focusing on 534 ischemic stroke patients and 499 controls . in a prospective study of intensive care patients in newcastle - upon - tyne conducted by baudouin et al . , haplogroup h seemed to confer an increased chance of long - term survival after sepsis than non - h haplogroups ( primarily the closely - related haplogroups j and t ) maybe for the linkage between mitochondrial dysfunctions and sepsis - induced multiple organ failure demonstrated by protti and singer . two possibilities have been proposed : haplogroup h might protect through greater heat generation ( because of higher electron transport rates or looser coupling ) , or through greater ros production ( because of tighter coupling and raised protonmotive force ) , which could reduce bacterial infection [ 18 , 19 ] . haplogroup r also was a strong independent predictor for survival advantage in severe sepsis with an important impact on long - term clinical outcome . haplogroup h has been associated with high spermatozoa motility , haplogroup t , instead , with asthenozoospermia , showing significant differences in their oxphos performance . did not confirm these findings demonstrating no systematic differences between haplotypes from asthenozoospermic samples and those from population surveys . haplogroup t could be related , instead , with a lower capacity to respond to endurance training , appearing as a marker negatively associated with the status of elite athletic endurance . within the western european , haplogroup j , haplogroups u and k were independently associated with a higher prevalence of age - related hearing loss as described by manwaring et al . on 912 patients of the blue mountains eye study cohort . mtdna haplogroup u , in mitochondrial patients carrying mtdna single macrodeletion , is associated with a higher relative risk of developing pigmentary retinal degeneration , short stature , dysphasia - dysarthria and cardiac conduction defects , modulating the clinical expression of mitochondrial encephalomyopathies due to mtdna macrodeletions . supporting such idea , changes in the number , shape , and function of mitochondria but also mutations in both the noncoding and coding regions of the mtdna have been reported in various types of cancers [ 2729 ] . in a recent study conducted in china , fang et al . found that macrohaplogroup m and its subhaplogroup d5 had an increased frequency in breast cancer patients relative to controls but haplogroup m was decreased in the metastatic group ; macrohaplogroup haplogroup d4a was associated with an increased risk of thyroid cancer , while no significant correlation has been detected between any mtdna haplogroups and colorectal cancer . these evidences suggest that mitochondrial haplogroups may have a tissue - specific , but also population - specific and stage - specific role in modulating cancer development . in 1992 , harman first proposed the mitochondrial - free - radical hypothesis , a mitochondrial theory of aging , supposing that accumulation of damage to mitochondria and mtdna leads to aging of humans and animals and affects longevity . the observation that mitochondrial function declines and mtdna mutation increases in tissue cells in an age - dependent manner supported his theory . age - related impairment in the respiratory enzymes not only decreases atp synthesis but also enhances production of ros through increased electron leakage in the respiratory chain . this association is probably population - specific , being evident in centenarians from northern italy ( haplogroup j increase the individual chance to attain longevity ) but not in those from southern italy . in nonagenarians / centenarians from ireland , finland , and japan , mtdna haplogroups and longevity are also associated . in a recent investigation on longevity in a japanese population , haplogroup d4b2b , d4a , and d5 have been found associated with centenarians ( over 100 years - of - age ) and haplogroup d4a with semisupercentenarians ( over 105 years - of - age ) , while another variant ( mt9055a , haplogroup uk ) was found to be significantly more frequent in french centenarians . a higher frequency of the j haplogroup and a significantly high frequency of three mtdna polymorphisms ( 150 t , 489c , 10398 g ) castri and colleagues found that the 5178a mutation in haplogroup d is associated with decreased longevity , whereas the 150 t mutation is associated with increased longevity in 152 subjects from costa rica . oxidative free radical damage is therefore involved in normal aging , but it is also generally viewed as an etiological cause of tissue degeneration , and ros damage has been reported within specific brain regions in many neurodegenerative conditions . basing on the evidence that mtdna variations lead to different oxphos performance , than to different oxidative stress profile , the association between haplogroups and neurodegenerative disorders has been long investigated ( see table 1 ) . van der walt et al . observed that haplotypes j and k reduced the incidence of parkinson 's disease ( pd ) by 50% . a further analysis revealed that the snp at 9055a of atp6 ( which defines haplogroup k ) reduced the risk in women , and the snp at 13708a of nd5 gene was protective in individuals older than 70 years ( haplogroup j ) . the cluster ukjt was associated with a 22% reduction in risk for pd but not with risk of alzheimer 's disease ( ad ) , confirming that the association with pd was disease - specific and not a general effect seen in all neurodegenerative diseases . the supercluster jtiwx , indeed , increased the risk of both pd and pd with dementia . this cluster was associated with a twofold increase in nonsynonymous substitutions in the mtdna genes encoding complex i subunits . in a large cohort of 620 italian pd patients , in contrast to these evidences , latsoudis et al . did not observe mtdna haplogroups that predisposed to pd in 224 pd patients and 383 controls from crete . a recent study of 2010 conducted on 168 multiplex pd families in which the proband and one parent were diagnosed with pd and 895 controls , in order to investigate the potential contribution of mtdna variants or mutations to the risk of pd , found no significant differences in the frequencies of mitochondrial haplogroups or of the 10398 g complex i gene polymorphism in pd patients compared to controls , and no significant associations with age of onset of pd . mitochondrial haplogroup and 10398 g polymorphism frequencies were similar in probands having an affected father as compared to probands having an affected mother . to investigate if specific genetic polymorphisms within the mtdna could act as susceptibility factors and contribute to the clinical expression of sporadic amyotrophic lateral sclerosis ( als ) , our group genotyped predefined european mtdna haplogroups in 222 patients of clear italian origin with sporadic als and 151 matched controls . age of onset , severity , and neurological system involved in the disease did not associate with haplogroups . in a comparison developed to test what makes this haplogroup i different from the other haplogroups tested , we found highly significant difference in 16391a and 10034c alleles . in accordance with the described study , mtdna polymorphisms might contribute to motor neuron degeneration , possibly interacting with unknown genetic or environmental factors . however , this finding was not confirmed by chinnery et al . , who studied large uk cohort of 504 als patients and 493 controls and found no evidence that mtdna haplogroups contribute to the risk of developing als . as far as regard friedreich 's ataxia ( fa ) , giacchetti et al . studied 99 fa patients and 48 control individuals , all from southern italy revealing that patients with haplogroup u class had a delay of 5 years in the disease onset and a lower rate of cardiomyopathy . however , no significant difference was found in the frequency distribution of haplogroups between patients and controls . huntington 's disease ( hd ) , as fa , is a trinucleotide expansion disorder , caused by a cag expansion in the it15 gene . on the basis of the giacchetti 's report , we wanted to determine whether mtdna polymorphisms play the role of modifier genes in hd . in this work , we have genotyped 51 patients with hd and 181 matched controls . only hd subjects with a similar number of triplets ( 49.3 5.3 , range 4560 ) were enrolled . the frequency of the haplogroups and haplogroup clusters did not differ between the two groups , and we did not observe correlation with gender , age of onset , and disease status . over the last 3 years , patients have undergone prospective evaluations , with a control every 6 months . no significant difference was observed between different haplogroups and haplogroup clusters in the cognitive or motor progression of the disease . therefore , our study did not support the association between mtdna haplogroup(s ) and hd . however , in a recent study , arning and colleagues demonstrate a significantly lower age at onset in patients carrying the most common haplogroup h . several lines of evidence suggest that mitochondrial genetic factors may influence susceptibility to multiple sclerosis ( ms ) . large scale screening of the mtdna revealed no pathogenic mutations but an association between haplogroups k and j with ms in caucasians . more recently , yu and colleagues observed that the frequency of haplogroup j was higher in patients than in controls . 13708a variant itself seemed to explain this association and might represent a susceptibility allele for ms . in the same year a study of ban et al . performed in order to explore this hypothesis further , resequenced the mitochondrial genome from 159 patients with ms and completed a haplogroup analysis including a further 835 patients and 1,506 controls . haplogroup analysis identified a trend towards an overrepresentation of superhaplogroup u. in addition they also found modest evidence of an association with variations in the nuclear gene ndufs2 . in 2009 , ghabaee and coauthors studied mtdna haplogroups , age , gender , clinical disability , course of the disease , and presenting symptoms of 52 ms patients . haplotype a was more prevalent in patients with younger age of onset and high proportion of haplogroup h was associated with optic nerve involvement . ad is a brain neurodegenerative disorder named for german physician alois alzheimer , who first described it at a scientific meeting in november 1906 , presenting the case of frau auguste d. a 51-year - old woman brought to see him in 1901 by her family . approximately 2 - 3% of ad cases are early onset and familial , with autosomal dominant inheritance . mutations in three genes are known to cause familial ad : mutations in the gene of amyloid precursor protein ( app ) , which is leaved sequentially by b- and c - secretases , and mutations in genes of presenilins 1 and 2 ( ps1 and ps2 ) , one or other of which is a component of each c - secretases complex . although these specific mutations have been associated with the relatively rare forms of familial ad , the causes of the much more frequently occurring sporadic ad remain unknown , and the mechanisms leading to neuronal death are still unclear . most forms of sporadic late - onset ad have probably a complex aetiology due to environmental and genetic factors , which taken alone , are not sufficient to develop the disease . presently , age is the major risk factor for sporadic ad , while the genetic major risk factor is recognized in the presence of allele 4 of apolipoprotein e ( apoe4 ) . however , in the majority of late - onset ad patients , the casual factors are still unknown and genetics factor probably interact with environmental factors or with other pathologic or physiologic conditions to exert the pathogenic effect , such as chronic inflammation , excitotoxicity damage , oxidative stress , and diminished brain metabolism . to date , the amyloid- cascade hypothesis , first proposed in 1992 , remains the main pathogenetic model of ad . however , although this cascade is potentially viable in familial ad cases with mutation in app and ps genes , its role in the sporadic ad is unclear . although the pathogenic mechanisms of neurodegeneration in ad are not clarified , in the past 15 years , numerous studies have been performed in order to better understand the possible involvement of mitochondria , accumulating evidences suggesting that mitochondrial dysfunction and oxidative stress occur in brain and peripheral tissues of ad patients and supporting the idea that mitochondria may trigger the abnormal onset of neuronal degeneration and death in ad . in order to study the importance and potential causes of mitochondrial dysfunction , the cytoplasmic hybrid ( cybrid ) technique , a technique in which mitochondria / mtdna from human ad and control platelets is transferred to culturable cells depleted of endogenous mtdna , has been applied . the resulting ad cybrids showed a transferred cox defect and revealed a number of consequences that recapitulate the pathology observed in ad brain [ 6567 ] . remarkably , ad cybrid lines had increased intracellular amyloid accumulation and increased amyloid secretion , as well as an increased proportion of morphologically abnormal mitochondria . trimmer et al . observed that ad cybrid lines show a bioenergetic defect that becomes more severe with passage in culture . ad cybrids also showed elevated spontaneous death with apoptotic nuclear morphology , increased cytoplasmic cytochrome c levels and caspase-3 activity , and elevated cleavage of caspase substrate not observed in non - ad cybrids . again , the spontaneous alterations in cell death and cell - death pathways observed spontaneously in ad cybrids can be reproduced by exposing non - ad cybrids to exogenous aggregated beta amyloid or oxidative stress [ 69 , 70 ] . despite these reports , a number of technical issues have confounded cybrid studies [ 71 , 72 ] . however , these studies on cybrid models reinforce the concept that primary mtdna changes could be responsible for the mitochondrial features of sporadic ad and could be the origin of the increased oxidative stress and a deposition found in sporadic ad brain . with the aim of better clarifing the involvement of mitochondria in the pathogenesis of ad , it has studied the possible association of mitochondrial haplogroups and ad susceptibility . as already discussed , inherited european mitochondrial haplogroups may be related to longevity [ 37 , 73 ] , as well as to neurodegeneration , ad risk , and , thus , death in caucasians . in a paper of chagnon et al . , haplogroup t is described underrepresented in ad , while haplogroup j overrepresented . van der walt et al . showed that males classified as haplogroup u had a significant increase in risk of ad , while females demonstrated a significant decrease in risk with the same u haplogroup . to assess the relationship between mtdna haplogroup and ad in an iranian population , fesahat sequenced the two mtdna hypervariable segments in 30 ad patients and 100 control subjects . they found that haplogroups h and u are significantly more abundant in ad patients , assuming that these two haplogroups might act synergistically to increase the penetrance of ad disease . by studying an italian sample of subjects , carrieri et al . hypothesized that k and u haplogroups may act by neutralizing the effect of the major ad risk factor apoe 4 allele . however , this association was not confirmed recently by a collaborative study performed by elson et al . , in which the authors analyzed the complete mtdna coding region sequences from more than 270 ad patients and normal controls . they also observed that for both synonymous and nonsilent changes , the overall numbers of nucleotide substitutions were the same for the ad and control sequences . regarding our experience , in our laboratory we did not find any evidence for an etiological role of haplogroups in ad risk . we studied the frequency of the european mtdna haplogroups in a clinically well - defined group of 209 unrelated patients and 191 controls , both with clear tuscan origin ( in order to minimize the risk of false associations between gene markers and disease ) . furthermore , there was no significant difference between genders as far as mtdna haplogroups distribution in both ad patients and control groups is concerned . takasaki , studying a japanese population of 96 ad patients , described an association between ad and the haplogroups g2a , b4c1 , and n9b1 . in addition , to compare mitochondrial haplogroups of the ad patients with those of other classes of japanese people , the relationships between four classes of japanese people ( 96 japanese centenarians , 96 parkinson 's disease ( pd ) patients , 96 type 2 diabetic ( t2d ) patients , and 96 nonobese young males ) and their haplogroups are also described . the four classes of people are associated with a set of haplogroups , therefore different from those of the ad japanese patients . a study conducted on a polish population found that hv cluster is significantly associated with the risk of ad , regardless of the apoe4 status , reporting no evidence for the involvement of haplogroup u , k , j , or t in ad risk . the most recent report about a possible link between ad and mtdna genotypes shows evidence for subhaplogroup h5 as a risk factor for late onset ad . santoro et al . analyzed 936 ad patients and 776 controls comparable for age and ethnicity from the central - northern regions of italy . h4 , h5 , and h6 are associated with an increased production of ros than those with haplogroup t. accordingly , it was hypothesized that subjects with haplogroup h could be more prone to oxidative stress than those with other haplogroups , and consequently more susceptible to neurodegenerative diseases . they found that subhaplogroup h5 appears to be associated with a higher risk of ad in both the total sample and the female group . they also found that subhaplogroup h5 interacts with age in modifying ad risk ; in fact h5 subjects younger than 75 years old had a higher ad risk than non - h5 subjects . age is a strong risk factor for ad and the authors hypothesized that age 75 could be considered as a threshold , under which risk factors such as subhaplogroup h5 and apoe could independently exert their major effect on the development of the disease , while over this age , other risk factors , such as ageing itself , would largely prevail . tanaka and colleagues also agree that inherited mtdna common polymorphisms could not be the single major causes of ad but that some rare variants in the protein - coding - region may have protective effects for high - risk populations with the apoe 4 allele . the authors however indicate that the np956 - 965 poly - c insertion and 856a > g variant might be a risk factor for ad . in the past 15 years , research has been directed at clarifying the involvement of mitochondria and defects in mitochondrial oxidative phosphorylation in several disorders . it has been speculated that mtdna mutations that accumulate with age might lead to impaired energy generation and to increased amount of ros , both resulting in cell damage . polymorphisms in mtdna may cause subtle differences in mitochondrial respiratory chain activity and free radical overproduction . this could predispose an individual , or a population sharing the same mtdna genotype , to an earlier onset of apoptotic processes , such as accumulation of somatic mtdna mutations and mitochondrial impairment . the opposite could be true for different polymorphism(s ) , which could be beneficial increasing mitochondrial respiration and/or reducing ros production [ 57 , 85 ] . a critical role for mitochondrial dysfunction and oxidative damage in neurodegenerative diseases has been greatly strengthened by recent findings . however , despite the morphological and biochemical evidence of mitochondrial dysfunctions in various tissues of patients with neurodegenerative disorders , the role of the mitochondrial genome and of its haplogroups as a risk factor is still debated . mtdna haplogroups have been associated with a number of different neurodegenerative diseases , but to date , the only disease consistently associated with a different mtdna haplogroup frequency is pd . although it has been suggested that inherited haplogroups k and u may influence ad risk in caucasians , this is still an unresolved question . to date , mtdna haplogroups do not seem to play a major role in ad . the mtdna alterations that cybrid models induce to hypothesize might be due to somatic factors . app , a-induced mitochondrial toxicity , oxidative stress , somatic mtdna damage , mitochondrial dysfunction and apoptosis seem to be interconnected in vicious manner that reinforces this dysfunction by causing mtdna damage , impairment of the mitochondrial respiration and oxidative stress leading to neurodegeneration and dementia . researches performed in order to allow the identification of ad risk factor also in preclinical stages might be useful in the development of more effective therapeutic interventions at a potentially reversible stage .

mitochondria , the powerhouse of the cell , play a critical role in several metabolic processes and apoptotic pathways . multiple evidences suggest that mitochondria may be crucial in ageing - related neurodegenerative diseases . moreover , mitochondrial haplogroups have been linked to multiple area of medicine , from normal ageing to diseases , including neurodegeneration . polymorphisms within the mitochondrial genome might lead to impaired energy generation and to increased amount of reactive oxygen species , having either susceptibility or protective role in several diseases . here , we highlight the role of the mitochondrial haplogroups in the pathogenetic cascade leading to diseases , with special attention to alzheimer 's disease .

1. Introduction 2. Mitochondrial Haplogroups and Medicine 3. Haplogroups and Aging 4. Haplogroups and Neurodegeneration 5. Haplogroups and Alzheimer's Disease 6. Conclusions

these regions include the outer membrane , the intermembrane space , the inner membrane , the cristae , and the matrix . cellular power plants because they produce most of the cell 's supply of adenosine triphosphate ( atp ) , by means of the oxidative phosphorylation ( oxphos ) machinery , which comprises electron transport chain ( etc ) and atp synthase ( complex v ) . the etc provides the cell with the most efficient energetic outcome in terms of atp production . it consists of four multimeric protein complexes ( complex i to iv ) located in the inner mitochondrial membrane together with complex v . etc dysfunction leads to reduced atp production , impaired calcium buffering , and increased generation of reactive oxygen species ( ros ) . mitochondria have their own dna , the mitochondrial dna ( mtdna ) , represented by a circular molecule of 16.5 kb without introns , constituting of a heavy chain ( h ) and a light chain ( l ) [ 3 , 5 ] . mtdna carries 37 genes : 22 encoding for mitochondrial transfer rnas ( trnas ) ( for the 20 standard amino acids , plus an extra gene for leucine and serine ) , 2 for ribosomal rnas ( rrnas ) and 13 encoding for polypeptides subunits of complexes of the respiratory chain system , 7 of them belonging to complex i or nadh dehydrogenase ( nd1 , nd2 , nd3 , nd4 , nd4l , nd5 , nd6 ) , 1 to complex iii or cytochrome c reductase , 3 to complex iv or cytochrome c oxidase ( cox i , cox ii and cox iii ) , and 2 to complex v or atp synthase ( atpase6 and atpase8 ) . the remaining mitochondrial proteins , including all the complex ii subunits , are encoded by nuclear dna . furthermore , human mtdna is characterized by a much greater evolutionary rate than that of the average of nuclear genes . ancestor mother , the mitochondrial eve , by identifying common polymorphisms that have been accumulated with time . these common polymorphisms describe classes of continent - specific genotypes , the haplogroups , evolved from the same ancestor , which can be detected by restriction fragment length polymorphism ( rflp ) analysis . because the process of molecular differentiation is relatively fast and occurred mainly during and after the recent process of dispersal into different parts of the world , the basal branching structure of mtdna variation in most parts of the world is now well understood . the major branches of the tree were usually restricted geographically , some to sub - saharan africans , others to east asians and yet others to europeans and near easterners . about 85000 years ago , probably in the horn of africa , changes in climate , the glacial interstadial phase 21 , triggered the rising of many descendant haplogroups from the root of haplogroup l3 , the first multifurcation node , probably because of some colonization event or local population growth [ 6 , 9 , 10 ] . non - african mtdna ( excluding migrations from africa within the past few thousand years ) descend from l3 and belong either to the m or n superclades . haplogroup n soon gave rise to haplogroup r. in the indian subcontinent and in southeast asia there is the richest basal variation in the three founder haplogroups m , n , and r , and this suggests a rapid colonization along the southern coast of asia , about 60000 years ago . over 30 subclades of the haplogroup m haplogroups a , b , c , d , and x have been found in the americas , coming mainly from asia . the expansions northwards occurred later , about 45000 years ago when technology and climatic conditions enabled the exploration of the interior of eurasia . one of the more marginal extensions eventually led to the peopling of europe . in europeans and near easterners ( who share a rather recent common ancestor ) , nine different mitochondrial haplogroups have been identified ( h , i , j , k , t , u , v , w , x ) . european mtdna variation is surprisingly impoverished in the number of independent basal lineages , compared with the south asian mtdna variation , maybe for the peripheral role that the pioneer migration into the near east and subsequently europe must have had in the broader out - of - africa scenario . complete mtdna sequencing and the increasing number of samples analyzed allowed subdividing haplogroups in smaller groups identifying younger branches on the mtdna evolution tree . it has been supposed that genetic polymorphisms within the mitochondrial genome might lead to impaired energy generation and to increased amount of ros , causing subtle differences in the encoded proteins and , thus , minimum changes in mitochondrial respiratory chain oxphos activity and free radical overproduction . accumulation of mtdna mutations enhances normal ageing , leads to oxidative damage , and causes energy failure and increased production of ros , in a vicious cycle . this could predispose to diseases or modify longevity in individual or in a population sharing the same mtdna genotype . several studies correlate mitochondrial haplogroups with disease 's susceptibility or confer them protective roles . furthermore , the lower ros production associated to j could also account for the accrual of this variant in elderly people consequent to a decreased oxidative damage . this mutation on the trnaleu gene ( uur ) is one of the most common mtdna mutation . one of the most common manifestation of some mitochondrial disease is the presence of retinal pigmentary changes , which are similar to those observed in age - related maculopathy ( arm ) . haplogroup h1 may be protective for ischemic stroke ; in fact , it was found to be significantly less frequent in stroke patients than in controls , when comparing each clade against all other haplogroups pooled together , in a paper of rosa et al . , haplogroup h seemed to confer an increased chance of long - term survival after sepsis than non - h haplogroups ( primarily the closely - related haplogroups j and t ) maybe for the linkage between mitochondrial dysfunctions and sepsis - induced multiple organ failure demonstrated by protti and singer . two possibilities have been proposed : haplogroup h might protect through greater heat generation ( because of higher electron transport rates or looser coupling ) , or through greater ros production ( because of tighter coupling and raised protonmotive force ) , which could reduce bacterial infection [ 18 , 19 ] . haplogroup h has been associated with high spermatozoa motility , haplogroup t , instead , with asthenozoospermia , showing significant differences in their oxphos performance . haplogroup t could be related , instead , with a lower capacity to respond to endurance training , appearing as a marker negatively associated with the status of elite athletic endurance . within the western european , haplogroup j , haplogroups u and k were independently associated with a higher prevalence of age - related hearing loss as described by manwaring et al . on 912 patients of the blue mountains eye study cohort . supporting such idea , changes in the number , shape , and function of mitochondria but also mutations in both the noncoding and coding regions of the mtdna have been reported in various types of cancers [ 2729 ] . found that macrohaplogroup m and its subhaplogroup d5 had an increased frequency in breast cancer patients relative to controls but haplogroup m was decreased in the metastatic group ; macrohaplogroup haplogroup d4a was associated with an increased risk of thyroid cancer , while no significant correlation has been detected between any mtdna haplogroups and colorectal cancer . these evidences suggest that mitochondrial haplogroups may have a tissue - specific , but also population - specific and stage - specific role in modulating cancer development . in 1992 , harman first proposed the mitochondrial - free - radical hypothesis , a mitochondrial theory of aging , supposing that accumulation of damage to mitochondria and mtdna leads to aging of humans and animals and affects longevity . age - related impairment in the respiratory enzymes not only decreases atp synthesis but also enhances production of ros through increased electron leakage in the respiratory chain . in a recent investigation on longevity in a japanese population , haplogroup d4b2b , d4a , and d5 have been found associated with centenarians ( over 100 years - of - age ) and haplogroup d4a with semisupercentenarians ( over 105 years - of - age ) , while another variant ( mt9055a , haplogroup uk ) was found to be significantly more frequent in french centenarians . a higher frequency of the j haplogroup and a significantly high frequency of three mtdna polymorphisms ( 150 t , 489c , 10398 g ) castri and colleagues found that the 5178a mutation in haplogroup d is associated with decreased longevity , whereas the 150 t mutation is associated with increased longevity in 152 subjects from costa rica . basing on the evidence that mtdna variations lead to different oxphos performance , than to different oxidative stress profile , the association between haplogroups and neurodegenerative disorders has been long investigated ( see table 1 ) . observed that haplotypes j and k reduced the incidence of parkinson 's disease ( pd ) by 50% . the cluster ukjt was associated with a 22% reduction in risk for pd but not with risk of alzheimer 's disease ( ad ) , confirming that the association with pd was disease - specific and not a general effect seen in all neurodegenerative diseases . this cluster was associated with a twofold increase in nonsynonymous substitutions in the mtdna genes encoding complex i subunits . a recent study of 2010 conducted on 168 multiplex pd families in which the proband and one parent were diagnosed with pd and 895 controls , in order to investigate the potential contribution of mtdna variants or mutations to the risk of pd , found no significant differences in the frequencies of mitochondrial haplogroups or of the 10398 g complex i gene polymorphism in pd patients compared to controls , and no significant associations with age of onset of pd . to investigate if specific genetic polymorphisms within the mtdna could act as susceptibility factors and contribute to the clinical expression of sporadic amyotrophic lateral sclerosis ( als ) , our group genotyped predefined european mtdna haplogroups in 222 patients of clear italian origin with sporadic als and 151 matched controls . age of onset , severity , and neurological system involved in the disease did not associate with haplogroups . in a comparison developed to test what makes this haplogroup i different from the other haplogroups tested , we found highly significant difference in 16391a and 10034c alleles . studied 99 fa patients and 48 control individuals , all from southern italy revealing that patients with haplogroup u class had a delay of 5 years in the disease onset and a lower rate of cardiomyopathy . however , no significant difference was found in the frequency distribution of haplogroups between patients and controls . huntington 's disease ( hd ) , as fa , is a trinucleotide expansion disorder , caused by a cag expansion in the it15 gene . on the basis of the giacchetti 's report , we wanted to determine whether mtdna polymorphisms play the role of modifier genes in hd . in this work , we have genotyped 51 patients with hd and 181 matched controls . the frequency of the haplogroups and haplogroup clusters did not differ between the two groups , and we did not observe correlation with gender , age of onset , and disease status . over the last 3 years , patients have undergone prospective evaluations , with a control every 6 months . no significant difference was observed between different haplogroups and haplogroup clusters in the cognitive or motor progression of the disease . several lines of evidence suggest that mitochondrial genetic factors may influence susceptibility to multiple sclerosis ( ms ) . in the same year a study of ban et al . performed in order to explore this hypothesis further , resequenced the mitochondrial genome from 159 patients with ms and completed a haplogroup analysis including a further 835 patients and 1,506 controls . haplogroup analysis identified a trend towards an overrepresentation of superhaplogroup u. in addition they also found modest evidence of an association with variations in the nuclear gene ndufs2 . in 2009 , ghabaee and coauthors studied mtdna haplogroups , age , gender , clinical disability , course of the disease , and presenting symptoms of 52 ms patients . approximately 2 - 3% of ad cases are early onset and familial , with autosomal dominant inheritance . mutations in three genes are known to cause familial ad : mutations in the gene of amyloid precursor protein ( app ) , which is leaved sequentially by b- and c - secretases , and mutations in genes of presenilins 1 and 2 ( ps1 and ps2 ) , one or other of which is a component of each c - secretases complex . although these specific mutations have been associated with the relatively rare forms of familial ad , the causes of the much more frequently occurring sporadic ad remain unknown , and the mechanisms leading to neuronal death are still unclear . presently , age is the major risk factor for sporadic ad , while the genetic major risk factor is recognized in the presence of allele 4 of apolipoprotein e ( apoe4 ) . however , in the majority of late - onset ad patients , the casual factors are still unknown and genetics factor probably interact with environmental factors or with other pathologic or physiologic conditions to exert the pathogenic effect , such as chronic inflammation , excitotoxicity damage , oxidative stress , and diminished brain metabolism . to date , the amyloid- cascade hypothesis , first proposed in 1992 , remains the main pathogenetic model of ad . however , although this cascade is potentially viable in familial ad cases with mutation in app and ps genes , its role in the sporadic ad is unclear . although the pathogenic mechanisms of neurodegeneration in ad are not clarified , in the past 15 years , numerous studies have been performed in order to better understand the possible involvement of mitochondria , accumulating evidences suggesting that mitochondrial dysfunction and oxidative stress occur in brain and peripheral tissues of ad patients and supporting the idea that mitochondria may trigger the abnormal onset of neuronal degeneration and death in ad . in order to study the importance and potential causes of mitochondrial dysfunction , the cytoplasmic hybrid ( cybrid ) technique , a technique in which mitochondria / mtdna from human ad and control platelets is transferred to culturable cells depleted of endogenous mtdna , has been applied . again , the spontaneous alterations in cell death and cell - death pathways observed spontaneously in ad cybrids can be reproduced by exposing non - ad cybrids to exogenous aggregated beta amyloid or oxidative stress [ 69 , 70 ] . however , these studies on cybrid models reinforce the concept that primary mtdna changes could be responsible for the mitochondrial features of sporadic ad and could be the origin of the increased oxidative stress and a deposition found in sporadic ad brain . with the aim of better clarifing the involvement of mitochondria in the pathogenesis of ad , it has studied the possible association of mitochondrial haplogroups and ad susceptibility . as already discussed , inherited european mitochondrial haplogroups may be related to longevity [ 37 , 73 ] , as well as to neurodegeneration , ad risk , and , thus , death in caucasians . hypothesized that k and u haplogroups may act by neutralizing the effect of the major ad risk factor apoe 4 allele . they also observed that for both synonymous and nonsilent changes , the overall numbers of nucleotide substitutions were the same for the ad and control sequences . regarding our experience , in our laboratory we did not find any evidence for an etiological role of haplogroups in ad risk . we studied the frequency of the european mtdna haplogroups in a clinically well - defined group of 209 unrelated patients and 191 controls , both with clear tuscan origin ( in order to minimize the risk of false associations between gene markers and disease ) . in addition , to compare mitochondrial haplogroups of the ad patients with those of other classes of japanese people , the relationships between four classes of japanese people ( 96 japanese centenarians , 96 parkinson 's disease ( pd ) patients , 96 type 2 diabetic ( t2d ) patients , and 96 nonobese young males ) and their haplogroups are also described . the four classes of people are associated with a set of haplogroups , therefore different from those of the ad japanese patients . a study conducted on a polish population found that hv cluster is significantly associated with the risk of ad , regardless of the apoe4 status , reporting no evidence for the involvement of haplogroup u , k , j , or t in ad risk . h4 , h5 , and h6 are associated with an increased production of ros than those with haplogroup t. accordingly , it was hypothesized that subjects with haplogroup h could be more prone to oxidative stress than those with other haplogroups , and consequently more susceptible to neurodegenerative diseases . age is a strong risk factor for ad and the authors hypothesized that age 75 could be considered as a threshold , under which risk factors such as subhaplogroup h5 and apoe could independently exert their major effect on the development of the disease , while over this age , other risk factors , such as ageing itself , would largely prevail . tanaka and colleagues also agree that inherited mtdna common polymorphisms could not be the single major causes of ad but that some rare variants in the protein - coding - region may have protective effects for high - risk populations with the apoe 4 allele . in the past 15 years , research has been directed at clarifying the involvement of mitochondria and defects in mitochondrial oxidative phosphorylation in several disorders . it has been speculated that mtdna mutations that accumulate with age might lead to impaired energy generation and to increased amount of ros , both resulting in cell damage . a critical role for mitochondrial dysfunction and oxidative damage in neurodegenerative diseases has been greatly strengthened by recent findings . however , despite the morphological and biochemical evidence of mitochondrial dysfunctions in various tissues of patients with neurodegenerative disorders , the role of the mitochondrial genome and of its haplogroups as a risk factor is still debated . mtdna haplogroups have been associated with a number of different neurodegenerative diseases , but to date , the only disease consistently associated with a different mtdna haplogroup frequency is pd . to date , mtdna haplogroups do not seem to play a major role in ad . app , a-induced mitochondrial toxicity , oxidative stress , somatic mtdna damage , mitochondrial dysfunction and apoptosis seem to be interconnected in vicious manner that reinforces this dysfunction by causing mtdna damage , impairment of the mitochondrial respiration and oxidative stress leading to neurodegeneration and dementia . researches performed in order to allow the identification of ad risk factor also in preclinical stages might be useful in the development of more effective therapeutic interventions at a potentially reversible stage .

prolonged exposure to hand - arm vibration is a cause of hand - arm vibration syndrome ( havs ) . this disorder is characterized by circulatory disturbances , neurological disorders , and bone - and - joint ( musculoskeletal ) changes in upper extremities . in cases of havs , the severity of peripheral neuropathy ( pn ) is evaluated by measuring vibrotactile perception thresholds ( vpts ) at the fingertips . however , to evaluate havs neuropathy with vpts , it is necessary first to rule out conditions such as ( 1 ) traumas ( burning , frostbite , and other causes ) , ( 2 ) raynaud 's phenomenon of nonvibratory causes , ( 3 ) thoracic outlet syndrome , ( 4 ) neuropathy / angiopathy caused by poisoning and related agents , ( 5 ) angiopathy caused by pulseless disease , diabetes , buerger 's disease and related disorders , ( 6 ) collagen vascular diseases ( e.g. , rheumatoid arthritis , systemic sclerosis , etc . ) , ( 7 ) gout , and ( 8) certain other chronic arthritides ( e.g. , tuberculosis - induced , etc . ) . some recent studies have shown associations between selected anthropometric factors and pn . in a study by skov et al . using vibrometry data gathered from workers in the united states who were exposed to organic solvents , older age and taller body were positively associated with higher vpt . specifically , the foot 's vpt was positively associated with body height , but the hand 's vpt was not . the greater effects of age and height on toe threshold , rather than finger threshold , is consistent with the hypothesis that the length of the nerve increases susceptibility to pn . the regrowth of injured axons is apparently as slow as 1 mm per day , and it takes a longer time for longer nerve fibers to recover if they get injured . chuang et al . did not find a significantly positive association between body height and vpt in a population exposed to lead . this negative finding was probably because the study subjects were shorter , on average , than those in the study by skov et al .. as for body weight , the associations of body weight and body mass index ( bmi ) with vpt were either negative or not evaluated . the prevalence of overweight or obesity ( bmi 25.0 kg / m ) is increasing , especially among males in japan . overweight or obesity itself is a risk factor for some chronic diseases such as hypertension , diabetes , and dyslipidemia . it is also reported that pn is observed among obese patients with or without diabetes . in other words , larger body size may be associated with an increased risk of pn . on the other hand , the surface - area - to - volume ratio ( sa : v ) of the human body is lower in those with a larger body volume ( the square - cube law ) . in other words , those with a larger body size will have a lower sa : v ; they radiate less body heat per unit of mass and stay warmer in cold climates . bergmann 's rule attests to this mechanism , which indicates that species of larger size are found in colder environments , and species of smaller size are found in warmer environments . higher body height , body weight , and bmi indicate larger body sizes that will stay warmer in cold environments . on the basis of this perspective , we hypothesize that large body size may be negatively ( i.e. , protectively ) associated with vpts . in other words , larger body size may be associated with a decreased risk of pn . thus , although some papers have reported the association between anthropometric factors and havs , as well as evidence to support the association between these factors and somatic disorders related to havs , only a limited number of studies have been conducted to evaluate the associations between anthropometric factors and vpt . in japan , health examinations are provided for industrial vibrating tool operators ( ivtos ) to evaluate havs , but to the best of our knowledge , the associations between anthropometric factors and vpt among ivtos remain unclear . if anthropometric factors are proven to be associated with havs - related pn , such information may be useful for preventing it , for example , by excluding high - risk populations from this type of physically hard work , or for advising ivtos to gain or lose weight to a certain level that would minimize the risk of havs - related pn . for this analysis , we recruited study participants from two groups of workers : group 1 , workers predominantly engaged in forestry , who were exposed to hand - arm vibration through hand - held vibrating tools , mainly chain saws ( n=336 , all males ) ; and group 2 , public servants engaged in road or farm maintenance , who were exposed to hand - arm vibration through handheld vibrating tools , mainly bush clearers ( n=92 , 91 males and 1 female ) . the examinations were conducted annually as a part of occupational health management , pursuant to a circular from the ministry of health , labour and welfare ( formerly the ministry of labour ) , government of japan . we included both groups 1 and 2 in the study to evaluate whether anthropometric factors affect pn regardless of the task performed . medical examinations that included the assessment of vpts were performed from november to december 2013 for group 1 and from january to february 2012 for group 2 . a total of 331 workers in group 1 ( 98.5% ) and 92 workers in group 2 ( 100% ) gave written consent to have their data from the medical examinations included in this study . workers were asked to complete a self - administered questionnaire about their conditions , including years engaged in operating industrial vibrating tools , days per year operating such tools , hours per day operating them , lifestyle factors ( smoking , alcohol consumption , etc . ) , medical history ( including occupational traumas ) , havs - related symptoms ( tingling , numbness , dysesthesia , pain , white finger , etc . ) , and other symptoms and complaints . total operating time ( tot ) ( hours ) was defined as daily operating time ( hours / day ) number of days exposed to vibration ( days / year)years exposed to vibration ( years ) . medical examinations were conducted in a quiet room , with the temperature set at 20 - 23c . workers were asked to abstain from smoking and drinking beverages with caffeine and alcohol for at least two hours beforehand . after acclimatization to the room temperature for at least 30 minutes , vpts at 125 hz were measured in four fingertips ( except thumbs ) of both hands in group 1 and in all five fingertips ( thumbs included ) of both hands in group 2 . vpts were measured in a sitting position with a vibrometer [ au-02 , rion co. , ltd . , tokyo , japan ( reference value : 0 db=308 mm / s ) ] . during measurement , the ascending threshold was observed ; vibration began at a low level ( -10.0 db in general ) and then gradually intensified at an interval of 2.5 db until the worker perceived it , which was taken as the vpt . after staying in a sitting position , they immersed their dominant hand or the hand with havs - related symptoms ( tingling , numbness , dysesthesia , pain , white finger , etc . ) , if any , in cold water to the wrist for a designated time period ( 10 minutes at 10c for group 1 and 5 minutes at 12c for group 2 ) . the 10c , 10-min method is a conventional test condition for evaluating havs in japan . the 12c , 5-min method is less invasive than the 10c , 10-min method and can be conducted easily among ivtos with exposure to a smaller amount of vibration . body height was measured with a stadiometer , with shoes removed . as for subjects whose body height was unavailable greater worker height served not only as factor representing body size but also as a proxy indicator for longer peripheral nerve fiber lengths . body weight was measured in both groups with light clothing and also with shoes removed . body mass index ( bmi ) ( kg / m ) was calculated as body weight in kilograms divided by the square of the height in meters . after finishing the cold - water immersion test , the subjects received physical and neurological examinations from a physician . after excluding ( a ) female workers ( n=1 in group 2 ) , ( b ) workers who reported a history of diabetes ( n=1 in group 1 , and n=5 in group 2 ) , ( c ) workers who did not participate in the cold - water immersion test ( n=2 in group 1 ) , and ( d ) workers with some missing data for age , body height , body weight , bmi , or years exposed to vibrating tools ( n=3 in group 1 , and n=18 in group 2 ) , male workers with complete data ( n=325 in group 1 and n=68 in group 2 ) were used for the statistical analyses . for both group 1 ( 10c , 10-min method ) and group 2 ( 12c , 5-min method ) , pn was defined as a vpt 17.5 db at 10 minutes after finishing the cold - water immersion . we reviewed public servants ' medical records and found that the vpt at 10 minutes after finishing immersion was virtually the same between group 2 public servants in january to february 2012 ( 12c , 5-min method ) and public servants of the same population who received a cold - water immersion test ( 10c , 10-min method ) in january to february 2010 , while the vpt before immersion was significantly different [ table 1 ] . therefore , we adopted the same cutoff value for the vpt at 10 minutes after immersion in both the groups . ( unfortunately , anthropometry was not conducted among the public servants in 2010 , and so we could not compare anthropometric data for them with those of group 1 workers in 2013 . ) the characteristics of ivtos were compared between group 1 and group 2 . continuous variables ( age , body height , body weight , bmi , years , days and hours exposed to vibrating tools , and tot ) were compared with mann - whitney 's u - test . categorical variables ( prevalence of pn ) were compared with the chi - square test . the odds ratio ( or ) for pn , along with its 95% confidence interval ( ci ) , was calculated as an outcome variable of each explanatory variable using a logistic regression model . in the univariate analysis , we evaluated independent exposure variables such as age , body height , body weight , bmi , years exposed to industrial vibrating tools , days per year exposed to industrial vibrating tools , hours per day exposed to industrial vibrating tools , and tot . the study subjects of each group were divided into quartiles according to each variable . in group 2 , due to the small number of subjects with pn , we merged more than one classes into a single class in some analyses . in the multivariate analysis , we included all variables with p<0.10 in the univariate analysis as well as body weight and bmi in group 1 . in group 2 , due to the small number of study subjects , we included variables with p<0.10 in the univariate analysis as well as variables included in the multivariate model of group 1 . then we performed additional analyses , removing body weight or bmi in a mutually exclusive manner , in order to control potential multicollinearity between bmi and body weight . a value of p<0.05 was considered significant , and a value of 0.05 p<0.10 was considered marginally significant . all analyses were conducted using the sas 9.3 software ( sas institute , inc . , cary , nc , usa ) . figures indicate the average ( standard deviation ) of vibrotactile perception thresholds unless otherwise specified . the unpaired t - test analysis of covariance was conducted between the year 2010 and year 2012 data , controlling for the vibrotactile perception threshold before immersion . for this analysis , we recruited study participants from two groups of workers : group 1 , workers predominantly engaged in forestry , who were exposed to hand - arm vibration through hand - held vibrating tools , mainly chain saws ( n=336 , all males ) ; and group 2 , public servants engaged in road or farm maintenance , who were exposed to hand - arm vibration through handheld vibrating tools , mainly bush clearers ( n=92 , 91 males and 1 female ) . the examinations were conducted annually as a part of occupational health management , pursuant to a circular from the ministry of health , labour and welfare ( formerly the ministry of labour ) , government of japan . we included both groups 1 and 2 in the study to evaluate whether anthropometric factors affect pn regardless of the task performed . medical examinations that included the assessment of vpts were performed from november to december 2013 for group 1 and from january to february 2012 for group 2 . a total of 331 workers in group 1 ( 98.5% ) and 92 workers in group 2 ( 100% ) gave written consent to have their data from the medical examinations included in this study . workers were asked to complete a self - administered questionnaire about their conditions , including years engaged in operating industrial vibrating tools , days per year operating such tools , hours per day operating them , lifestyle factors ( smoking , alcohol consumption , etc . ) , medical history ( including occupational traumas ) , havs - related symptoms ( tingling , numbness , dysesthesia , pain , white finger , etc . ) , and other symptoms and complaints . total operating time ( tot ) ( hours ) was defined as daily operating time ( hours / day ) number of days exposed to vibration ( days / year)years exposed to vibration ( years ) . medical examinations were conducted in a quiet room , with the temperature set at 20 - 23c . workers were asked to abstain from smoking and drinking beverages with caffeine and alcohol for at least two hours beforehand . after acclimatization to the room temperature for at least 30 minutes , vpts at 125 hz were measured in four fingertips ( except thumbs ) of both hands in group 1 and in all five fingertips ( thumbs included ) of both hands in group 2 . vpts were measured in a sitting position with a vibrometer [ au-02 , rion co. , ltd . , tokyo , japan ( reference value : 0 db=308 mm / s ) ] . during measurement , the ascending threshold was observed ; vibration began at a low level ( -10.0 db in general ) and then gradually intensified at an interval of 2.5 db until the worker perceived it , which was taken as the vpt . after staying in a sitting position , they immersed their dominant hand or the hand with havs - related symptoms ( tingling , numbness , dysesthesia , pain , white finger , etc . ) , if any , in cold water to the wrist for a designated time period ( 10 minutes at 10c for group 1 and 5 minutes at 12c for group 2 ) . the 10c , 10-min method is a conventional test condition for evaluating havs in japan . the 12c , 5-min method is less invasive than the 10c , 10-min method and can be conducted easily among ivtos with exposure to a smaller amount of vibration . body height was measured with a stadiometer , with shoes removed . as for subjects whose body height was unavailable greater worker height served not only as factor representing body size but also as a proxy indicator for longer peripheral nerve fiber lengths . body weight was measured in both groups with light clothing and also with shoes removed . body mass index ( bmi ) ( kg / m ) was calculated as body weight in kilograms divided by the square of the height in meters . after finishing the cold - water immersion test , the subjects received physical and neurological examinations from a physician . after excluding ( a ) female workers ( n=1 in group 2 ) , ( b ) workers who reported a history of diabetes ( n=1 in group 1 , and n=5 in group 2 ) , ( c ) workers who did not participate in the cold - water immersion test ( n=2 in group 1 ) , and ( d ) workers with some missing data for age , body height , body weight , bmi , or years exposed to vibrating tools ( n=3 in group 1 , and n=18 in group 2 ) , male workers with complete data ( n=325 in group 1 and n=68 in group 2 ) were used for the statistical analyses . for both group 1 ( 10c , 10-min method ) and group 2 ( 12c , 5-min method ) , pn was defined as a vpt 17.5 db at 10 minutes after finishing the cold - water immersion . we reviewed public servants ' medical records and found that the vpt at 10 minutes after finishing immersion was virtually the same between group 2 public servants in january to february 2012 ( 12c , 5-min method ) and public servants of the same population who received a cold - water immersion test ( 10c , 10-min method ) in january to february 2010 , while the vpt before immersion was significantly different [ table 1 ] . therefore , we adopted the same cutoff value for the vpt at 10 minutes after immersion in both the groups . ( unfortunately , anthropometry was not conducted among the public servants in 2010 , and so we could not compare anthropometric data for them with those of group 1 workers in 2013 . ) the characteristics of ivtos were compared between group 1 and group 2 . continuous variables ( age , body height , body weight , bmi , years , days and hours exposed to vibrating tools , and tot ) were compared with mann - whitney 's u - test . categorical variables ( prevalence of pn ) were compared with the chi - square test . the odds ratio ( or ) for pn , along with its 95% confidence interval ( ci ) , was calculated as an outcome variable of each explanatory variable using a logistic regression model . in the univariate analysis , we evaluated independent exposure variables such as age , body height , body weight , bmi , years exposed to industrial vibrating tools , days per year exposed to industrial vibrating tools , hours per day exposed to industrial vibrating tools , and tot . the study subjects of each group were divided into quartiles according to each variable . in group 2 , due to the small number of subjects with pn , we merged more than one classes into a single class in some analyses . in the multivariate analysis , we included all variables with p<0.10 in the univariate analysis as well as body weight and bmi in group 1 . in group 2 , due to the small number of study subjects , we included variables with p<0.10 in the univariate analysis as well as variables included in the multivariate model of group 1 . then we performed additional analyses , removing body weight or bmi in a mutually exclusive manner , in order to control potential multicollinearity between bmi and body weight . a value of p<0.05 was considered significant , and a value of 0.05 p<0.10 was considered marginally significant . all analyses were conducted using the sas 9.3 software ( sas institute , inc . , figures indicate the average ( standard deviation ) of vibrotactile perception thresholds unless otherwise specified . the unpaired t - test analysis of covariance was conducted between the year 2010 and year 2012 data , controlling for the vibrotactile perception threshold before immersion . table 2 shows the characteristics of the ivtos . there was no significant difference in age , body height , and body weight between groups 1 and 2 . their body statures were also similar to those of the average of japanese male in the fifth decade of life in 2012 ( height , 170.9 cm ; weight , 70.5 kg ) . workers in group 1 had a lower number of years exposed to vibration but a higher number of days and hours exposed to vibrating tools as well as a larger tot . the prevalence of pn was 24.3% ( 79/325 ) in group 1 and 13.2% ( 9/68 ) in group 2 . peripheral neuropathy was defined as a vibrotactile perception threshold 17.5 db at 10 minutes after finishing cold - water immersion ( 10 minutes at 10c for group 1 , and 5 minutes at 12c for group 2 ) . n=307 . n=305 . tables 3 shows the ors for pn in group 1 . in the univariate analysis , higher body weight ( or 0.39 , 95% ci 0.17 - 0.85 in quartile 4 ( q4 ) versus quartile 1 ( q1 ) ; trend p=0.021 ) and bmi ( or 0.37 , 95% ci 0.17 - 0.80 in q4 versus q1 ; trend p=0.009 ) were significantly negatively associated with pn , whereas older age ( or 4.98 , 95% ci 2.18 - 11.3 in q4 versus q1 ; trend p=0.000 ) and higher number of years exposed to vibration ( or 2.78 , 95% ci 1.35 - 5.75 in q4 versus q1 ; trend p=0.008 ) were positively associated with pn . operating days , operating hours , and tot did not show any significant association with pn . in the multivariate analysis , age and bmi remained significant , while the statistically significant association between body weight and operating years and pn disappeared . removal of bmi from the model resulted in a negative association between body weight and pn with marginally statistical significance . removal of body weight from the model instead did not cause bmi to lose its negative association with pn . pn , peripheral neuropathy ; or , odds ratio ; ci , confidence interval ; bmi , body mass index ; tot , total operating time . full model : adjusted for all variables with p<0.10 in the univariate analysis ( age , weight , bmi , and years exposed to vibration ) . likewise , table 4 shows the ors for pn in group 2 . in the univariate analysis , higher body weight ( or 0.10 , 95% ci 0.01 - 0.97 in quartiles 3 and 4 versus q1 ; trend p=0.034 ) and bmi ( or 0.06 , 95% ci 0.006 - 0.53 in quartiles 3 and 4 versus q1 ; trend p=0.007 ) were significantly associated with a decreased risk of pn , just like in group 1 . age did not show no positive association with pn ( or 2.05 , 95% ci 0.41 - 10.2 in q4 versus q1 ; trend p=0.307 ) . higher numbers of years , days and hours exposed to vibrating tools as well as tot were associated with higher ors but were not significantly associated with pn , unlike in group 1 . in the multivariate analysis , the negative ( protective ) association found between bmi and pn remained , whereas the negative association between body weight and pn was no longer significant . furthermore , by removing body weight or bmi in a mutually exclusive manner , bmi still decreased the risk of pn , while the pn - preventing effect of body weight reemerged but did not reach significance . the risk of pn among workers exposed to vibration for 25 years or longer was seven times as high as for those with vibration exposure for less than 15 years , although it was not significant . in this study , both body weight and bmi were associated with a decreased risk of pn . in the multivariate model , on the other hand , age was positively associated with an increased risk of pn , but this was only consistently observed in group 1 . years exposed to vibration showed a risk - increasing effect on pn in the univariate analysis of group 1 only . the negative ( preventive ) association between body weight and bmi and pn is consistent with the hypothesis that higher body heat production per unit of mass accelerates the recovery of hand warmth and vpt . given the greater preventive effects of body weight and bmi in group 2 , these anthropometric factors might be more effective among populations with lower exposure to vibration . our findings showed the association between body weight / bmi and pn in a population of japan . from our findings , people with greater body weight / bmi such as western people will obtain greater benefit than japanese people . however , these findings should not encourage ivtos to gain weight simply to prevent havs - related pn . overweight itself is associated with some chronic diseases , such as hypertension , dyslipidemia , and diabetes . therefore , a combination of interventions such as dietary modifications and physical exercise has been advocated to help control obesity , lose weight , and limit progressive decline in lean tissues with age . by contrast , a recent review shows that up to 30% of obese patients are metabolically healthy ; they have insulin sensitivity similar to healthy lean individuals , lower liver fat content and carotid artery intima / media thicknesses than the majority of metabolically " unhealthy " obese patients . the effects of obesity , metabolism , and lean tissues on vpts should be investigated in the future . the positive association between older age and pn may reflect aging and/or chronic exposure to vibration . this may be due to the greater range of age among group 1 subjects ( 21 - 75 years ) than among group 2 subjects ( 33 - 73 years ) . years exposed to vibration showed statistical significance only in the univariate analysis of group 1 with the greater range of age . total operating time , which indicates occupational vibration exposure more directly than operating years , showed no association with pn , unlike operating years in the univariate analysis of group 1 . if a higher number of years or tot indicates a greater amount of vibration exposure , these factors may not appropriately represent the vibration dose in this study ; various kinds of recall bias may be involved here ( see the first limitation shown below ) . in a study by skov et al . , a j - shaped increase in finger vpt was demonstrated with age ; there was no increase up to age 35 , and a linear increase was seen thereafter . for finger vpt , the data were sparser for the toe threshold , but they suggested a linear increase with both age and height . the researchers concluded that the greater effect of age and height on toe threshold , rather than finger threshold , was consistent with the hypothesis that the length of a nerve increases susceptibility to pn . on the other hand , a study by chuang et al . the average ( standard deviation ) of body height among study subjects in skov 's study was 170.1 ( 9.1 ) cm , which was similar to our population 's body height , while it was 158.7 ( 8.4 ) cm among chuang 's study subjects . we did not measure toe vpts or show a significant effect of body height in our population , neither as a factor representing body size nor as a proxy indicator for peripheral nerve fiber lengths . however , as for body weight and bmi , unlike skov 's study , where these factors did not show consistent associations with vpt , we showed their preventive effects on havs - related pn , which will provide some information for the prevention of pn caused by occupational exposure . firstly , information on workers ' exposure to vibration was collected only by self - report using a self - administered questionnaire in a cross - sectional manner . hours per day and days per year exposed to vibrating tools fluctuated , as workers ' task types changed during their careers , and workers may not correctly remember information about their vibration exposures ( recall bias ) . however , if failure to recall the history of exposure to vibration correctly occurs to all ivtos equally , this will be non - differential misclassification of exposure , resulting in the underestimation of the effect of exposure to vibration on vpt . secondly , as we evaluated vibration dose , we did not take into consideration factors such as different types of tools operated , nor did we have available their acceleration values . we previously reported the dose - response relationship between hand - transmitted vibration and havs in a tropical environment , concluding that lifetime vibration dose and cumulative exposure index , which were indices calculated from both time exposed to vibration and acceleration values of vibrating tools , were more useful than total operating time ( tot ) , which was an index calculated from time exposed to vibration only . the current study was based on annual regular health examinations for havs , in which acceleration values of vibrating tools were not measured . to evaluate exposure to vibration more accurately thirdly , diabetes was evaluated based only on self - reporting by workers , and more objective measures such as fasting plasma glucose or glycosylated hemoglobin ( hba1c ) were not available . further studies utilizing blood samples in evaluation of the effect of diabetes on pn are desirable . fourthly , workers ' body compositions ( e.g. , lean body weight ) were not considered in this investigation . besides , due to the nature of cross - sectional studies , longitudinal changes in anthropometric factors these factors should also be studied in the future , possibly in a cohort study . some advantages in this study should also be mentioned . firstly , as far as we know , this study is the first to show the protective effects of higher body weight and bmi against pn among ivtos . secondly , this study was based on a population representing one of the largest active study fields of havs in japan . findings from our study may help decrease havs by considering anthropometric factors of workers and identifying workers at high risk of havs . among ivtos , factors reflecting body heat production , such as body weight and bmi , were associated with a decreased risk of pn defined by the vpt , regardless of the task performed . ( a part of this study was presented at the 86th annual meeting of the japan society for occupational health on may 14 - 17 , 2013 , the 87th annual meeting of the japan society for occupational health on may 21 - 24 , 2014 , and the 22nd japan conference on human response to vibration ( jchrv2014 ) on august 25 - 27 , 2014 . )

objectives : the effect of anthropometric factors on the fingertip vibrotactile perception threshold ( vpt ) of industrial vibrating tool operators ( ivtos ) is not well known . the purpose of this study was to investigate the associations between anthropometric factors and fingertip vpt . methods : we included for analysis two groups of ivtos : group 1 , predominantly forestry workers ( n=325 ) ; and group 2 , public servants ( n=68 ) . these ivtos regularly received medical examinations to evaluate hand - arm vibration syndrome . in the examination , measurements of their fingertip vpts were taken before and after cold - water immersion ( 10 minutes at 10c for group 1 and 5 minutes at 12c for group 2 ) . their body height and weight were measured to calculate the body mass index ( bmi ) . the presence of peripheral neuropathy ( pn ) was defined as a vpt 17.5 db at 10 minutes after finishing immersion . results : in the univariate analysis , weight and bmi were associated with a decreased risk of pn in both groups 1 and 2 . the negative association between bmi and pn remained in the multivariate analysis consistently , but weight reached marginal significance only in the multivariate analysis without bmi in both the groups . age was positively associated with pn consistently in group 1 but not in group 2 . years exposed to vibration showed positive association with pn only in the univariate analysis of group 1 . conclusions : among ivtos , factors reflecting body heat production , such as weight and bmi , were associated with a decreased risk of vpt - defined pn , regardless of the task engaged .

Introduction Subjects and Methods Study subjects Procedure Statistical analysis Results Discussion Conclusion

prolonged exposure to hand - arm vibration is a cause of hand - arm vibration syndrome ( havs ) . in cases of havs , the severity of peripheral neuropathy ( pn ) is evaluated by measuring vibrotactile perception thresholds ( vpts ) at the fingertips . however , to evaluate havs neuropathy with vpts , it is necessary first to rule out conditions such as ( 1 ) traumas ( burning , frostbite , and other causes ) , ( 2 ) raynaud 's phenomenon of nonvibratory causes , ( 3 ) thoracic outlet syndrome , ( 4 ) neuropathy / angiopathy caused by poisoning and related agents , ( 5 ) angiopathy caused by pulseless disease , diabetes , buerger 's disease and related disorders , ( 6 ) collagen vascular diseases ( e.g. some recent studies have shown associations between selected anthropometric factors and pn . using vibrometry data gathered from workers in the united states who were exposed to organic solvents , older age and taller body were positively associated with higher vpt . specifically , the foot 's vpt was positively associated with body height , but the hand 's vpt was not . did not find a significantly positive association between body height and vpt in a population exposed to lead . this negative finding was probably because the study subjects were shorter , on average , than those in the study by skov et al .. as for body weight , the associations of body weight and body mass index ( bmi ) with vpt were either negative or not evaluated . in other words , larger body size may be associated with an increased risk of pn . on the other hand , the surface - area - to - volume ratio ( sa : v ) of the human body is lower in those with a larger body volume ( the square - cube law ) . in other words , larger body size may be associated with a decreased risk of pn . thus , although some papers have reported the association between anthropometric factors and havs , as well as evidence to support the association between these factors and somatic disorders related to havs , only a limited number of studies have been conducted to evaluate the associations between anthropometric factors and vpt . in japan , health examinations are provided for industrial vibrating tool operators ( ivtos ) to evaluate havs , but to the best of our knowledge , the associations between anthropometric factors and vpt among ivtos remain unclear . if anthropometric factors are proven to be associated with havs - related pn , such information may be useful for preventing it , for example , by excluding high - risk populations from this type of physically hard work , or for advising ivtos to gain or lose weight to a certain level that would minimize the risk of havs - related pn . for this analysis , we recruited study participants from two groups of workers : group 1 , workers predominantly engaged in forestry , who were exposed to hand - arm vibration through hand - held vibrating tools , mainly chain saws ( n=336 , all males ) ; and group 2 , public servants engaged in road or farm maintenance , who were exposed to hand - arm vibration through handheld vibrating tools , mainly bush clearers ( n=92 , 91 males and 1 female ) . we included both groups 1 and 2 in the study to evaluate whether anthropometric factors affect pn regardless of the task performed . medical examinations that included the assessment of vpts were performed from november to december 2013 for group 1 and from january to february 2012 for group 2 . a total of 331 workers in group 1 ( 98.5% ) and 92 workers in group 2 ( 100% ) gave written consent to have their data from the medical examinations included in this study . total operating time ( tot ) ( hours ) was defined as daily operating time ( hours / day ) number of days exposed to vibration ( days / year)years exposed to vibration ( years ) . after acclimatization to the room temperature for at least 30 minutes , vpts at 125 hz were measured in four fingertips ( except thumbs ) of both hands in group 1 and in all five fingertips ( thumbs included ) of both hands in group 2 . vpts were measured in a sitting position with a vibrometer [ au-02 , rion co. , ltd . , if any , in cold water to the wrist for a designated time period ( 10 minutes at 10c for group 1 and 5 minutes at 12c for group 2 ) . body mass index ( bmi ) ( kg / m ) was calculated as body weight in kilograms divided by the square of the height in meters . after finishing the cold - water immersion test , the subjects received physical and neurological examinations from a physician . after excluding ( a ) female workers ( n=1 in group 2 ) , ( b ) workers who reported a history of diabetes ( n=1 in group 1 , and n=5 in group 2 ) , ( c ) workers who did not participate in the cold - water immersion test ( n=2 in group 1 ) , and ( d ) workers with some missing data for age , body height , body weight , bmi , or years exposed to vibrating tools ( n=3 in group 1 , and n=18 in group 2 ) , male workers with complete data ( n=325 in group 1 and n=68 in group 2 ) were used for the statistical analyses . for both group 1 ( 10c , 10-min method ) and group 2 ( 12c , 5-min method ) , pn was defined as a vpt 17.5 db at 10 minutes after finishing the cold - water immersion . we reviewed public servants ' medical records and found that the vpt at 10 minutes after finishing immersion was virtually the same between group 2 public servants in january to february 2012 ( 12c , 5-min method ) and public servants of the same population who received a cold - water immersion test ( 10c , 10-min method ) in january to february 2010 , while the vpt before immersion was significantly different [ table 1 ] . therefore , we adopted the same cutoff value for the vpt at 10 minutes after immersion in both the groups . ( unfortunately , anthropometry was not conducted among the public servants in 2010 , and so we could not compare anthropometric data for them with those of group 1 workers in 2013 . ) the characteristics of ivtos were compared between group 1 and group 2 . continuous variables ( age , body height , body weight , bmi , years , days and hours exposed to vibrating tools , and tot ) were compared with mann - whitney 's u - test . in the univariate analysis , we evaluated independent exposure variables such as age , body height , body weight , bmi , years exposed to industrial vibrating tools , days per year exposed to industrial vibrating tools , hours per day exposed to industrial vibrating tools , and tot . in group 2 , due to the small number of subjects with pn , we merged more than one classes into a single class in some analyses . in the multivariate analysis , we included all variables with p<0.10 in the univariate analysis as well as body weight and bmi in group 1 . in group 2 , due to the small number of study subjects , we included variables with p<0.10 in the univariate analysis as well as variables included in the multivariate model of group 1 . the unpaired t - test analysis of covariance was conducted between the year 2010 and year 2012 data , controlling for the vibrotactile perception threshold before immersion . for this analysis , we recruited study participants from two groups of workers : group 1 , workers predominantly engaged in forestry , who were exposed to hand - arm vibration through hand - held vibrating tools , mainly chain saws ( n=336 , all males ) ; and group 2 , public servants engaged in road or farm maintenance , who were exposed to hand - arm vibration through handheld vibrating tools , mainly bush clearers ( n=92 , 91 males and 1 female ) . we included both groups 1 and 2 in the study to evaluate whether anthropometric factors affect pn regardless of the task performed . medical examinations that included the assessment of vpts were performed from november to december 2013 for group 1 and from january to february 2012 for group 2 . a total of 331 workers in group 1 ( 98.5% ) and 92 workers in group 2 ( 100% ) gave written consent to have their data from the medical examinations included in this study . total operating time ( tot ) ( hours ) was defined as daily operating time ( hours / day ) number of days exposed to vibration ( days / year)years exposed to vibration ( years ) . after acclimatization to the room temperature for at least 30 minutes , vpts at 125 hz were measured in four fingertips ( except thumbs ) of both hands in group 1 and in all five fingertips ( thumbs included ) of both hands in group 2 . , if any , in cold water to the wrist for a designated time period ( 10 minutes at 10c for group 1 and 5 minutes at 12c for group 2 ) . body mass index ( bmi ) ( kg / m ) was calculated as body weight in kilograms divided by the square of the height in meters . after finishing the cold - water immersion test , the subjects received physical and neurological examinations from a physician . after excluding ( a ) female workers ( n=1 in group 2 ) , ( b ) workers who reported a history of diabetes ( n=1 in group 1 , and n=5 in group 2 ) , ( c ) workers who did not participate in the cold - water immersion test ( n=2 in group 1 ) , and ( d ) workers with some missing data for age , body height , body weight , bmi , or years exposed to vibrating tools ( n=3 in group 1 , and n=18 in group 2 ) , male workers with complete data ( n=325 in group 1 and n=68 in group 2 ) were used for the statistical analyses . for both group 1 ( 10c , 10-min method ) and group 2 ( 12c , 5-min method ) , pn was defined as a vpt 17.5 db at 10 minutes after finishing the cold - water immersion . we reviewed public servants ' medical records and found that the vpt at 10 minutes after finishing immersion was virtually the same between group 2 public servants in january to february 2012 ( 12c , 5-min method ) and public servants of the same population who received a cold - water immersion test ( 10c , 10-min method ) in january to february 2010 , while the vpt before immersion was significantly different [ table 1 ] . therefore , we adopted the same cutoff value for the vpt at 10 minutes after immersion in both the groups . ( unfortunately , anthropometry was not conducted among the public servants in 2010 , and so we could not compare anthropometric data for them with those of group 1 workers in 2013 . ) the characteristics of ivtos were compared between group 1 and group 2 . continuous variables ( age , body height , body weight , bmi , years , days and hours exposed to vibrating tools , and tot ) were compared with mann - whitney 's u - test . in the univariate analysis , we evaluated independent exposure variables such as age , body height , body weight , bmi , years exposed to industrial vibrating tools , days per year exposed to industrial vibrating tools , hours per day exposed to industrial vibrating tools , and tot . in group 2 , due to the small number of subjects with pn , we merged more than one classes into a single class in some analyses . in the multivariate analysis , we included all variables with p<0.10 in the univariate analysis as well as body weight and bmi in group 1 . in group 2 , due to the small number of study subjects , we included variables with p<0.10 in the univariate analysis as well as variables included in the multivariate model of group 1 . then we performed additional analyses , removing body weight or bmi in a mutually exclusive manner , in order to control potential multicollinearity between bmi and body weight . the unpaired t - test analysis of covariance was conducted between the year 2010 and year 2012 data , controlling for the vibrotactile perception threshold before immersion . there was no significant difference in age , body height , and body weight between groups 1 and 2 . workers in group 1 had a lower number of years exposed to vibration but a higher number of days and hours exposed to vibrating tools as well as a larger tot . the prevalence of pn was 24.3% ( 79/325 ) in group 1 and 13.2% ( 9/68 ) in group 2 . peripheral neuropathy was defined as a vibrotactile perception threshold 17.5 db at 10 minutes after finishing cold - water immersion ( 10 minutes at 10c for group 1 , and 5 minutes at 12c for group 2 ) . tables 3 shows the ors for pn in group 1 . in the univariate analysis , higher body weight ( or 0.39 , 95% ci 0.17 - 0.85 in quartile 4 ( q4 ) versus quartile 1 ( q1 ) ; trend p=0.021 ) and bmi ( or 0.37 , 95% ci 0.17 - 0.80 in q4 versus q1 ; trend p=0.009 ) were significantly negatively associated with pn , whereas older age ( or 4.98 , 95% ci 2.18 - 11.3 in q4 versus q1 ; trend p=0.000 ) and higher number of years exposed to vibration ( or 2.78 , 95% ci 1.35 - 5.75 in q4 versus q1 ; trend p=0.008 ) were positively associated with pn . in the multivariate analysis , age and bmi remained significant , while the statistically significant association between body weight and operating years and pn disappeared . removal of bmi from the model resulted in a negative association between body weight and pn with marginally statistical significance . removal of body weight from the model instead did not cause bmi to lose its negative association with pn . pn , peripheral neuropathy ; or , odds ratio ; ci , confidence interval ; bmi , body mass index ; tot , total operating time . full model : adjusted for all variables with p<0.10 in the univariate analysis ( age , weight , bmi , and years exposed to vibration ) . likewise , table 4 shows the ors for pn in group 2 . in the univariate analysis , higher body weight ( or 0.10 , 95% ci 0.01 - 0.97 in quartiles 3 and 4 versus q1 ; trend p=0.034 ) and bmi ( or 0.06 , 95% ci 0.006 - 0.53 in quartiles 3 and 4 versus q1 ; trend p=0.007 ) were significantly associated with a decreased risk of pn , just like in group 1 . age did not show no positive association with pn ( or 2.05 , 95% ci 0.41 - 10.2 in q4 versus q1 ; trend p=0.307 ) . higher numbers of years , days and hours exposed to vibrating tools as well as tot were associated with higher ors but were not significantly associated with pn , unlike in group 1 . in the multivariate analysis , the negative ( protective ) association found between bmi and pn remained , whereas the negative association between body weight and pn was no longer significant . furthermore , by removing body weight or bmi in a mutually exclusive manner , bmi still decreased the risk of pn , while the pn - preventing effect of body weight reemerged but did not reach significance . the risk of pn among workers exposed to vibration for 25 years or longer was seven times as high as for those with vibration exposure for less than 15 years , although it was not significant . in this study , both body weight and bmi were associated with a decreased risk of pn . in the multivariate model , on the other hand , age was positively associated with an increased risk of pn , but this was only consistently observed in group 1 . years exposed to vibration showed a risk - increasing effect on pn in the univariate analysis of group 1 only . the negative ( preventive ) association between body weight and bmi and pn is consistent with the hypothesis that higher body heat production per unit of mass accelerates the recovery of hand warmth and vpt . given the greater preventive effects of body weight and bmi in group 2 , these anthropometric factors might be more effective among populations with lower exposure to vibration . our findings showed the association between body weight / bmi and pn in a population of japan . overweight itself is associated with some chronic diseases , such as hypertension , dyslipidemia , and diabetes . the positive association between older age and pn may reflect aging and/or chronic exposure to vibration . years exposed to vibration showed statistical significance only in the univariate analysis of group 1 with the greater range of age . total operating time , which indicates occupational vibration exposure more directly than operating years , showed no association with pn , unlike operating years in the univariate analysis of group 1 . the average ( standard deviation ) of body height among study subjects in skov 's study was 170.1 ( 9.1 ) cm , which was similar to our population 's body height , while it was 158.7 ( 8.4 ) cm among chuang 's study subjects . we did not measure toe vpts or show a significant effect of body height in our population , neither as a factor representing body size nor as a proxy indicator for peripheral nerve fiber lengths . however , as for body weight and bmi , unlike skov 's study , where these factors did not show consistent associations with vpt , we showed their preventive effects on havs - related pn , which will provide some information for the prevention of pn caused by occupational exposure . however , if failure to recall the history of exposure to vibration correctly occurs to all ivtos equally , this will be non - differential misclassification of exposure , resulting in the underestimation of the effect of exposure to vibration on vpt . we previously reported the dose - response relationship between hand - transmitted vibration and havs in a tropical environment , concluding that lifetime vibration dose and cumulative exposure index , which were indices calculated from both time exposed to vibration and acceleration values of vibrating tools , were more useful than total operating time ( tot ) , which was an index calculated from time exposed to vibration only . to evaluate exposure to vibration more accurately thirdly , diabetes was evaluated based only on self - reporting by workers , and more objective measures such as fasting plasma glucose or glycosylated hemoglobin ( hba1c ) were not available . firstly , as far as we know , this study is the first to show the protective effects of higher body weight and bmi against pn among ivtos . among ivtos , factors reflecting body heat production , such as body weight and bmi , were associated with a decreased risk of pn defined by the vpt , regardless of the task performed . ( a part of this study was presented at the 86th annual meeting of the japan society for occupational health on may 14 - 17 , 2013 , the 87th annual meeting of the japan society for occupational health on may 21 - 24 , 2014 , and the 22nd japan conference on human response to vibration ( jchrv2014 ) on august 25 - 27 , 2014 . )

oncology practice has dramatically changed in the last ten to fifteen years ( 1 ) . genomic assessment of tumors has become commonplace , and there has been a paradigm shift toward the use of targeted therapies directed against the specific molecular drivers of tumors in individual patients where possible . this personalized approach to cancer treatment has profoundly altered patient management , in terms of the available treatment armamentarium , logical sequencing of agents , and optimized use of the targeted therapies to maximize survival and limit toxicities . at many timepoints along the care continuum , imaging interpretation plays a critical role , especially in terms of assessment of patients ' response to molecular targeted therapy ( mtt ) or lack thereof , and in detection of disease and treatment - related complications . as oncology care becomes increasingly diverse and complex , it is a challenge to remain up - to - date on the state of the art and standards within the field . for example , there are now seven vascular endothelial growth factor ( vegf)-targeted agents approved for use in advanced or metastatic renal cell carcinoma ( rcc ) by the united states food and drug administration ( fda ) , two of which were approved in the last six months . innovations in genomic characterization of cancer , targeted drug discoveries and new approvals occur continuously , and it is difficult to keep abreast of changes directly impacting cancer patients treated in both academic and community practices . to fully integrate into the multidisciplinary team and provide best care for patients , radiologists must communicate with oncologists using a common language . therefore , we must be familiar with routinely employed mtts and the range of imaging findings associated with their use . the radiologist may be the first provider to suggest an atypical response pattern or a subclinical toxicity , which can significantly alter study interpretation and contribute to patient management . the purpose of this review is to provide a summary of the commonly used mtts relevant to imaging interpretation , including the rationale of the various anti - angiogenic and non - anti - angiogenic treatments , indications for use , response assessment methods and characteristic toxicities . immune checkpoint blocker agents will not be addressed in this report since these represent a separate class of targeted immune therapies covered separately in this special issue . folkman ( 2 ) described the link between solid tumor growth and neovascularization in 1971 , and he proposed the term anti - angiogenesis , referring to the prevention of new vessel sprouts from penetrating into an early tumor implant as a means of cancer treatment . since that time , it has been shown that a number of cancers , including colon , renal and lung , are associated with increased vegf expression , and that vegf expression levels have prognostic significance in many instances ( 3456 ) . a number of anti - angiogenic agents , also known as vegf - targeted agents , have since become fda - approved , and have been shown to prolong progression - free and overall survival in various malignancies . there are multiple subtypes of vegf ( a , b , c , d , and placental growth factor ) and multiple vegf - receptor ( vegfr ) tyrosine kinases ( vegfr 1 , 2 , and 3 ) , and antiangiogenic drugs may target one or more specific circulating vegf subtypes or vegfr . the first angiogenesis inhibitor to be approved by the fda , bevacizumab ( avastin ; genentech inc . , south san francisco , ca , usa ) , is a recombinant , humanized monoclonal antibody to circulating vegf - a . approved indications for use of bevacizumab include , but are not limited to : metastatic colorectal cancer , unresectable or metastatic non - squamous , non - small cell lung cancer , metastatic rcc , recurrrent glioma , recurrent or metastatic cervical cancer , as well as platinum - resistant ovarian or primary peritoneal cancer , often in combination with chemotherapy ( 789101112 ) . , bridgewater , nj and tarrytown , ny , usa ) is an agent which also binds vegf , representing a humanized recombinant fusion protein comprised of portions of the extracellular domains of vegf receptors fused to the fc portion of human immunoglobulin g1 ( 13 ) . ziv - aflibercept is approved for use in metatstatic colorectal cancer in combination with 5-fluorouracil , leucovorin , and irinotecan following treatment with an oxaliplatin - based regimen ( 14 ) . small molecule inhibitors of receptor tyrosine kinases inhibit ( tki ) the vegf - receptor and other transmembrane kinases , and include sunitinib ( sutent ; pfizer , new york , ny , usa ) , sorafenib ( nexavar ; bayer pharmaceuticals corporation , wayne , nj and onyx pharmaceuticals inc . , emeryville , ca , usa ) , pazopanib ( votrient ; glaxosmithkline , middlesex , united kingdom ) and others ( 15 ) . tkis are approved in a variety of malignancies , including metastatic rcc , hepatocellular carcinoma and gastrointestinal stromal tumor , amongst others ( 161718 ) . since anti - angiogenic agents exert their effects via distinct mechanisms of action , it follows that response patterns differ from those seen with traditional cytotoxic therapies ( fig . tumor stabilization has been acknowledged as an endpoint of treatment ( 19 ) . according to the response evaluation criteria in solid tumors ( recist ) , which was developed during the age of cytotoxic chemotherapy , a 30% decrease in the sum long axis diameter of target lesions is required for partial response ( 20 ) . in the era of anti - angiogenic targeted therapy , response rates according to recist are sometimes low , ranging from 1040% in rcc trials , for example ( 917 ) . a rather large number of rcc patients achieve a best response of stable disease on treatment , a heterogeneous categorization including patients who may or may not experience prolonged progression free survival ( 21 ) . several drugs have gained fda - approval for indications on the basis of prolonged progression - free survival or clinical benefit in treated patients . kerbel and folkman ( 22 ) acknowledged the challenges in determining the most reliable end points indicative of therapeutic efficacy in anti - angiogenic treatments . many investigators have examined a variety of alternative response criteria , aimed at optimizing response assessment in drug and cancer - specific treatment settings ( 23 ) . for example , the response assessment in neuro - oncology ( rano ) criteria have been developed to assess high grade gliomas treated with anti - angiogenic agents , and to address limitations of the previously used macdonald criteria , including underestimation of non - enhancing tumor and overestimation of response related to changes in enhancement ( 24 ) . similarly , computed tomography ( ct)-based morphologic criteria have been developed to address morphologic changes in liver metastases which correlate with pathologic changes and overall survival in patients with metastatic colorectal cancer treated with bevacizumab - containing regimens ( fig . changes of cavitation have been incorporated into modified recist ( mrecist ) assessment of non - small - cell lung cancer treated with an anti - angiogenic agent ( 26 ) . in the setting of hepatocellular carcinoma treated with sorafenib , adjustments to recist include measurement of the arterially enhancing components of tumors in mrecist and other changes in irregular tumors using response evaluation criteria in cancer of the liver ( fig . a variety of alternative assessment methods have been tested to better differentiate responders and non - responders to anti - angiogenic treatments , including choi criteria , morphology , attenuation , size and structure criteria , and alternate tumor shrinkage thresholds ( 21293031 ) . anti - angiogenic or vegf - targeted therapies are associated with several treatment - specific complications of which radiologists must be aware , since imaging manifestations of the drug toxicity may occur before a patient develops symptoms . some of the most important , potentially life - threatening adverse events associated with anti - angiogenic therapy are bowel perforation , pneumatosis and/or tumor bowel fistula , which may have subtle or overt imaging appearances ( fig . 4 ) . while these events are infrequent , occurring in 1.7% of 1442 patients treated with bevacizumab according to one report ( 32 ) , prompt detection is important , as pneumatosis and perforation are often managed conservatively via discontinuation of therapy and supportive care . the radiologist may be the first provider to encounter these toxicities , as 70.8% of cancer patients with pneumatosis and/or bowel perforation were asymptomatic in one study ( 33 ) . other potential abdominal complications in anti - angiogenic treatment include colitis , cholecystitis , pancreatitis and hepatic steatosis ( 34 ) . hemorrhage , thrombosis and hypertension are other potential toxicities related to vegf - targeted therapy . hemorrhage is reported in up to 30% of patients with various cancers treated with bevacizumab , severe in fewer than 5% of patients ( 35 ) . while common presentations include epistaxis , gastrointestinal bleeding and hemoptysis , it is particularly relevant for radiologists to be cognizant of potential intratumoral hemorrhage , which can result in increased size of metastases and lead to erroneous interpretation of progression ( pseudoprogression ) . a clue to potential pseudoprogression is development of fluid - fluid levels in metastases , and mri is a helpful problem solving tool to detect hemorrhagic ( t1-hyperintense ) contents in challenging cases . arterial thromboembolic events are increased in patients treated with vegf - targeted therapies , with overall relative risk of 1.46 in bevacizumab treated patients and 3.03 in sunitinib or sorafenib treated patients , compared to controls ( 3637 ) . risk of venous thromboembolic events in this setting is considered controversial , with increased risk in one meta - analysis and no increased risk in another ( 3839 ) . hypertension is another adverse effect and potential biomarker in patients treated with anti - angiogenic agents , since improved response rates and survival parameters have been documented in patients who develop hypertension on treatment ( 40 ) . posterior reversible encephalopathy syndrome is a rare complication of anti - angiogenic therapy ( 41 ) . when reporting imaging studies of patients treated with anti - angiogenic or vegf - targeted therapy , it is important for the radiologist to pay close attention to the study indication ( including disease and specific therapy ) , to use appropriate cancer - specific and/or treatment - specific response criteria when available , to consider the possibility of an atypical response , and to evaluate for known toxicities of the treatment regimen on imaging . reporting of cancer imaging studies can be facilitated by the use of cancer imaging radiology report templates ( 42 ) . for example , in a patient with metastatic colon cancer to liver treated with a bevacizumab - containing regimen , it is relevant to consider response according to ct - based morphologic criteria . atypical response patterns should be considered , including potential increased size but decreased density / greater homogeneity in liver metastases , and/or apparent new lesions which could have been isodense at baseline ( 43 ) . in difficult cases , correlation may be made with tumor markers ( e.g. , carcinoembryonic antigen ) and patient 's performance status through discussion with the referring oncologist . in addition , it is important to scrutinize the imaging study for treatment related complications and toxicities , such as pneumatosis , thromboembolism and hepatic steatosis . as reviewed , anti - angiogenic agents are approved for use in a variety of malignancies . also during the last decade , multiple other mtts have been developed to target molecular pathways other than / in addition to the vegf - axis . many of these are approved for use in cancer - specific treatment settings , with or without associated documentation of a cancer 's driver mutation via fda - approved genetic tests . using lung , renal and breast cancer as representative examples , several commonly used mtts will be discussed , including an overview of their treatment rationale , expected response patterns and imaging manifestations of toxicity . epidermal growth factor receptor ( egfr ) tyrosine kinase is a protein involved in cell proliferation , apoptosis , angiogenesis and invasion , overexpressed in non - small - cell lung cancer ( 44 ) . activating mutations in the egfr - tyrosine kinase domain have been documented in approximately 10% of non - small cell lung cancer patients of north american / western european descent and 2550% of non - small cell lung cancer patients of asian descent ( 45 ) . presence of an activating mutation in egfr has been associated with dramatic response to targeted egfr tki gefitinib ( iressa ; astrazeneca , pharmaceuticals , wilmington , de , usa ) and erlotinib ( tarceva ; genentech , south san francisco , ca , usa ) ( fig . this discovery , reported in 2004 , was a major advance in the lung cancer treatment paradigm ; prior to this , all patients with advanced non - small cell lung cancer were treated with a platinum - based chemotherapy regimen . in the new era , patient 's tumors are prospectively tested for sensitizing mutations amenable to targeted treatments such as egfr tkis . clinical features associated with the sensitizing mutations and responsiveness to egfr tkis include asian descent , female sex and never smoker status . another genomically characterized lung cancer involves anaplastic lymphoma kinase ( alk ) rearrangement , occurring in 27% of non - small - cell lung cancer patients ( 49 ) . in this setting , the echinoderm microtubule - associated protein - like 4 ( emf4 ) gene clinical features associated with alk rearranged lung cancer include young age and never to light smoking history . patients with alk rearrangements are highly sensitive to alk inhibitors such as crizotinib ( xalkori ; pfizer , new york , ny , usa ) and ceritinib ( zykadia ; novartis , east hanover , nj , usa ) ( fig . 6 ) ( 4950 ) . in lung cancer patients with selected driver mutations , for example , response rates to egfr tkis are reportedly 5575% in the first line setting , and median progression free survival is on the order of 9 months ( 515253 ) . similarly , the phase iii overall response rate to crizotinib was 65% and median progression free survival approximately 8 months ( 54 ) . conventional size based response criteria ( such as recist ) are typically employed in these settings , which capture response in many cases , though other methods of assessment including ct tumor volumetry have been proposed ( 55 ) . as many patients demonstrate dramatic tumor shrinkage , the nadir target lesion diameter may be quite small and modest increases in diameter upon acquired resistance ( for example , via acquired t790 m mutation or another mechanism in egfr mutant patients ) may represent recist disease progression ( 23 ) . if targeted therapy is withdrawn , a flare phenomenon can occur , with dramatic increase in tumor burden upon withdrawal of the targeted agent ( fig . many in the field realize the clinical benefit to continuing egfr - targeted therapy beyond recist progression in some patients , either alone or in combination with chemotherapy . alternative criteria are sought to objectively differentiate patients with slow progression amenable to targeted agent continuation from those without treatment benefit ( 5758 ) . tumor volume models to define tumor growth have been investigated in this regard ( 59 ) . the appropriate time to initiate a treatment change will become even more complicated as therapies targeted to resistance mechanisms become available . for example , osimertinib ( tagrisso ; astrazenecae , pharmaceuticals , wilmington , de , usa ) was approved for patients with acquired egfr t790 m mutation - positive metastatic non - small cell lung cancer in november 2015 ( 60 ) . when reporting imaging studies of patients with genomically characterized lung cancer , it is often helpful for the radiologist to know the specific mutation and targeted therapy . furthermore , it may be useful to know the date of the baseline scan , the tumor burden at baseline , as well as have an awareness of the nadir tumor burden , if reached . familiarity with the treatment course and the expected time of resistance development may help to detect subtle changes in tumor size and shape indicative of early resistance . it is also important to assess such patients for radiologic evidence of drug toxicity , including pneumonitis in those treated with egfr tki or alk inhibitors . four patterns of pneumonitis have been associated with egfr tki according to one report , including : 1 ) nonspecific area of groundglass attenuation , 2 ) multifocal airspace consolidation , 3 ) patchy ground - glass attenuation accompanied by interlobar septal thickening , and 4 ) extensive bilateral ground - glass attenuation or airspace consolidation with traction bronchiectasis ( 61 ) . pattern a was the most common in this study , involving 44% of 70 patients with gefitinib - related lung disease , while pattern d was associated with the highest mortality , involving 75% of patients in this category . in phase iii clinical trials of erlotinib and crizotinib , interstitial lung disease / pneumonitis resulted in 5 deaths ( 1% ) and 2 deaths ( ~1% ) in the treatment groups , respectively ( 5462 ) . radiologists should also be aware of other potential imaging manifestions of egfr tki toxicity , including diarrhea / colitis , pneumatosis and periportal edema related to hepatic failure ( 63 ) . diarrhea / colitis is commonly reported , evidenced by fluid filled colon , dilation , wall thickening and/or mesenteric hyperemia ( 63 ) . in contrast , pneumatosis and periportal edema related to hepatic failure are rare events ( 63 ) . it is worthwhile to mention that each of the egfr tkis toxicities can be seen in patients treated with these agents in other cancer settings ( e.g. , erlotinib treatment of pancreatic cancer ; cetuximab ( erbitux ; bristol - myers squibb , new york , ny , usa ) treatment of advanced squamous cell carcinoma of the head and neck ) ( 6465 ) . development of complex renal cysts has been associated with crizotinib therapy , and cysts regress when therapy is discontinued ( 66 ) . rapid onset hypogonadism with low testosterone has also been reported with crizotinib therapy ( fig . a drug - based imaging toxicity checklist or template may assist radiologists in reporting pertinent findings ( 42 ) . in rcc , the vegf - axis is a common therapeutic target due to inactivation of the von hippel lindau gene in the majority of clear cell rccs , leading to accumulation of hypoxia inducible factor and overexpression of vegf . however , the phosphatidylinositol-3-kinase - akt - mammalian target of rapamycin ( mtor ) pathway is another active intracellular molecular signaling pathway in rcc amenable to targeted treatment . mtor is a serine / threonine kinase , and its activation ultimately promotes protein synthesis and cell survival ( 6869 ) . everolimus ( afinitor ; novartis , east hanover , nj , usa ) and temsirolimus ( torisel pfizer , new york , ny , usa ) are inhibitors of mtor approved in metastatic rcc ( 7071 ) . both agents were associated with modest response rates ranging from 19% in phase iii trials , though progression free survival was prolonged by both agents and overall survival prolonged by the latter ( 7071 ) . everolimus is also approved for use in subependymal giant cell astrocytoma , advanced hormone receptor positive human epidermal growth factor receptor 2 ( her2)-negative breast cancer , and pancreatic neuroendocrine tumors . clinical trials of everolimus in waldenstrom 's macroglobulinemia have been reported , with promising results ( 72 ) . given the high prevalence of stable disease according to recist in rcc patients treated with mtor inhibitors , alternative criteria have been sought to better differentiate responders and non - responders . according to an analysis of phase iii study data in everolimus - treated patients , a reduction of greater than 5% in the sum long diameter measurements of target lesions predicted benefit in progression free survival better than partial response ( 30% reduction ) ( 73 ) . however , it is possible that 5% reduction in the sum long diameter measurements of target lesions is within the range of measurement variability , therefore , the clinical utility of this threshold is questionable ( 74 ) . modified choi criteria have been applied to rcc patients treated with everolimus , and modified choi assessments in three dimensions enabled identification of patients with progression free survival benefit from everolimus according to one study ( 75 ) . given the limitations and the complexity of these alternative criteria , response assessment remains largely according to recist in this clinical setting . radiographic evidence of pneumonitis occurs in up to a third of patients , represented by ground - glass opacities and consolidation ( fig . ( 76 ) , 31% of patients with radiographic evidence of pneumonitis were symptomatic ( with cough , shortness of breath and/or oxygen requirement ) , highlighting the important role of the radiologist in detecting early / asymptomatic lung toxicity . pneumonitis has been proposed as a marker of therapeutic benefit , since patients in one report with imaging evidence of pneumonitis more frequently demonstrated stable disease according to recist than those without pneumonitis ( 77 ) . in patients with other malignancies treated with mtor inhibitors , as many as 58% of patients with waldenstrom 's macroglobulinemia demonstrated imaging findings of pneumonitis , while 21% of patients with pancreatic neuroendocrine tumors did , most commonly manifesting as cryptogenic organizing pneumonia and non - specific interstitial pneumonia patterns ( 7980 ) . cholecystitis , pancreatitis and enteritis are less commonly seen mtor toxicities on imaging ( 818283 ) . while molecular targeted therapies are commonly employed in a variety of advanced / metastatic cancers , targeted treatment in breast cancer may be applied in neoadjuvant and adjuvant settings , as well as in advanced / metastatic disease . according to the national comprehensive cancer network ( nccn ) clinical practice guidelines in oncology , adjuvant therapy is recommended in patients with tumors greater than 1 cm or with lymph node involvement ( 84 ) . traditional cytotoxic chemotherapies including taxanes and anthracyclines are commonly used in first line treatment ( 84 ) . in addition to stage , hormone receptor status and her2 status are also important factors influencing treatment , and hormonal and/or targeted agents are part of the armamentarium . these are amenable to treatment with hormonal agents , including selective estrogen receptor modulators ( serms ) , like tamoxifen , and/or aromatase inhibitors ( ais ) , including anastrozole , letrozole , exemestane . serms act by an anti - estrogenic effect in the breast tissue and ais reduce conversion of androgens to estrogens , the latter commonly used in post - menopausal patients . given their relative safety profile , adjuvant hormonal therapy may be continued for 510 years after diagnosis . in metastatic disease , hormonal agents may be used in the first line setting in the absence of rapidly progressing visceral metastases , in light of the preferable side effect profiles of these drugs compared to cytotoxic chemotherapy . hormonal agents may be used alone or in combination with other agents , including mtts ( fig . for example , in february 2015 , the cyclin dependent kinase ( cdk ) inhibitor palbociclib ( ibrance ; pfizer ) was approved in combination with letrozole for initial treatment of advanced hormone receptor positive , her2 negative breast cancer in postmenopausal patients ( 85 ) . palbociclib is a small molecule inhibitor of cdk4 and 6 , which blocks progression of cells from the g1 into s phase of the cell cycle , resulting in growth arrest . in the phase ii study of letrozole plus palbociclib compared to letrozole alone , median progression free survival was doubled in patients treated with combination therapy ( 20.2 months vs. 10.2 months , p = 0.0004 ) and partial response rates were higher among patients with measurable disease treated with combination therapy ( 54% vs. 39% , p = 0.047 ) ( 85 ) . in february 2016 , palbociclib was approved in combination with fulvestrant , a complete estrogen receptor antagonist , for postmenopausal patients with hormone receptor positive , her2 negative advanced breast cancer after progression on endocrine therapy ( 86 ) . the mtor inhibitor everolimus is also approved in combination with exemestane for patients with advanced , hormone receptor positive , her2 negative breast cancer who have progressed on letrozole or anastrozole ( 87 ) . response to the combination of hormonal agents and mtts is typically assessed according to recist criteria in breast cancer . for example , in the case of serm or tamoxifen use , proestrogenic effects are observed in the uterus , including benign entities ( endometrial polyps , hyperplasia , adenomyosis , and fibroids ) and malignant disease ( endometrial carcinoma , and sarcoma ) ( 88 ) . human epidermal growth factor receptor 2 positive breast cancer represents up to 20% of breast cancer cases . positive breast cancers overexpress her2 , also known as erbb2 , a receptor tyrosine kinase involved in cell growth and proliferation . positivity is a biomarker of disease aggressiveness but is also predictive of responsiveness to her2 targeted treatments . , south san francisco , ca , usa ) is a monoclonal antibody targeted to her2 used in adjuvant and metastatic breast cancer treatment of her2 positive tumors , often in combination with other drugs ( 90 ) . toxicities associated with trastuzumab therapy include cardiotoxicity which may be identified on echocardiogram or on multiple - gated acquisition scan . pertuzumab ( perjeta ; genentech inc . ) is another her2 targeted therapy , aimed at inhibiting her2 dimerization . it is approved in combination with trastuzumab and docetaxel in the neoadjuvant and metastatic settings ( 91 ) . other targeted therapies are fda - approved for patients who have progressed on trastuzumab - containing regimens . in 2007 , the dual her2/egfr inhibitor lapatinib ( tykerb ; glaxosmithkline , middlesex , united kingdom ) was approved in her2 overexpressing breast cancer in combination with capecitabine in patients who have progressed on prior therapy containing trastuzumab ( 92 ) . in 2010 , lapatinib was approved in combination with letrozole in hormone receptor positive , her2 positive disease . lapatinib is a small molecule which may penetrate the blood brain barrier , an important feature in her2 positive disease , since one third or more patients with metastatic her2 positive breast cancer develop brain metastases ( 93 ) . in 2013 , the drug - antibody conjugate known as tdm-1 or ado - trastuzumab emtansine ( kadcyla ; genentech inc . ) was approved for patients with her2 positive disease after progression on trastuzumab and a taxane , separately or in combination ( 94 ) . in this therapy , dm-1 is an antimicrotubule agent which is linked to trastuzumab for selective delivery into cancer cells . response assessment is according to recist in the various her2 targeted treatments in breast cancer . epidermal growth factor receptor ( egfr ) tyrosine kinase is a protein involved in cell proliferation , apoptosis , angiogenesis and invasion , overexpressed in non - small - cell lung cancer ( 44 ) . activating mutations in the egfr - tyrosine kinase domain have been documented in approximately 10% of non - small cell lung cancer patients of north american / western european descent and 2550% of non - small cell lung cancer patients of asian descent ( 45 ) . presence of an activating mutation in egfr has been associated with dramatic response to targeted egfr tki gefitinib ( iressa ; astrazeneca , pharmaceuticals , wilmington , de , usa ) and erlotinib ( tarceva ; genentech , south san francisco , ca , usa ) ( fig . this discovery , reported in 2004 , was a major advance in the lung cancer treatment paradigm ; prior to this , all patients with advanced non - small cell lung cancer were treated with a platinum - based chemotherapy regimen . in the new era , patient 's tumors are prospectively tested for sensitizing mutations amenable to targeted treatments such as egfr tkis . clinical features associated with the sensitizing mutations and responsiveness to egfr tkis include asian descent , female sex and never smoker status . another genomically characterized lung cancer involves anaplastic lymphoma kinase ( alk ) rearrangement , occurring in 27% of non - small - cell lung cancer patients ( 49 ) . in this setting , the echinoderm microtubule - associated protein - like 4 ( emf4 ) gene clinical features associated with alk rearranged lung cancer include young age and never to light smoking history . patients with alk rearrangements are highly sensitive to alk inhibitors such as crizotinib ( xalkori ; pfizer , new york , ny , usa ) and ceritinib ( zykadia ; novartis , east hanover , nj , usa ) ( fig . for example , response rates to egfr tkis are reportedly 5575% in the first line setting , and median progression free survival is on the order of 9 months ( 515253 ) . similarly , the phase iii overall response rate to crizotinib was 65% and median progression free survival approximately 8 months ( 54 ) . conventional size based response criteria ( such as recist ) are typically employed in these settings , which capture response in many cases , though other methods of assessment including ct tumor volumetry have been proposed ( 55 ) . as many patients demonstrate dramatic tumor shrinkage , the nadir target lesion diameter may be quite small and modest increases in diameter upon acquired resistance ( for example , via acquired t790 m mutation or another mechanism in egfr mutant patients ) may represent recist disease progression ( 23 ) . if targeted therapy is withdrawn , a flare phenomenon can occur , with dramatic increase in tumor burden upon withdrawal of the targeted agent ( fig . many in the field realize the clinical benefit to continuing egfr - targeted therapy beyond recist progression in some patients , either alone or in combination with chemotherapy . alternative criteria are sought to objectively differentiate patients with slow progression amenable to targeted agent continuation from those without treatment benefit ( 5758 ) . tumor volume models to define tumor growth have been investigated in this regard ( 59 ) . the appropriate time to initiate a treatment change will become even more complicated as therapies targeted to resistance mechanisms become available . for example , osimertinib ( tagrisso ; astrazenecae , pharmaceuticals , wilmington , de , usa ) was approved for patients with acquired egfr t790 m mutation - positive metastatic non - small cell lung cancer in november 2015 ( 60 ) . when reporting imaging studies of patients with genomically characterized lung cancer , it is often helpful for the radiologist to know the specific mutation and targeted therapy . furthermore , it may be useful to know the date of the baseline scan , the tumor burden at baseline , as well as have an awareness of the nadir tumor burden , if reached . familiarity with the treatment course and the expected time of resistance development may help to detect subtle changes in tumor size and shape indicative of early resistance . it is also important to assess such patients for radiologic evidence of drug toxicity , including pneumonitis in those treated with egfr tki or alk inhibitors . four patterns of pneumonitis have been associated with egfr tki according to one report , including : 1 ) nonspecific area of groundglass attenuation , 2 ) multifocal airspace consolidation , 3 ) patchy ground - glass attenuation accompanied by interlobar septal thickening , and 4 ) extensive bilateral ground - glass attenuation or airspace consolidation with traction bronchiectasis ( 61 ) . pattern a was the most common in this study , involving 44% of 70 patients with gefitinib - related lung disease , while pattern d was associated with the highest mortality , involving 75% of patients in this category . in phase iii clinical trials of erlotinib and crizotinib , interstitial lung disease / pneumonitis resulted in 5 deaths ( 1% ) and 2 deaths ( ~1% ) in the treatment groups , respectively ( 5462 ) . radiologists should also be aware of other potential imaging manifestions of egfr tki toxicity , including diarrhea / colitis , pneumatosis and periportal edema related to hepatic failure ( 63 ) . diarrhea / colitis is commonly reported , evidenced by fluid filled colon , dilation , wall thickening and/or mesenteric hyperemia ( 63 ) . in contrast , pneumatosis and periportal edema related to hepatic failure are rare events ( 63 ) . it is worthwhile to mention that each of the egfr tkis toxicities can be seen in patients treated with these agents in other cancer settings ( e.g. , erlotinib treatment of pancreatic cancer ; cetuximab ( erbitux ; bristol - myers squibb , new york , ny , usa ) treatment of advanced squamous cell carcinoma of the head and neck ) ( 6465 ) . development of complex renal cysts has been associated with crizotinib therapy , and cysts regress when therapy is discontinued ( 66 ) . rapid onset hypogonadism with low testosterone has also been reported with crizotinib therapy ( fig . a drug - based imaging toxicity checklist or template may assist radiologists in reporting pertinent findings ( 42 ) . in rcc , the vegf - axis is a common therapeutic target due to inactivation of the von hippel lindau gene in the majority of clear cell rccs , leading to accumulation of hypoxia inducible factor and overexpression of vegf . however , the phosphatidylinositol-3-kinase - akt - mammalian target of rapamycin ( mtor ) pathway is another active intracellular molecular signaling pathway in rcc amenable to targeted treatment . mtor is a serine / threonine kinase , and its activation ultimately promotes protein synthesis and cell survival ( 6869 ) . everolimus ( afinitor ; novartis , east hanover , nj , usa ) and temsirolimus ( torisel pfizer , new york , ny , usa ) are inhibitors of mtor approved in metastatic rcc ( 7071 ) . both agents were associated with modest response rates ranging from 19% in phase iii trials , though progression free survival was prolonged by both agents and overall survival prolonged by the latter ( 7071 ) . everolimus is also approved for use in subependymal giant cell astrocytoma , advanced hormone receptor positive human epidermal growth factor receptor 2 ( her2)-negative breast cancer , and pancreatic neuroendocrine tumors . clinical trials of everolimus in waldenstrom 's macroglobulinemia have been reported , with promising results ( 72 ) . given the high prevalence of stable disease according to recist in rcc patients treated with mtor inhibitors , alternative criteria have been sought to better differentiate responders and non - responders . according to an analysis of phase iii study data in everolimus - treated patients , a reduction of greater than 5% in the sum long diameter measurements of target lesions predicted benefit in progression free survival better than partial response ( 30% reduction ) ( 73 ) . however , it is possible that 5% reduction in the sum long diameter measurements of target lesions is within the range of measurement variability , therefore , the clinical utility of this threshold is questionable ( 74 ) . modified choi criteria have been applied to rcc patients treated with everolimus , and modified choi assessments in three dimensions enabled identification of patients with progression free survival benefit from everolimus according to one study ( 75 ) . given the limitations and the complexity of these alternative criteria , response assessment remains largely according to recist in this clinical setting . radiographic evidence of pneumonitis occurs in up to a third of patients , represented by ground - glass opacities and consolidation ( fig . ( 76 ) , 31% of patients with radiographic evidence of pneumonitis were symptomatic ( with cough , shortness of breath and/or oxygen requirement ) , highlighting the important role of the radiologist in detecting early / asymptomatic lung toxicity . pneumonitis has been proposed as a marker of therapeutic benefit , since patients in one report with imaging evidence of pneumonitis more frequently demonstrated stable disease according to recist than those without pneumonitis ( 77 ) . in patients with other malignancies treated with mtor inhibitors , as many as 58% of patients with waldenstrom 's macroglobulinemia demonstrated imaging findings of pneumonitis , while 21% of patients with pancreatic neuroendocrine tumors did , most commonly manifesting as cryptogenic organizing pneumonia and non - specific interstitial pneumonia patterns ( 7980 ) . cholecystitis , pancreatitis and enteritis are less commonly seen mtor toxicities on imaging ( 818283 ) . while molecular targeted therapies are commonly employed in a variety of advanced / metastatic cancers , targeted treatment in breast cancer may be applied in neoadjuvant and adjuvant settings , as well as in advanced / metastatic disease . according to the national comprehensive cancer network ( nccn ) clinical practice guidelines in oncology , adjuvant therapy is recommended in patients with tumors greater than 1 cm or with lymph node involvement ( 84 ) . traditional cytotoxic chemotherapies including taxanes and anthracyclines are commonly used in first line treatment ( 84 ) . in addition to stage , hormone receptor status and her2 status are also important factors influencing treatment , and hormonal and/or targeted agents are part of the armamentarium . these are amenable to treatment with hormonal agents , including selective estrogen receptor modulators ( serms ) , like tamoxifen , and/or aromatase inhibitors ( ais ) , including anastrozole , letrozole , exemestane . serms act by an anti - estrogenic effect in the breast tissue and ais reduce conversion of androgens to estrogens , the latter commonly used in post - menopausal patients . given their relative safety profile , adjuvant hormonal therapy may be continued for 510 years after diagnosis . in metastatic disease , hormonal agents may be used in the first line setting in the absence of rapidly progressing visceral metastases , in light of the preferable side effect profiles of these drugs compared to cytotoxic chemotherapy . hormonal agents may be used alone or in combination with other agents , including mtts ( fig . for example , in february 2015 , the cyclin dependent kinase ( cdk ) inhibitor palbociclib ( ibrance ; pfizer ) was approved in combination with letrozole for initial treatment of advanced hormone receptor positive , her2 negative breast cancer in postmenopausal patients ( 85 ) . palbociclib is a small molecule inhibitor of cdk4 and 6 , which blocks progression of cells from the g1 into s phase of the cell cycle , resulting in growth arrest . in the phase ii study of letrozole plus palbociclib compared to letrozole alone , median progression free survival was doubled in patients treated with combination therapy ( 20.2 months vs. 10.2 months , p = 0.0004 ) and partial response rates were higher among patients with measurable disease treated with combination therapy ( 54% vs. 39% , p = 0.047 ) ( 85 ) . in february 2016 , palbociclib was approved in combination with fulvestrant , a complete estrogen receptor antagonist , for postmenopausal patients with hormone receptor positive , her2 negative advanced breast cancer after progression on endocrine therapy ( 86 ) . the mtor inhibitor everolimus is also approved in combination with exemestane for patients with advanced , hormone receptor positive , her2 negative breast cancer who have progressed on letrozole or anastrozole ( 87 ) . response to the combination of hormonal agents and mtts is typically assessed according to recist criteria in breast cancer . for example , in the case of serm or tamoxifen use , proestrogenic effects are observed in the uterus , including benign entities ( endometrial polyps , hyperplasia , adenomyosis , and fibroids ) and malignant disease ( endometrial carcinoma , and sarcoma ) ( 88 ) . . human epidermal growth factor receptor 2 positive breast cancer represents up to 20% of breast cancer cases . her2 positive breast cancers overexpress her2 , also known as erbb2 , a receptor tyrosine kinase involved in cell growth and proliferation . positivity is a biomarker of disease aggressiveness but is also predictive of responsiveness to her2 targeted treatments . , south san francisco , ca , usa ) is a monoclonal antibody targeted to her2 used in adjuvant and metastatic breast cancer treatment of her2 positive tumors , often in combination with other drugs ( 90 ) . toxicities associated with trastuzumab therapy include cardiotoxicity which may be identified on echocardiogram or on multiple - gated acquisition scan . pertuzumab ( perjeta ; genentech inc . ) is another her2 targeted therapy , aimed at inhibiting her2 dimerization . it is approved in combination with trastuzumab and docetaxel in the neoadjuvant and metastatic settings ( 91 ) . other targeted therapies are fda - approved for patients who have progressed on trastuzumab - containing regimens . in 2007 , the dual her2/egfr inhibitor lapatinib ( tykerb ; glaxosmithkline , middlesex , united kingdom ) was approved in her2 overexpressing breast cancer in combination with capecitabine in patients who have progressed on prior therapy containing trastuzumab ( 92 ) . in 2010 , lapatinib was approved in combination with letrozole in hormone receptor positive , her2 positive disease . lapatinib is a small molecule which may penetrate the blood brain barrier , an important feature in her2 positive disease , since one third or more patients with metastatic her2 positive breast cancer develop brain metastases ( 93 ) . in 2013 , the drug - antibody conjugate known as tdm-1 or ado - trastuzumab emtansine ( kadcyla ; genentech inc . ) was approved for patients with her2 positive disease after progression on trastuzumab and a taxane , separately or in combination ( 94 ) . in this therapy , dm-1 is an antimicrotubule agent which is linked to trastuzumab for selective delivery into cancer cells . response assessment is according to recist in the various her2 targeted treatments in breast cancer . interpretation of imaging studies in oncology patients has become progressively more complex in the context of evolving genomic cancer characterization and increasing number and types of therapies available to patients . radiologists should be familiar with anti - angiogenic treatments and the wide variety of non - anti - angiogenic molecular targeted therapies associated with typical and atypical patterns of tumor response , resistance , growth and toxicity . it is a formidable task to keep abreast of developments in oncology , but it is necessary to provide optimal diagnoses , render appropriate response assessments , and identify important drug toxicities which may impact patient management .

oncology is a rapidly evolving field with a shift toward personalized cancer treatment . the use of therapies targeted to the molecular features of individual tumors and the tumor microenvironment has become much more common . in this review , anti - angiogenic and other molecular targeted therapies are discussed , with a focus on typical and atypical response patterns and imaging manifestations of drug toxicities .

INTRODUCTION Anti-Angiogenic (VEGF-Targeted) Therapies Non-Anti-Angiogenic Molecular Targeted Therapies EGFR-Mutant and EMF4-ALK-Rearranged Non-Small Cell Lung Cancer Renal Cell Carcinoma Hormone Receptor Positive and HER2 Positive Breast Cancer CONCLUSION

genomic assessment of tumors has become commonplace , and there has been a paradigm shift toward the use of targeted therapies directed against the specific molecular drivers of tumors in individual patients where possible . this personalized approach to cancer treatment has profoundly altered patient management , in terms of the available treatment armamentarium , logical sequencing of agents , and optimized use of the targeted therapies to maximize survival and limit toxicities . at many timepoints along the care continuum , imaging interpretation plays a critical role , especially in terms of assessment of patients ' response to molecular targeted therapy ( mtt ) or lack thereof , and in detection of disease and treatment - related complications . as oncology care becomes increasingly diverse and complex , it is a challenge to remain up - to - date on the state of the art and standards within the field . to fully integrate into the multidisciplinary team and provide best care for patients , radiologists must communicate with oncologists using a common language . therefore , we must be familiar with routinely employed mtts and the range of imaging findings associated with their use . the radiologist may be the first provider to suggest an atypical response pattern or a subclinical toxicity , which can significantly alter study interpretation and contribute to patient management . the purpose of this review is to provide a summary of the commonly used mtts relevant to imaging interpretation , including the rationale of the various anti - angiogenic and non - anti - angiogenic treatments , indications for use , response assessment methods and characteristic toxicities . immune checkpoint blocker agents will not be addressed in this report since these represent a separate class of targeted immune therapies covered separately in this special issue . folkman ( 2 ) described the link between solid tumor growth and neovascularization in 1971 , and he proposed the term anti - angiogenesis , referring to the prevention of new vessel sprouts from penetrating into an early tumor implant as a means of cancer treatment . a number of anti - angiogenic agents , also known as vegf - targeted agents , have since become fda - approved , and have been shown to prolong progression - free and overall survival in various malignancies . , south san francisco , ca , usa ) , is a recombinant , humanized monoclonal antibody to circulating vegf - a . approved indications for use of bevacizumab include , but are not limited to : metastatic colorectal cancer , unresectable or metastatic non - squamous , non - small cell lung cancer , metastatic rcc , recurrrent glioma , recurrent or metastatic cervical cancer , as well as platinum - resistant ovarian or primary peritoneal cancer , often in combination with chemotherapy ( 789101112 ) . , bridgewater , nj and tarrytown , ny , usa ) is an agent which also binds vegf , representing a humanized recombinant fusion protein comprised of portions of the extracellular domains of vegf receptors fused to the fc portion of human immunoglobulin g1 ( 13 ) . small molecule inhibitors of receptor tyrosine kinases inhibit ( tki ) the vegf - receptor and other transmembrane kinases , and include sunitinib ( sutent ; pfizer , new york , ny , usa ) , sorafenib ( nexavar ; bayer pharmaceuticals corporation , wayne , nj and onyx pharmaceuticals inc . since anti - angiogenic agents exert their effects via distinct mechanisms of action , it follows that response patterns differ from those seen with traditional cytotoxic therapies ( fig . according to the response evaluation criteria in solid tumors ( recist ) , which was developed during the age of cytotoxic chemotherapy , a 30% decrease in the sum long axis diameter of target lesions is required for partial response ( 20 ) . in the era of anti - angiogenic targeted therapy , response rates according to recist are sometimes low , ranging from 1040% in rcc trials , for example ( 917 ) . kerbel and folkman ( 22 ) acknowledged the challenges in determining the most reliable end points indicative of therapeutic efficacy in anti - angiogenic treatments . for example , the response assessment in neuro - oncology ( rano ) criteria have been developed to assess high grade gliomas treated with anti - angiogenic agents , and to address limitations of the previously used macdonald criteria , including underestimation of non - enhancing tumor and overestimation of response related to changes in enhancement ( 24 ) . changes of cavitation have been incorporated into modified recist ( mrecist ) assessment of non - small - cell lung cancer treated with an anti - angiogenic agent ( 26 ) . in the setting of hepatocellular carcinoma treated with sorafenib , adjustments to recist include measurement of the arterially enhancing components of tumors in mrecist and other changes in irregular tumors using response evaluation criteria in cancer of the liver ( fig . a variety of alternative assessment methods have been tested to better differentiate responders and non - responders to anti - angiogenic treatments , including choi criteria , morphology , attenuation , size and structure criteria , and alternate tumor shrinkage thresholds ( 21293031 ) . anti - angiogenic or vegf - targeted therapies are associated with several treatment - specific complications of which radiologists must be aware , since imaging manifestations of the drug toxicity may occur before a patient develops symptoms . some of the most important , potentially life - threatening adverse events associated with anti - angiogenic therapy are bowel perforation , pneumatosis and/or tumor bowel fistula , which may have subtle or overt imaging appearances ( fig . other potential abdominal complications in anti - angiogenic treatment include colitis , cholecystitis , pancreatitis and hepatic steatosis ( 34 ) . a clue to potential pseudoprogression is development of fluid - fluid levels in metastases , and mri is a helpful problem solving tool to detect hemorrhagic ( t1-hyperintense ) contents in challenging cases . arterial thromboembolic events are increased in patients treated with vegf - targeted therapies , with overall relative risk of 1.46 in bevacizumab treated patients and 3.03 in sunitinib or sorafenib treated patients , compared to controls ( 3637 ) . risk of venous thromboembolic events in this setting is considered controversial , with increased risk in one meta - analysis and no increased risk in another ( 3839 ) . hypertension is another adverse effect and potential biomarker in patients treated with anti - angiogenic agents , since improved response rates and survival parameters have been documented in patients who develop hypertension on treatment ( 40 ) . posterior reversible encephalopathy syndrome is a rare complication of anti - angiogenic therapy ( 41 ) . when reporting imaging studies of patients treated with anti - angiogenic or vegf - targeted therapy , it is important for the radiologist to pay close attention to the study indication ( including disease and specific therapy ) , to use appropriate cancer - specific and/or treatment - specific response criteria when available , to consider the possibility of an atypical response , and to evaluate for known toxicities of the treatment regimen on imaging . reporting of cancer imaging studies can be facilitated by the use of cancer imaging radiology report templates ( 42 ) . for example , in a patient with metastatic colon cancer to liver treated with a bevacizumab - containing regimen , it is relevant to consider response according to ct - based morphologic criteria . atypical response patterns should be considered , including potential increased size but decreased density / greater homogeneity in liver metastases , and/or apparent new lesions which could have been isodense at baseline ( 43 ) . as reviewed , anti - angiogenic agents are approved for use in a variety of malignancies . also during the last decade , multiple other mtts have been developed to target molecular pathways other than / in addition to the vegf - axis . many of these are approved for use in cancer - specific treatment settings , with or without associated documentation of a cancer 's driver mutation via fda - approved genetic tests . using lung , renal and breast cancer as representative examples , several commonly used mtts will be discussed , including an overview of their treatment rationale , expected response patterns and imaging manifestations of toxicity . epidermal growth factor receptor ( egfr ) tyrosine kinase is a protein involved in cell proliferation , apoptosis , angiogenesis and invasion , overexpressed in non - small - cell lung cancer ( 44 ) . this discovery , reported in 2004 , was a major advance in the lung cancer treatment paradigm ; prior to this , all patients with advanced non - small cell lung cancer were treated with a platinum - based chemotherapy regimen . in this setting , the echinoderm microtubule - associated protein - like 4 ( emf4 ) gene clinical features associated with alk rearranged lung cancer include young age and never to light smoking history . if targeted therapy is withdrawn , a flare phenomenon can occur , with dramatic increase in tumor burden upon withdrawal of the targeted agent ( fig . tumor volume models to define tumor growth have been investigated in this regard ( 59 ) . the appropriate time to initiate a treatment change will become even more complicated as therapies targeted to resistance mechanisms become available . furthermore , it may be useful to know the date of the baseline scan , the tumor burden at baseline , as well as have an awareness of the nadir tumor burden , if reached . familiarity with the treatment course and the expected time of resistance development may help to detect subtle changes in tumor size and shape indicative of early resistance . it is also important to assess such patients for radiologic evidence of drug toxicity , including pneumonitis in those treated with egfr tki or alk inhibitors . four patterns of pneumonitis have been associated with egfr tki according to one report , including : 1 ) nonspecific area of groundglass attenuation , 2 ) multifocal airspace consolidation , 3 ) patchy ground - glass attenuation accompanied by interlobar septal thickening , and 4 ) extensive bilateral ground - glass attenuation or airspace consolidation with traction bronchiectasis ( 61 ) . pattern a was the most common in this study , involving 44% of 70 patients with gefitinib - related lung disease , while pattern d was associated with the highest mortality , involving 75% of patients in this category . radiologists should also be aware of other potential imaging manifestions of egfr tki toxicity , including diarrhea / colitis , pneumatosis and periportal edema related to hepatic failure ( 63 ) . development of complex renal cysts has been associated with crizotinib therapy , and cysts regress when therapy is discontinued ( 66 ) . in rcc , the vegf - axis is a common therapeutic target due to inactivation of the von hippel lindau gene in the majority of clear cell rccs , leading to accumulation of hypoxia inducible factor and overexpression of vegf . mtor is a serine / threonine kinase , and its activation ultimately promotes protein synthesis and cell survival ( 6869 ) . clinical trials of everolimus in waldenstrom 's macroglobulinemia have been reported , with promising results ( 72 ) . given the limitations and the complexity of these alternative criteria , response assessment remains largely according to recist in this clinical setting . in patients with other malignancies treated with mtor inhibitors , as many as 58% of patients with waldenstrom 's macroglobulinemia demonstrated imaging findings of pneumonitis , while 21% of patients with pancreatic neuroendocrine tumors did , most commonly manifesting as cryptogenic organizing pneumonia and non - specific interstitial pneumonia patterns ( 7980 ) . cholecystitis , pancreatitis and enteritis are less commonly seen mtor toxicities on imaging ( 818283 ) . while molecular targeted therapies are commonly employed in a variety of advanced / metastatic cancers , targeted treatment in breast cancer may be applied in neoadjuvant and adjuvant settings , as well as in advanced / metastatic disease . according to the national comprehensive cancer network ( nccn ) clinical practice guidelines in oncology , adjuvant therapy is recommended in patients with tumors greater than 1 cm or with lymph node involvement ( 84 ) . serms act by an anti - estrogenic effect in the breast tissue and ais reduce conversion of androgens to estrogens , the latter commonly used in post - menopausal patients . palbociclib is a small molecule inhibitor of cdk4 and 6 , which blocks progression of cells from the g1 into s phase of the cell cycle , resulting in growth arrest . response to the combination of hormonal agents and mtts is typically assessed according to recist criteria in breast cancer . positivity is a biomarker of disease aggressiveness but is also predictive of responsiveness to her2 targeted treatments . , south san francisco , ca , usa ) is a monoclonal antibody targeted to her2 used in adjuvant and metastatic breast cancer treatment of her2 positive tumors , often in combination with other drugs ( 90 ) . other targeted therapies are fda - approved for patients who have progressed on trastuzumab - containing regimens . lapatinib is a small molecule which may penetrate the blood brain barrier , an important feature in her2 positive disease , since one third or more patients with metastatic her2 positive breast cancer develop brain metastases ( 93 ) . in this therapy , dm-1 is an antimicrotubule agent which is linked to trastuzumab for selective delivery into cancer cells . epidermal growth factor receptor ( egfr ) tyrosine kinase is a protein involved in cell proliferation , apoptosis , angiogenesis and invasion , overexpressed in non - small - cell lung cancer ( 44 ) . this discovery , reported in 2004 , was a major advance in the lung cancer treatment paradigm ; prior to this , all patients with advanced non - small cell lung cancer were treated with a platinum - based chemotherapy regimen . another genomically characterized lung cancer involves anaplastic lymphoma kinase ( alk ) rearrangement , occurring in 27% of non - small - cell lung cancer patients ( 49 ) . in this setting , the echinoderm microtubule - associated protein - like 4 ( emf4 ) gene clinical features associated with alk rearranged lung cancer include young age and never to light smoking history . if targeted therapy is withdrawn , a flare phenomenon can occur , with dramatic increase in tumor burden upon withdrawal of the targeted agent ( fig . tumor volume models to define tumor growth have been investigated in this regard ( 59 ) . the appropriate time to initiate a treatment change will become even more complicated as therapies targeted to resistance mechanisms become available . furthermore , it may be useful to know the date of the baseline scan , the tumor burden at baseline , as well as have an awareness of the nadir tumor burden , if reached . familiarity with the treatment course and the expected time of resistance development may help to detect subtle changes in tumor size and shape indicative of early resistance . it is also important to assess such patients for radiologic evidence of drug toxicity , including pneumonitis in those treated with egfr tki or alk inhibitors . pattern a was the most common in this study , involving 44% of 70 patients with gefitinib - related lung disease , while pattern d was associated with the highest mortality , involving 75% of patients in this category . in rcc , the vegf - axis is a common therapeutic target due to inactivation of the von hippel lindau gene in the majority of clear cell rccs , leading to accumulation of hypoxia inducible factor and overexpression of vegf . however , the phosphatidylinositol-3-kinase - akt - mammalian target of rapamycin ( mtor ) pathway is another active intracellular molecular signaling pathway in rcc amenable to targeted treatment . mtor is a serine / threonine kinase , and its activation ultimately promotes protein synthesis and cell survival ( 6869 ) . everolimus ( afinitor ; novartis , east hanover , nj , usa ) and temsirolimus ( torisel pfizer , new york , ny , usa ) are inhibitors of mtor approved in metastatic rcc ( 7071 ) . clinical trials of everolimus in waldenstrom 's macroglobulinemia have been reported , with promising results ( 72 ) . given the high prevalence of stable disease according to recist in rcc patients treated with mtor inhibitors , alternative criteria have been sought to better differentiate responders and non - responders . according to an analysis of phase iii study data in everolimus - treated patients , a reduction of greater than 5% in the sum long diameter measurements of target lesions predicted benefit in progression free survival better than partial response ( 30% reduction ) ( 73 ) . given the limitations and the complexity of these alternative criteria , response assessment remains largely according to recist in this clinical setting . while molecular targeted therapies are commonly employed in a variety of advanced / metastatic cancers , targeted treatment in breast cancer may be applied in neoadjuvant and adjuvant settings , as well as in advanced / metastatic disease . according to the national comprehensive cancer network ( nccn ) clinical practice guidelines in oncology , adjuvant therapy is recommended in patients with tumors greater than 1 cm or with lymph node involvement ( 84 ) . serms act by an anti - estrogenic effect in the breast tissue and ais reduce conversion of androgens to estrogens , the latter commonly used in post - menopausal patients . in metastatic disease , hormonal agents may be used in the first line setting in the absence of rapidly progressing visceral metastases , in light of the preferable side effect profiles of these drugs compared to cytotoxic chemotherapy . palbociclib is a small molecule inhibitor of cdk4 and 6 , which blocks progression of cells from the g1 into s phase of the cell cycle , resulting in growth arrest . response to the combination of hormonal agents and mtts is typically assessed according to recist criteria in breast cancer . positivity is a biomarker of disease aggressiveness but is also predictive of responsiveness to her2 targeted treatments . , south san francisco , ca , usa ) is a monoclonal antibody targeted to her2 used in adjuvant and metastatic breast cancer treatment of her2 positive tumors , often in combination with other drugs ( 90 ) . is another her2 targeted therapy , aimed at inhibiting her2 dimerization . other targeted therapies are fda - approved for patients who have progressed on trastuzumab - containing regimens . lapatinib is a small molecule which may penetrate the blood brain barrier , an important feature in her2 positive disease , since one third or more patients with metastatic her2 positive breast cancer develop brain metastases ( 93 ) . in this therapy , dm-1 is an antimicrotubule agent which is linked to trastuzumab for selective delivery into cancer cells . interpretation of imaging studies in oncology patients has become progressively more complex in the context of evolving genomic cancer characterization and increasing number and types of therapies available to patients . radiologists should be familiar with anti - angiogenic treatments and the wide variety of non - anti - angiogenic molecular targeted therapies associated with typical and atypical patterns of tumor response , resistance , growth and toxicity . it is a formidable task to keep abreast of developments in oncology , but it is necessary to provide optimal diagnoses , render appropriate response assessments , and identify important drug toxicities which may impact patient management .

statistical society of this research consisted of male freshman , junior and senior high school students in city of uremia in 2010 - 2011 school year ( n=10286 ) . one thousand students , approximately 10 percent of this statistical society , were randomly selected for screening traumatic events ( current and previous injuries , hospitalizations , domestic violence , community violence , disasters , accidents , physical abuse ) and psychological symptoms . fifty five students refused to respond to the inventories , 129 of the subjects ( 13/63% ) were exposed to at least one traumatic event based on children traumatic events screening inventory and had a score of higher than 90 in the symptom checklist-90 ( scl-90 ) . of the subjects , 60 were randomly selected . then , clinical interview was conducted , and the selected sample was randomly assigned in to three groups of cognitive processing therapy , holographic reprocessing and control . traumatic events screening inventory- self report form ( tesi - sr ) : tesi is a 24 item scale developed by ford et al . ( 2002 ) for the purpose of probing the history of exposure to traumatic events and distinguishing these events from other negative life experiences in children and adolescents aged 6 18 . the tesi inquires about a variety of traumatic events , including current and previous injuries , hospitalizations , domestic violence , community violence , disasters , accidents , physical , and sexual abuse . the issue of whether the child 's reactions raised to the level of criterion b of ptsd is evaluated in this inventory . it should be noted that in this research , items related to sexual abuse were omitted from the inventory by harasat office located in education department . this coefficiency was obtained to be 0/76 in the initial sample ( n=946 ) of this research . structured clinical interview : the structured clinical interview for dsm - iv ( scid ) is a well - established semi - structured interview for the use of establishing axis i and ii diagnoses which is consistent with the dsm - iv . this interview was used to diagnose disorders related to trauma , and to confirm diagnosis of scl-90 . previous versions of the scid have adequate reliability and validity for clinical and research purposes ( 22 ) . symptom check - list 90-revised ( scl90-r ) : the scl90-r consists of 90 items which measures nine symptom domains and provides a global index of emotional distress and the global severity index . internal consistency of this scale ranges from 0/71 to 0/84 across all nine scales , and its test retest reliability ranges from 0/67 to0/91 ( 23 ) . internal consistency of this scale in iranian sample ranged from 0/77 for psychos to 0/90 for depression ( 24 ) . cronbach 's alpha of this scale in the current sample ranged from 0/69 for phobia to 0/86 for depression . posttraumatic cognitions inventory ( ptci):the ptci is a 36 item self - report measure which assesses trauma related thoughts and beliefs . individuals rated the occurrence of thoughts and beliefs using a seven - point likert scale . the measure loads on three factors of negative cognitions about self , negative cognitions about the world , and self - blame . the ptci discriminated the traumatized individuals with and without ptsd more effectively compared to the world assumptions scale and the personal beliefs and reactions scale which do not include cognitions specific to the sequel of traumatic events ( 25 ) . alpha coefficients of the three subscales were reported to be 0/87 for the negative cognitions about self , 0/88 for the negative cognitions about the world , and 0/86 for self - blame ( 25 ) . in the current study , for the total scale , these coefficients were 0/86 , 0/67 , 0/70 and 0/88 respectively for the three groups . data were collected after recognizing 129 subjects ( 13/63% ) as being exposed to at least one traumatic event based on children traumatic events screening inventory and had a score of higher than 90 in the symptom checklist-90(scl-90 ) . then , the selected sample was randomly assigned in to three equal groups of cognitive processing therapy ( n=20 ) , holographic reprocessing ( n=20 ) and control group ( n=20 ) . at this stage , then , cognitive processing therapy and holographic reprocessing were performed as the therapeutic interventions on the experimental groups . it is important to note that because these therapeutic methods were not used for iranian samples to date , the manuals of these methods were translated to farsi . next , based on the opinion of two psychotherapists about the effectiveness of these therapeutic methods on the posttraumatic symptoms and their correspondence with iranian culture , they were applied in this research . because the control group was attention only control group , therefore , six 30- minute sessions of group conversation about the six following issues were provided : adolescence period ; education ; communication with school staffs ; counseling ; learning and studying methods ; and relationship between girls and boys . these interventions were conducted by one of the researchers of the current study . to examine the effectiveness of therapeutic intervention , differences of pre - post test scores were analyzed using one way anova and scheffe test . the details of each intervention are as follows : cognitive processing therapy ( cpt ) : according to manual developed by resick et al . basic issues of the sessions are as follows : first and second sessions : after rapport was built with subjects , the necessary information about their current problems was provided to them , their symptoms were evaluated , and the structure of therapeutic sessions was determined . existent disorders of the subjects were explained to them based on the cognitive viewpoint and treatment was offered to them . at the end of the second session , subjects were instructed to write about the meaning of their tramatization experience , and how it affected their views of themselves , others and the world . third and fourth sessions : in these sessions , the meaning of traumatic events was established , and written exposure was applied based on what subjects had written at the impact statement . furthermore , abc method was used to address the relationship between subjects ' thoughts and affections . fifth and sixth sessions : subjects were helped to reappraise their beliefs about the experienced traumatic event . cognitive restructuring techniques were introduced to them and they were questioned about their beliefs , using the list of challenging questions . then , the subjects were asked to address their thoughts and beliefs by self monitoring orderly and challenged them by using the challenging questions . seventh to twelfth sessions : subjects ' excessive and over - generalized beliefs about self were recognized , and they were helped to deal with them . furthermore , subjects were helped to recognize , reappraise and review beliefs related to safety , trust , control , self esteem and intimacy . at the end of the twelfth session , the meaning of traumatic event was viewed again , and subjects were asked to write a new report about their traumatic event , and review the previously rewritten report . finally , subjects were asked to think about their achievement and reward themselves , as they have learned to cope with their experience of traumatic event . holographic reprocessing ( hr ) : according to manual developed by katz ( 2005 ) , this method was conducted in three stages ( two step in each stage ) . in sum , therapy was offered in six sequential steps . since the total therapeutic sessions consisted of nine 45 minute sessions , three sessions were allocated to each stage . step 1 ) establishing a therapeutic alliance : according to therapeutic manual , the following techniques were used to establish therapeutic alliance : 1 ) establish a good feeling ; 2 ) educate , normalize and make the client right ; 3 ) address resistance , transference and counter - transference . step 2 ) ensuring a foundation of coping skills : at this step , the following coping skills were taught to the subjects : 1 ) relaxation techniques ( signal breath , cleansing breath , relaxation sandwich , counting exercise , and personal biofeedback machine ) ; 2 ) detecting feelings ( body scan / emotional scan , deciphering messages ) ; 3 ) tolerating the experienced affects ( by mindful observation , deliberate distraction , five ways to express feelings ) ; 4 ) shifting feelings ; 5 ) building emotional resiliency ( maypole of support ) ; 6 ) addressing such symptoms as nightmares , intrusive thoughts and panic ; 7 ) creating a feelings plan step 3 ) engaging in experiential discovery : at this step , the subjects were helped to engage in experiential discovery by using the following techniques : 1 ) connecting with the experience ; 2 ) identifying the feeling ; 3 ) free association with images , memories , or other feelings step 4 ) mapping the experiential hologram : at this step , a simple image of a pot on a stove is offered to help the subjects organize and explain the components of the experiential hologram . in this analogy , each component is matched with an aspect of the image ( the acquired motivation is matched with turning on the stove ; the core violation is matched with the hot burner ; the boiling contents inside the pot correspond to the personal truth including the associated feelings , assumptions , and beliefs that boil to the surface ; the lid on the pot attempts to contain the boiling contents ; the lid represents coping strategies ; keeping the lid on tight to avoid the steam is the avoidance strategy ; and keeping the lid on loosely to release the pressure of the steam is the compensation strategy ; the steam that escapes from the pot corresponds to the residual negative emotions that linger in between cycles of the hologram ) . step 5 ) reprocessing the experiential hologram : the following techniques were used to reprocess experiential hologram : 1 ) nine steps for reprocessing ( contextualize scene , ask for permission , induce relaxation , approach the scene , set the stage , assess for level of distress , rescript the scene , complete and debrief ) ; 2 ) techniques to recontextualize a scene ( hindsight advantage , age comparison ) ; 3 ) techniques to ensure emotional distance(observer vantage point , remaining current age , changing attributes ) ; 4 ) goals for reprocessing ( completing communication , releasing and integrating affect , gaining a new perception , increasing power and safety);5 ) reprocessing techniques ( imagery rescripting , fantasy , role - playing , story board , emotionally connected conversation , guided imagery ) . step 6 ) establishing new patterns : at this step , subjects were briefed on the impact of reprocessing and its application in their future . they were also guided to develop the skills to set new goals , develop new self - concept and countering images . statistical society of this research consisted of male freshman , junior and senior high school students in city of uremia in 2010 - 2011 school year ( n=10286 ) . one thousand students , approximately 10 percent of this statistical society , were randomly selected for screening traumatic events ( current and previous injuries , hospitalizations , domestic violence , community violence , disasters , accidents , physical abuse ) and psychological symptoms . fifty five students refused to respond to the inventories , 129 of the subjects ( 13/63% ) were exposed to at least one traumatic event based on children traumatic events screening inventory and had a score of higher than 90 in the symptom checklist-90 ( scl-90 ) . of the subjects , 60 were randomly selected . then , clinical interview was conducted , and the selected sample was randomly assigned in to three groups of cognitive processing therapy , holographic reprocessing and control . traumatic events screening inventory- self report form ( tesi - sr ) : tesi is a 24 item scale developed by ford et al . ( 2002 ) for the purpose of probing the history of exposure to traumatic events and distinguishing these events from other negative life experiences in children and adolescents aged 6 18 . the tesi inquires about a variety of traumatic events , including current and previous injuries , hospitalizations , domestic violence , community violence , disasters , accidents , physical , and sexual abuse . the issue of whether the child 's reactions raised to the level of criterion b of ptsd is evaluated in this inventory . it should be noted that in this research , items related to sexual abuse were omitted from the inventory by harasat office located in education department . this coefficiency was obtained to be 0/76 in the initial sample ( n=946 ) of this research . structured clinical interview : the structured clinical interview for dsm - iv ( scid ) is a well - established semi - structured interview for the use of establishing axis i and ii diagnoses which is consistent with the dsm - iv . this interview was used to diagnose disorders related to trauma , and to confirm diagnosis of scl-90 . previous versions of the scid have adequate reliability and validity for clinical and research purposes ( 22 ) . symptom check - list 90-revised ( scl90-r ) : the scl90-r consists of 90 items which measures nine symptom domains and provides a global index of emotional distress and the global severity index . the high scores correspond to greater severity of symptoms . internal consistency of this scale ranges from 0/71 to 0/84 across all nine scales , and its test retest reliability ranges from 0/67 to0/91 ( 23 ) . internal consistency of this scale in iranian sample ranged from 0/77 for psychos to 0/90 for depression cronbach 's alpha of this scale in the current sample ranged from 0/69 for phobia to 0/86 for depression . posttraumatic cognitions inventory ( ptci):the ptci is a 36 item self - report measure which assesses trauma related thoughts and beliefs . individuals rated the occurrence of thoughts and beliefs using a seven - point likert scale . the measure loads on three factors of negative cognitions about self , negative cognitions about the world , and self - blame . the ptci discriminated the traumatized individuals with and without ptsd more effectively compared to the world assumptions scale and the personal beliefs and reactions scale which do not include cognitions specific to the sequel of traumatic events ( 25 ) . alpha coefficients of the three subscales were reported to be 0/87 for the negative cognitions about self , 0/88 for the negative cognitions about the world , and 0/86 for self - blame ( 25 ) . in the current study , for the total scale , these coefficients were 0/86 , 0/67 , 0/70 and 0/88 respectively for the three groups . data were collected after recognizing 129 subjects ( 13/63% ) as being exposed to at least one traumatic event based on children traumatic events screening inventory and had a score of higher than 90 in the symptom checklist-90(scl-90 ) . then , the selected sample was randomly assigned in to three equal groups of cognitive processing therapy ( n=20 ) , holographic reprocessing ( n=20 ) and control group ( n=20 ) . at this stage , then , cognitive processing therapy and holographic reprocessing were performed as the therapeutic interventions on the experimental groups . it is important to note that because these therapeutic methods were not used for iranian samples to date , the manuals of these methods were translated to farsi . next , based on the opinion of two psychotherapists about the effectiveness of these therapeutic methods on the posttraumatic symptoms and their correspondence with iranian culture , they were applied in this research . because the control group was attention only control group , therefore , six 30- minute sessions of group conversation about the six following issues were provided : adolescence period ; education ; communication with school staffs ; counseling ; learning and studying methods ; and relationship between girls and boys . these interventions were conducted by one of the researchers of the current study . to examine the effectiveness of therapeutic intervention , differences of pre - post test scores were analyzed using one way anova and scheffe test . the details of each intervention are as follows : cognitive processing therapy ( cpt ) : according to manual developed by resick et al . basic issues of the sessions are as follows : first and second sessions : after rapport was built with subjects , the necessary information about their current problems was provided to them , their symptoms were evaluated , and the structure of therapeutic sessions was determined . existent disorders of the subjects were explained to them based on the cognitive viewpoint and treatment was offered to them . at the end of the second session , subjects were instructed to write about the meaning of their tramatization experience , and how it affected their views of themselves , others and the world . third and fourth sessions : in these sessions , the meaning of traumatic events was established , and written exposure was applied based on what subjects had written at the impact statement . furthermore , abc method was used to address the relationship between subjects ' thoughts and affections . fifth and sixth sessions : subjects were helped to reappraise their beliefs about the experienced traumatic event . cognitive restructuring techniques were introduced to them and they were questioned about their beliefs , using the list of challenging questions . then , the subjects were asked to address their thoughts and beliefs by self monitoring orderly and challenged them by using the challenging questions . seventh to twelfth sessions : subjects ' excessive and over - generalized beliefs about self were recognized , and they were helped to deal with them . furthermore , subjects were helped to recognize , reappraise and review beliefs related to safety , trust , control , self esteem and intimacy . at the end of the twelfth session , the meaning of traumatic event was viewed again , and subjects were asked to write a new report about their traumatic event , and review the previously rewritten report . finally , subjects were asked to think about their achievement and reward themselves , as they have learned to cope with their experience of traumatic event . holographic reprocessing ( hr ) : according to manual developed by katz ( 2005 ) , this method was conducted in three stages ( two step in each stage ) . in sum , therapy was offered in six sequential steps . since the total therapeutic sessions consisted of nine 45 minute sessions step 1 ) establishing a therapeutic alliance : according to therapeutic manual , the following techniques were used to establish therapeutic alliance : 1 ) establish a good feeling ; 2 ) educate , normalize and make the client right ; 3 ) address resistance , transference and counter - transference . step 2 ) ensuring a foundation of coping skills : at this step , the following coping skills were taught to the subjects : 1 ) relaxation techniques ( signal breath , cleansing breath , relaxation sandwich , counting exercise , and personal biofeedback machine ) ; 2 ) detecting feelings ( body scan / emotional scan , deciphering messages ) ; 3 ) tolerating the experienced affects ( by mindful observation , deliberate distraction , five ways to express feelings ) ; 4 ) shifting feelings ; 5 ) building emotional resiliency ( maypole of support ) ; 6 ) addressing such symptoms as nightmares , intrusive thoughts and panic ; 7 ) creating a feelings plan step 3 ) engaging in experiential discovery : at this step , the subjects were helped to engage in experiential discovery by using the following techniques : 1 ) connecting with the experience ; 2 ) identifying the feeling ; 3 ) free association with images , memories , or other feelings step 4 ) mapping the experiential hologram : at this step , a simple image of a pot on a stove is offered to help the subjects organize and explain the components of the experiential hologram . in this analogy , each component is matched with an aspect of the image ( the acquired motivation is matched with turning on the stove ; the core violation is matched with the hot burner ; the boiling contents inside the pot correspond to the personal truth including the associated feelings , assumptions , and beliefs that boil to the surface ; the lid on the pot attempts to contain the boiling contents ; the lid represents coping strategies ; keeping the lid on tight to avoid the steam is the avoidance strategy ; and keeping the lid on loosely to release the pressure of the steam is the compensation strategy ; the steam that escapes from the pot corresponds to the residual negative emotions that linger in between cycles of the hologram ) . step 5 ) reprocessing the experiential hologram : the following techniques were used to reprocess experiential hologram : 1 ) nine steps for reprocessing ( contextualize scene , ask for permission , induce relaxation , approach the scene , set the stage , assess for level of distress , rescript the scene , complete and debrief ) ; 2 ) techniques to recontextualize a scene ( hindsight advantage , age comparison ) ; 3 ) techniques to ensure emotional distance(observer vantage point , remaining current age , changing attributes ) ; 4 ) goals for reprocessing ( completing communication , releasing and integrating affect , gaining a new perception , increasing power and safety);5 ) reprocessing techniques ( imagery rescripting , fantasy , role - playing , story board , emotionally connected conversation , guided imagery ) . step 6 ) establishing new patterns : at this step , subjects were briefed on the impact of reprocessing and its application in their future . they were also guided to develop the skills to set new goals , develop new self - concept and countering images . step 1 ) establishing a therapeutic alliance : according to therapeutic manual , the following techniques were used to establish therapeutic alliance : 1 ) establish a good feeling ; 2 ) educate , normalize and make the client right ; 3 ) address resistance , transference and counter - transference . step 2 ) ensuring a foundation of coping skills : at this step , the following coping skills were taught to the subjects : 1 ) relaxation techniques ( signal breath , cleansing breath , relaxation sandwich , counting exercise , and personal biofeedback machine ) ; 2 ) detecting feelings ( body scan / emotional scan , deciphering messages ) ; 3 ) tolerating the experienced affects ( by mindful observation , deliberate distraction , five ways to express feelings ) ; 4 ) shifting feelings ; 5 ) building emotional resiliency ( maypole of support ) ; 6 ) addressing such symptoms as nightmares , intrusive thoughts and panic ; 7 ) creating a feelings plan step 3 ) engaging in experiential discovery : at this step , the subjects were helped to engage in experiential discovery by using the following techniques : 1 ) connecting with the experience ; 2 ) identifying the feeling ; 3 ) free association with images , memories , or other feelings step 4 ) mapping the experiential hologram : at this step , a simple image of a pot on a stove is offered to help the subjects organize and explain the components of the experiential hologram . in this analogy , each component is matched with an aspect of the image ( the acquired motivation is matched with turning on the stove ; the core violation is matched with the hot burner ; the boiling contents inside the pot correspond to the personal truth including the associated feelings , assumptions , and beliefs that boil to the surface ; the lid on the pot attempts to contain the boiling contents ; the lid represents coping strategies ; keeping the lid on tight to avoid the steam is the avoidance strategy ; and keeping the lid on loosely to release the pressure of the steam is the compensation strategy ; the steam that escapes from the pot corresponds to the residual negative emotions that linger in between cycles of the hologram ) . step 5 ) reprocessing the experiential hologram : the following techniques were used to reprocess experiential hologram : 1 ) nine steps for reprocessing ( contextualize scene , ask for permission , induce relaxation , approach the scene , set the stage , assess for level of distress , rescript the scene , complete and debrief ) ; 2 ) techniques to recontextualize a scene ( hindsight advantage , age comparison ) ; 3 ) techniques to ensure emotional distance(observer vantage point , remaining current age , changing attributes ) ; 4 ) goals for reprocessing ( completing communication , releasing and integrating affect , gaining a new perception , increasing power and safety);5 ) reprocessing techniques ( imagery rescripting , fantasy , role - playing , story board , emotionally connected conversation , guided imagery ) . step 6 ) establishing new patterns : at this step , subjects were briefed on the impact of reprocessing and its application in their future . they were also guided to develop the skills to set new goals , develop new self - concept and countering images . sixty students who had experienced at least one traumatic event , and had a score of higher than 90 in scl-90 r were studied in three groups of cognitive processing therapy , holographic reprocessing and control ; each group consisted of 20 subjects ( n= 20 ) . four subjects in the cognitive processing group , 2 in the holographic reprocessing group , and 5 in the control group did not complete the intervention programs , thus they were excluded from this study . demographic features of participants in the three groups are demonstrated in table 1 . according to this table , the results of 2 test for the variables of educational field and educational level , and the one way anova test for age variable demonstrated no significant difference between the groups in these variables . demographic features of the participants and tests of significant difference among groups table 2 demonstrates the mean and standard deviation of pre- test and post- test scores in the three groups . mean and standard deviations of pre test and post test scores in posttraumatic cognitions inventory table 3 showed the results of anova for comparing the means of differences between pre - test and post - test scores in posttraumatic cognition inventory . as observed in this table , a significant difference exists between the three groups in the subscales of negative cognitions about self ( f=6/70 , p<0/003 ) , self - blame ( f=18/40 , p<0/001 ) and the total scale ( f= 8/66 , p<0/001 ) . anova results to compare three groups furthermore , the results showed that the three groups did not differ significantly from one another with respect to negative cognitions about the world . according to table 4 , the results of scheffe test showed that in the components of negative cognitions about self and total score of this inventory , the two therapeutic groups ( cpt and hr ) did not differ significantly . however , a significant difference was observed between the two groups ( cpt and hr ) and the control group . in the component of self - blame , cognitive processing therapy group significantly differed from hr and control groups , but no significant difference was observed between cognitive processing and control groups . results of scheffe test to compare the means of difference between pre test post test scores in three groups based on f and significant level this research was conducted to examine the effectiveness of cognitive processing therapy and holographic reprocessing on the reduction of posttraumatic cognitions in students exposed to trauma . the results of this study showed no significant difference between cpt and hr groups at the differences of pretest furthermore , these two therapeutic methods were equally effective on the reduction of posttraumatic negative cognitions , particularly negative cognition about self and self blame . these results are consistent with the results of resick and schnicke ( 13 ) , resick et al . ( 18 ) , in relation to the effectiveness of cognitive processing therapy , and are also in accordance with the results of katz et al . ( 20 ) in relation to the effectiveness of holographic reprocessing on the reduction of posttraumatic symptoms . foa and colleagues have emphasized the role of cognitions about self and the world in natural recovery from trauma , the development of posttraumatic disorders , and the cbt processes ameliorating these trauma symptoms ( 25 ) ; they correspond with the following ideas : the effects of these therapeutic methods on the negative cognitions can free the traumatized individual from other trauma symptoms . the effectiveness of these therapeutic methods on the reduction of negative cognitions can be attributed to the congruence of logic of these methods with the features of adolescence period . each of these therapeutic methods was considered an integrative therapy with both the exposure therapy and psycho social education and the cognitive techniques for the coping with dysfunctional cognitions . furthermore , it can be noted that altering negative cognitions in adolescence period , where these cognitions are formed , is easier than other periods of life . similarly , both cpt and hr are trauma- focused cognitive behavioral therapies ; therefore , the effect of these methods on negative cognitions was expected . on the subscales of posttraumatic cognitions inventory , the obtained results showed no significant difference between cognitive processing and holographic reprocessing groups on the negative cognitions about self , but these groups differed significantly from the control group on this variable ; meaning that each of these methods were equally effective on the reduction of negative cognitions about self . this result is consistent with the results of foa and rauch research ( 12 ) on reduction of negative cognitions about self , following prolonged exposure therapy , and with the results of katz et al . ( 20 ) study on the effectiveness of holographic reprocessing on the reduction of negative cognitions . because cognitive processing therapy has targeted some essential components of self such as trust , safety , intimacy , control and respect , and holographic reprocessing is also based on intellectualizing the experiential self , it was expected that these therapeutic methods be effective for the negative cognitions about self more than controlled interventions . as the psychological identity and self are formed in the adolescence period , the experience of trauma can lead to development of maladaptive and irrational beliefs in adolescents . cpt addresses these negative beliefs by cognitive reconstruction and hr by mapping the experientional hologram . therefore , receiving these therapies along with experiencing the safe environment of therapy can help modulate generalized and negative beliefs about self . on the self - blame subscale , the obtained results showed that holographic reprocessing group differed from both cognitive processing and control groups . furthermore , holographic reprocessing therapy was effective for self - blame more than cognitive processing therapy and control intervention . these results also showed that cognitive processing group does not differ significantly from control group in this variable . the results of this study are not consistent with the results of those studies supporting the effectiveness of cognitive processing therapy on the symptoms of ptsd and comorbid disorders ( 13 , 14 , 15 , 16 , 17 , 18 ) , while they are consistent with katz 's opinion ( 19 ) about holographic reprocessing therapy being effective for the reduction of trauma related symptoms more than other therapeutic methods , because it interactively uses the basic techniques of psychodynamic , cognitive- behavioral and experiential therapies . similarly , after the experience of trauma , many people begin to blame their younger version of self for how they behaved at that time . hr can encounter a person with his / her own ego version by mapping experientional hologram ; in this state , the person let go of the painful feeling about the younger version of self , and finally is free of the burden of self blame . moreover , the results of this research showed no significant difference between the three groups in the subscale of negative cognitions about the world , meaning that therapeutic methods were not effective for these cognitions . these results are not consistent with the results of foa and rauch ( 12 ) , resick and schnicke ( 13 ) , resick et al . ( 18 ) in respect to the effectiveness of cognitive processing therapy , and results of katz et al . negative beliefs about the world were targeted in both cpt and hr , but the ineffectiveness of these methods for thecognitions can be due to the occurrence of some traumatic events such as crashing the airplane of tehran - uremia , and assassination of nucleus scientists shortly before conducting this research . in conclusion , the results of this research revealed that cognitive processing therapy and holographic reprocessing , as the trauma related psychotherapies , were equally effective for the reduction of posttraumatic cognitions in trauma - exposed students . thus , it appears that these therapeutic methods which had been originally developed and tested for sexually assaulted females can also be applied for the victims of other traumatic events ( death of a parent , parental divorce , physical abuse etc ) particularly during the adolescence period . therefore , the results of the current study provide important evidence in the support of these two trauma- focused cognitive behavioral therapies for use in clinical setting . firstly , because two months after conducting this research , high school examination time and summer holidays began , it was not possible to follow up the study . secondly , these three therapeutic interventions were applied only by one researcher . thirdly , no data were available on validity and reliability of traumatic events screening inventory , and posttraumatic cognitions inventory for iranians . thus , it is recommended that in the future researches , follow up evaluations be conducted to study the constancy of the therapies and interventions applied by different specialists to increase the validity of these therapies . furthermore , it is suggested that these methods be used for the victims of different traumas in all ages and in both genders .

objectivethis research was conducted to examine the effect of cognitive processing therapy and holographic reprocessing on the reduction of posttraumatic cognitions in students exposed to trauma.methodthis was an experimental study with spread pretest - posttest randomized groups design . statistical society of this research consisted of male freshman , junior and senior high school students of uremia ( n=10286 ) . utilizing traumatic events screening inventory , and scl-90 r on 1000 randomly selected high school students , 129 students were recognized as having experienced traumatic events . of the subjects , 60 were selected randomly . then , clinical interview was conducted , and the selected sample was randomly assigned in to three groups of cognitive processing therapy , holographic reprocessing and control . these groups responded to posttraumatic cognitions inventory in pretest and post test . differences of pre - post test scores were analyzed using one way anova and scheffe test.resultsthe results demonstrated significant differences between the three groups in total score of the posttraumatic cognition inventory . difference was also observed in negative cognitions on self and self - blame dimensions . furthermore , these two therapeutic methods were equally effective in the reduction of posttraumatic cognitions.conclusionit appears that cognitive processing therapy and holographic reprocessing which had been originally developed and tested for sexually assaulted females , can also be applied for the victims of other traumatic events , particularly adolescents .

Materials and Method Participants Measures First stage: safety Second stage: discovery Third stage: reprocessing Results Discussion

statistical society of this research consisted of male freshman , junior and senior high school students in city of uremia in 2010 - 2011 school year ( n=10286 ) . one thousand students , approximately 10 percent of this statistical society , were randomly selected for screening traumatic events ( current and previous injuries , hospitalizations , domestic violence , community violence , disasters , accidents , physical abuse ) and psychological symptoms . fifty five students refused to respond to the inventories , 129 of the subjects ( 13/63% ) were exposed to at least one traumatic event based on children traumatic events screening inventory and had a score of higher than 90 in the symptom checklist-90 ( scl-90 ) . of the subjects , 60 were randomly selected . then , clinical interview was conducted , and the selected sample was randomly assigned in to three groups of cognitive processing therapy , holographic reprocessing and control . the tesi inquires about a variety of traumatic events , including current and previous injuries , hospitalizations , domestic violence , community violence , disasters , accidents , physical , and sexual abuse . this coefficiency was obtained to be 0/76 in the initial sample ( n=946 ) of this research . this interview was used to diagnose disorders related to trauma , and to confirm diagnosis of scl-90 . posttraumatic cognitions inventory ( ptci):the ptci is a 36 item self - report measure which assesses trauma related thoughts and beliefs . the measure loads on three factors of negative cognitions about self , negative cognitions about the world , and self - blame . alpha coefficients of the three subscales were reported to be 0/87 for the negative cognitions about self , 0/88 for the negative cognitions about the world , and 0/86 for self - blame ( 25 ) . in the current study , for the total scale , these coefficients were 0/86 , 0/67 , 0/70 and 0/88 respectively for the three groups . data were collected after recognizing 129 subjects ( 13/63% ) as being exposed to at least one traumatic event based on children traumatic events screening inventory and had a score of higher than 90 in the symptom checklist-90(scl-90 ) . then , the selected sample was randomly assigned in to three equal groups of cognitive processing therapy ( n=20 ) , holographic reprocessing ( n=20 ) and control group ( n=20 ) . at this stage , then , cognitive processing therapy and holographic reprocessing were performed as the therapeutic interventions on the experimental groups . it is important to note that because these therapeutic methods were not used for iranian samples to date , the manuals of these methods were translated to farsi . next , based on the opinion of two psychotherapists about the effectiveness of these therapeutic methods on the posttraumatic symptoms and their correspondence with iranian culture , they were applied in this research . to examine the effectiveness of therapeutic intervention , differences of pre - post test scores were analyzed using one way anova and scheffe test . basic issues of the sessions are as follows : first and second sessions : after rapport was built with subjects , the necessary information about their current problems was provided to them , their symptoms were evaluated , and the structure of therapeutic sessions was determined . existent disorders of the subjects were explained to them based on the cognitive viewpoint and treatment was offered to them . at the end of the second session , subjects were instructed to write about the meaning of their tramatization experience , and how it affected their views of themselves , others and the world . seventh to twelfth sessions : subjects ' excessive and over - generalized beliefs about self were recognized , and they were helped to deal with them . step 2 ) ensuring a foundation of coping skills : at this step , the following coping skills were taught to the subjects : 1 ) relaxation techniques ( signal breath , cleansing breath , relaxation sandwich , counting exercise , and personal biofeedback machine ) ; 2 ) detecting feelings ( body scan / emotional scan , deciphering messages ) ; 3 ) tolerating the experienced affects ( by mindful observation , deliberate distraction , five ways to express feelings ) ; 4 ) shifting feelings ; 5 ) building emotional resiliency ( maypole of support ) ; 6 ) addressing such symptoms as nightmares , intrusive thoughts and panic ; 7 ) creating a feelings plan step 3 ) engaging in experiential discovery : at this step , the subjects were helped to engage in experiential discovery by using the following techniques : 1 ) connecting with the experience ; 2 ) identifying the feeling ; 3 ) free association with images , memories , or other feelings step 4 ) mapping the experiential hologram : at this step , a simple image of a pot on a stove is offered to help the subjects organize and explain the components of the experiential hologram . in this analogy , each component is matched with an aspect of the image ( the acquired motivation is matched with turning on the stove ; the core violation is matched with the hot burner ; the boiling contents inside the pot correspond to the personal truth including the associated feelings , assumptions , and beliefs that boil to the surface ; the lid on the pot attempts to contain the boiling contents ; the lid represents coping strategies ; keeping the lid on tight to avoid the steam is the avoidance strategy ; and keeping the lid on loosely to release the pressure of the steam is the compensation strategy ; the steam that escapes from the pot corresponds to the residual negative emotions that linger in between cycles of the hologram ) . statistical society of this research consisted of male freshman , junior and senior high school students in city of uremia in 2010 - 2011 school year ( n=10286 ) . one thousand students , approximately 10 percent of this statistical society , were randomly selected for screening traumatic events ( current and previous injuries , hospitalizations , domestic violence , community violence , disasters , accidents , physical abuse ) and psychological symptoms . fifty five students refused to respond to the inventories , 129 of the subjects ( 13/63% ) were exposed to at least one traumatic event based on children traumatic events screening inventory and had a score of higher than 90 in the symptom checklist-90 ( scl-90 ) . of the subjects , 60 were randomly selected . then , clinical interview was conducted , and the selected sample was randomly assigned in to three groups of cognitive processing therapy , holographic reprocessing and control . the tesi inquires about a variety of traumatic events , including current and previous injuries , hospitalizations , domestic violence , community violence , disasters , accidents , physical , and sexual abuse . this coefficiency was obtained to be 0/76 in the initial sample ( n=946 ) of this research . this interview was used to diagnose disorders related to trauma , and to confirm diagnosis of scl-90 . internal consistency of this scale ranges from 0/71 to 0/84 across all nine scales , and its test retest reliability ranges from 0/67 to0/91 ( 23 ) . posttraumatic cognitions inventory ( ptci):the ptci is a 36 item self - report measure which assesses trauma related thoughts and beliefs . the measure loads on three factors of negative cognitions about self , negative cognitions about the world , and self - blame . alpha coefficients of the three subscales were reported to be 0/87 for the negative cognitions about self , 0/88 for the negative cognitions about the world , and 0/86 for self - blame ( 25 ) . in the current study , for the total scale , these coefficients were 0/86 , 0/67 , 0/70 and 0/88 respectively for the three groups . data were collected after recognizing 129 subjects ( 13/63% ) as being exposed to at least one traumatic event based on children traumatic events screening inventory and had a score of higher than 90 in the symptom checklist-90(scl-90 ) . then , the selected sample was randomly assigned in to three equal groups of cognitive processing therapy ( n=20 ) , holographic reprocessing ( n=20 ) and control group ( n=20 ) . at this stage , then , cognitive processing therapy and holographic reprocessing were performed as the therapeutic interventions on the experimental groups . next , based on the opinion of two psychotherapists about the effectiveness of these therapeutic methods on the posttraumatic symptoms and their correspondence with iranian culture , they were applied in this research . to examine the effectiveness of therapeutic intervention , differences of pre - post test scores were analyzed using one way anova and scheffe test . basic issues of the sessions are as follows : first and second sessions : after rapport was built with subjects , the necessary information about their current problems was provided to them , their symptoms were evaluated , and the structure of therapeutic sessions was determined . existent disorders of the subjects were explained to them based on the cognitive viewpoint and treatment was offered to them . at the end of the second session , subjects were instructed to write about the meaning of their tramatization experience , and how it affected their views of themselves , others and the world . step 2 ) ensuring a foundation of coping skills : at this step , the following coping skills were taught to the subjects : 1 ) relaxation techniques ( signal breath , cleansing breath , relaxation sandwich , counting exercise , and personal biofeedback machine ) ; 2 ) detecting feelings ( body scan / emotional scan , deciphering messages ) ; 3 ) tolerating the experienced affects ( by mindful observation , deliberate distraction , five ways to express feelings ) ; 4 ) shifting feelings ; 5 ) building emotional resiliency ( maypole of support ) ; 6 ) addressing such symptoms as nightmares , intrusive thoughts and panic ; 7 ) creating a feelings plan step 3 ) engaging in experiential discovery : at this step , the subjects were helped to engage in experiential discovery by using the following techniques : 1 ) connecting with the experience ; 2 ) identifying the feeling ; 3 ) free association with images , memories , or other feelings step 4 ) mapping the experiential hologram : at this step , a simple image of a pot on a stove is offered to help the subjects organize and explain the components of the experiential hologram . in this analogy , each component is matched with an aspect of the image ( the acquired motivation is matched with turning on the stove ; the core violation is matched with the hot burner ; the boiling contents inside the pot correspond to the personal truth including the associated feelings , assumptions , and beliefs that boil to the surface ; the lid on the pot attempts to contain the boiling contents ; the lid represents coping strategies ; keeping the lid on tight to avoid the steam is the avoidance strategy ; and keeping the lid on loosely to release the pressure of the steam is the compensation strategy ; the steam that escapes from the pot corresponds to the residual negative emotions that linger in between cycles of the hologram ) . step 2 ) ensuring a foundation of coping skills : at this step , the following coping skills were taught to the subjects : 1 ) relaxation techniques ( signal breath , cleansing breath , relaxation sandwich , counting exercise , and personal biofeedback machine ) ; 2 ) detecting feelings ( body scan / emotional scan , deciphering messages ) ; 3 ) tolerating the experienced affects ( by mindful observation , deliberate distraction , five ways to express feelings ) ; 4 ) shifting feelings ; 5 ) building emotional resiliency ( maypole of support ) ; 6 ) addressing such symptoms as nightmares , intrusive thoughts and panic ; 7 ) creating a feelings plan step 3 ) engaging in experiential discovery : at this step , the subjects were helped to engage in experiential discovery by using the following techniques : 1 ) connecting with the experience ; 2 ) identifying the feeling ; 3 ) free association with images , memories , or other feelings step 4 ) mapping the experiential hologram : at this step , a simple image of a pot on a stove is offered to help the subjects organize and explain the components of the experiential hologram . in this analogy , each component is matched with an aspect of the image ( the acquired motivation is matched with turning on the stove ; the core violation is matched with the hot burner ; the boiling contents inside the pot correspond to the personal truth including the associated feelings , assumptions , and beliefs that boil to the surface ; the lid on the pot attempts to contain the boiling contents ; the lid represents coping strategies ; keeping the lid on tight to avoid the steam is the avoidance strategy ; and keeping the lid on loosely to release the pressure of the steam is the compensation strategy ; the steam that escapes from the pot corresponds to the residual negative emotions that linger in between cycles of the hologram ) . sixty students who had experienced at least one traumatic event , and had a score of higher than 90 in scl-90 r were studied in three groups of cognitive processing therapy , holographic reprocessing and control ; each group consisted of 20 subjects ( n= 20 ) . four subjects in the cognitive processing group , 2 in the holographic reprocessing group , and 5 in the control group did not complete the intervention programs , thus they were excluded from this study . demographic features of participants in the three groups are demonstrated in table 1 . according to this table , the results of 2 test for the variables of educational field and educational level , and the one way anova test for age variable demonstrated no significant difference between the groups in these variables . demographic features of the participants and tests of significant difference among groups table 2 demonstrates the mean and standard deviation of pre- test and post- test scores in the three groups . mean and standard deviations of pre test and post test scores in posttraumatic cognitions inventory table 3 showed the results of anova for comparing the means of differences between pre - test and post - test scores in posttraumatic cognition inventory . as observed in this table , a significant difference exists between the three groups in the subscales of negative cognitions about self ( f=6/70 , p<0/003 ) , self - blame ( f=18/40 , p<0/001 ) and the total scale ( f= 8/66 , p<0/001 ) . anova results to compare three groups furthermore , the results showed that the three groups did not differ significantly from one another with respect to negative cognitions about the world . according to table 4 , the results of scheffe test showed that in the components of negative cognitions about self and total score of this inventory , the two therapeutic groups ( cpt and hr ) did not differ significantly . however , a significant difference was observed between the two groups ( cpt and hr ) and the control group . in the component of self - blame , cognitive processing therapy group significantly differed from hr and control groups , but no significant difference was observed between cognitive processing and control groups . results of scheffe test to compare the means of difference between pre test post test scores in three groups based on f and significant level this research was conducted to examine the effectiveness of cognitive processing therapy and holographic reprocessing on the reduction of posttraumatic cognitions in students exposed to trauma . the results of this study showed no significant difference between cpt and hr groups at the differences of pretest furthermore , these two therapeutic methods were equally effective on the reduction of posttraumatic negative cognitions , particularly negative cognition about self and self blame . ( 18 ) , in relation to the effectiveness of cognitive processing therapy , and are also in accordance with the results of katz et al . ( 20 ) in relation to the effectiveness of holographic reprocessing on the reduction of posttraumatic symptoms . foa and colleagues have emphasized the role of cognitions about self and the world in natural recovery from trauma , the development of posttraumatic disorders , and the cbt processes ameliorating these trauma symptoms ( 25 ) ; they correspond with the following ideas : the effects of these therapeutic methods on the negative cognitions can free the traumatized individual from other trauma symptoms . the effectiveness of these therapeutic methods on the reduction of negative cognitions can be attributed to the congruence of logic of these methods with the features of adolescence period . each of these therapeutic methods was considered an integrative therapy with both the exposure therapy and psycho social education and the cognitive techniques for the coping with dysfunctional cognitions . furthermore , it can be noted that altering negative cognitions in adolescence period , where these cognitions are formed , is easier than other periods of life . similarly , both cpt and hr are trauma- focused cognitive behavioral therapies ; therefore , the effect of these methods on negative cognitions was expected . on the subscales of posttraumatic cognitions inventory , the obtained results showed no significant difference between cognitive processing and holographic reprocessing groups on the negative cognitions about self , but these groups differed significantly from the control group on this variable ; meaning that each of these methods were equally effective on the reduction of negative cognitions about self . this result is consistent with the results of foa and rauch research ( 12 ) on reduction of negative cognitions about self , following prolonged exposure therapy , and with the results of katz et al . ( 20 ) study on the effectiveness of holographic reprocessing on the reduction of negative cognitions . because cognitive processing therapy has targeted some essential components of self such as trust , safety , intimacy , control and respect , and holographic reprocessing is also based on intellectualizing the experiential self , it was expected that these therapeutic methods be effective for the negative cognitions about self more than controlled interventions . on the self - blame subscale , the obtained results showed that holographic reprocessing group differed from both cognitive processing and control groups . furthermore , holographic reprocessing therapy was effective for self - blame more than cognitive processing therapy and control intervention . the results of this study are not consistent with the results of those studies supporting the effectiveness of cognitive processing therapy on the symptoms of ptsd and comorbid disorders ( 13 , 14 , 15 , 16 , 17 , 18 ) , while they are consistent with katz 's opinion ( 19 ) about holographic reprocessing therapy being effective for the reduction of trauma related symptoms more than other therapeutic methods , because it interactively uses the basic techniques of psychodynamic , cognitive- behavioral and experiential therapies . hr can encounter a person with his / her own ego version by mapping experientional hologram ; in this state , the person let go of the painful feeling about the younger version of self , and finally is free of the burden of self blame . moreover , the results of this research showed no significant difference between the three groups in the subscale of negative cognitions about the world , meaning that therapeutic methods were not effective for these cognitions . ( 18 ) in respect to the effectiveness of cognitive processing therapy , and results of katz et al . negative beliefs about the world were targeted in both cpt and hr , but the ineffectiveness of these methods for thecognitions can be due to the occurrence of some traumatic events such as crashing the airplane of tehran - uremia , and assassination of nucleus scientists shortly before conducting this research . in conclusion , the results of this research revealed that cognitive processing therapy and holographic reprocessing , as the trauma related psychotherapies , were equally effective for the reduction of posttraumatic cognitions in trauma - exposed students . thus , it appears that these therapeutic methods which had been originally developed and tested for sexually assaulted females can also be applied for the victims of other traumatic events ( death of a parent , parental divorce , physical abuse etc ) particularly during the adolescence period . therefore , the results of the current study provide important evidence in the support of these two trauma- focused cognitive behavioral therapies for use in clinical setting . thirdly , no data were available on validity and reliability of traumatic events screening inventory , and posttraumatic cognitions inventory for iranians . thus , it is recommended that in the future researches , follow up evaluations be conducted to study the constancy of the therapies and interventions applied by different specialists to increase the validity of these therapies . furthermore , it is suggested that these methods be used for the victims of different traumas in all ages and in both genders .

the ability to detect whether one is being addressed is of critical importance to successfully communicate with others . before decoding the message that is conveyed by another person , one must pick up on signals that indicate that the message is in fact directed at the self ( i.e. , self - referential ) . the most common way to initiate intentional communication is by using so - called ostensive signals such as calling someone 's name and looking directly at someone to establish eye contact . although these two ostensive signals differ in modality and most low - level perceptual features , they both serve to make the intention to communicate manifest to the receiver ( sperber and wilson , 1995 ; csibra , 2010 ) . adults are highly sensitive to eye contact and name cues , which powerfully grab their attention even when they are engaged in cognitively demanding tasks ( moray , 1959 ; von grnau and anston , 1995 ) . critically , it has been argued that mentalizing , the ability to attribute mental states such as intentions to others ( frith and frith , 1999 , 2006 ) , is involved in understanding communication already at the stage at which a sender initiates communication through eye contact or calling someone 's name . accordingly , kampe et al . ( 2003 ) predicted that brain regions implicated in mentalizing would be engaged when detecting communicative signals as referring to the self . ( 2003 ) have shown that , in adults , calling a person 's name or making eye contact activate common brain regions that are implicated in attributing mental states to others ( frith and frith , 1999 , 2006 ) . more specifically , the brain regions adults employ in processing these communicative signals are localized in the right medial prefrontal cortex and the left temporal pole ( kampe et al . , 2003 ) . these findings suggest that , in adults , the communicative intention of another person directed toward oneself is detected by the same brain region , regardless of modality . this process occurs without deliberate attention ( participants were asked to detect faces with eyes closed rather than direct gaze or to detect last names rather than their own name ) and it also does not depend on subsequent interaction between participants . more support for the view that mentalizing is critical for understanding ostensive communication cues comes from individuals with autism who have been shown to be impaired in their mentalizing skills and also have severe difficulties in recognizing when they are being addressed ( baron - cohen , 2000 ) . indeed , lack of orienting to name and eye contact is perhaps one of the earliest signs of autism identifiable in childhood ( osterling et al . , 2002 ) . taken together , this poses the important ontogenetic question of whether and when typically developing infants are sensitive to eye contact and own name as ostensive signals of communicative intent and more specifically , whether infants would engage brain regions ( and thus cognitive processes ) similar to adults when they respond to ostensive signals across modalities . findings as to whether young infants already engage in adult - like brain processes associated with mentalizing when they interpret communicative signals can inform theories of early social - cognitive development and may add to recent work that has shown that infants understand certain kinds of intentions and other mental states surprisingly early in development ( woodward , 2009 ; baillargeon et al . , 2010 ) . it is known that infants are sensitive to eye contact from birth ( farroni et al . , 2002 , 2005 ) , suggesting that eye contact is a potentially hard - wired and pre - specified cue detected without or at least with only very little prior experience . first names on the other hand are arbitrary auditory signals chosen by the parents that must be learned by the infant . sensitivity to own name emerges around 4.5 months of age when infants have been shown to first orient to their name ( mandel et al . , , we measured prefrontal cortex responses in 5-month - old infants using near - infrared spectroscopy ( nirs ) permitting spatial localization of brain activation by measuring hemodynamic responses to examine the neural basis and developmental origins of detecting ostensive communicative signals across modalities ( see lloyd - fox et al . , 2010 , for a detailed review of nirs and its use with infants ) . other neuroimaging techniques that are well established in adults are limited in their use with infants because of methodological concerns . for example , positron emission tomography ( pet ) exposes participants to radioisotopes , and fmri requires the participant to remain very still and exposes them to a noisy environment . although both pet and fmri have been used with infants , this work is restricted to the study of sleeping , sedated or very young infants . nirs is better suited for infant research because it can accommodate a good degree of movement from the infants , enabling them to sit upright on their parent 's lap and behave relatively freely while watching or listening to certain stimuli . in addition , unlike pet and fmri , nirs systems are portable . finally , despite its inferior spatial resolution , nirs , like fmri , measures localized patterns of hemodynamic responses , thus allowing for a comparison of infant nirs data with adult fmri data ( see strangman et al . , 2002 , for evidence of a strong correlation between hemodynamic responses measured with fmri and nirs ) . we studied 5-month - old infants because , as mentioned above , at this age they have been shown to be sensitive to eye contact as well as name cues . moreover , we focused on prefrontal cortical responses since this is one of the parts of the brain that showed common activation to eye contact and name cues in adults ( kampe , et al . , 2003 ) , and previous infant nirs studies have successfully obtained specialized prefrontal cortex responses in social tasks ( grossmann , et al . , 2008 ; grossmann and johnson , 2010 ) . infants watched human faces that either signaled eye contact or directed their gaze away from the infant , and they also listened to voices that addressed them with their own name or with the name of another infant . twenty 5-month - old infants were included in the final sample ( 11 girls , range 139166 days , m = 151.8 days ) . nine additional infants were tested but not included in the final sample because of fussiness ( n = 4 ) , too many motion artifacts ( n = 3 ) , technical problems or experimenter errors ( n = 2 ) . all infants contributed a total of four trials for each experimental condition . the required minimum number of artifact - free trials per condition was 2 ( see section data acquisition and analysis for artifact treatment ) . please note that an attrition rate at this level is within the normal range for an infant nirs study ( lloyd - fox et al . , 2010 ) . all infants were born full - term ( 3742 weeks gestation ) and with normal birthweight ( > 2500 g ) . visual stimuli were static pictures of a smiling face depicting mutual or averted gaze ( left or right ) . the faces subtended a visual angle of 22 17 at a viewing distance of 60 cm . each infant heard her own name and a stranger 's name , matched for syllable number and always differing from the infant 's name in its first phoneme . infants sat on their parent 's lap while watching the stimuli on a computer monitor within an acoustically shielded , dimly lit room . the experiment consisted of two sessions balanced across participants : a visual and an auditory session . each trial consisted of three presentations of the stimulus , each presentation lasting for 1000 ms for the pictures and an average of 790 ms for the names ( ranging from 394 to 1228 ms ) . a star appeared on the center of the screen for 300 ms before and after each stimulus . the two experimental conditions within each session were randomly distributed , with no more than two trials of the same condition occurring in a row . the inter - trial interval varied randomly between 10 and 12 s. non - social moving visual stimuli were presented during the inter - trial interval to keep infants attention . the nirs method relies on the optical determination of changes in hemoglobin concentrations in cerebral cortex which result from increased regional cerebral blood flow . two custom - built arrays consisting of nine optodes ( five sources , four detectors ) in a 12-channel ( source detector pairs ) arrangement with an inter - optode separation of 25 mm were placed over the frontal lobe on each hemisphere using an easycap ( www.easycap.de ) . our current measurement technique did not provide us with information about the depth of the activation ( see blasi et al . , 2007 , for nirs methodology that allows for the measurement of depth - dependent hemodynamic responses in infants ) . after calculation of the oxyhb concentration changes , pulse - related signal changes and overall trends were eliminated by low - pass filtering ( butterworth , fifth order , lower cutoff 0.5 hz ) . movement artifacts were corrected by an established procedure ( see grossmann et al . , 2010 ) , which allows marking of artifacts and then padding the contaminated data segments by linear interpolation . after visual inspection of the time course of the concentration changes a time window around the peak of the hemodynamic response ( between 5 and 7 s after stimulus onset ) was chosen for statistical analysis . cortical responses were assessed by comparing average concentration changes ( oxyhb ) within trials for this time window between the two experimental conditions . paired t - test were used in order to compare between conditions within each modality : eye contact versus averted gaze in the visual modality and infant 's own name versus stranger 's name in the auditory modality . please note that similar to previous nirs studies ( see lloyd - fox et al . , 2010 ) p - values twenty 5-month - old infants were included in the final sample ( 11 girls , range 139166 days , m = 151.8 days ) . nine additional infants were tested but not included in the final sample because of fussiness ( n = 4 ) , too many motion artifacts ( n = 3 ) , technical problems or experimenter errors ( n = 2 ) . all infants contributed a total of four trials for each experimental condition . the required minimum number of artifact - free trials per condition was 2 ( see section data acquisition and analysis for artifact treatment ) . please note that an attrition rate at this level is within the normal range for an infant nirs study ( lloyd - fox et al . , 2010 ) . all infants were born full - term ( 3742 weeks gestation ) and with normal birthweight ( > 2500 g ) . visual stimuli were static pictures of a smiling face depicting mutual or averted gaze ( left or right ) . the faces subtended a visual angle of 22 17 at a viewing distance of 60 cm . each infant heard her own name and a stranger 's name , matched for syllable number and always differing from the infant 's name in its first phoneme . infants sat on their parent 's lap while watching the stimuli on a computer monitor within an acoustically shielded , dimly lit room . the experiment consisted of two sessions balanced across participants : a visual and an auditory session . each trial consisted of three presentations of the stimulus , each presentation lasting for 1000 ms for the pictures and an average of 790 ms for the names ( ranging from 394 to 1228 ms ) . a star appeared on the center of the screen for 300 ms before and after each stimulus . the two experimental conditions within each session were randomly distributed , with no more than two trials of the same condition occurring in a row . the inter - trial interval varied randomly between 10 and 12 s. non - social moving visual stimuli were presented during the inter - trial interval to keep infants attention . the nirs method relies on the optical determination of changes in hemoglobin concentrations in cerebral cortex which result from increased regional cerebral blood flow . two custom - built arrays consisting of nine optodes ( five sources , four detectors ) in a 12-channel ( source detector pairs ) arrangement with an inter - optode separation of 25 mm were placed over the frontal lobe on each hemisphere using an easycap ( www.easycap.de ) . our current measurement technique did not provide us with information about the depth of the activation ( see blasi et al . , 2007 , for nirs methodology that allows for the measurement of depth - dependent hemodynamic responses in infants ) . after calculation of the oxyhb concentration changes , pulse - related signal changes and overall trends were eliminated by low - pass filtering ( butterworth , fifth order , lower cutoff 0.5 hz ) . movement artifacts were corrected by an established procedure ( see grossmann et al . , 2010 ) , which allows marking of artifacts and then padding the contaminated data segments by linear interpolation . after visual inspection of the time course of the concentration changes a time window around the peak of the hemodynamic response ( between 5 and 7 s after stimulus onset ) was chosen for statistical analysis . cortical responses were assessed by comparing average concentration changes ( oxyhb ) within trials for this time window between the two experimental conditions . paired t - test were used in order to compare between conditions within each modality : eye contact versus averted gaze in the visual modality and infant 's own name versus stranger 's name in the auditory modality . please note that similar to previous nirs studies ( see lloyd - fox et al . , 2010 ) our analysis of 5-month - old infants prefrontal brain responses revealed two adjacent regions in the left hemisphere sensitive to the communicative signals conveyed to the infant ( see figure 1 ) . specifically , a left dorsal prefrontal region showed sensitivity to infants own name as indexed by a significantly increased oxyhb concentration ( average of concentration change between 5 and 7 s after stimulus onset ) when own name was compared to the control name ( channel 9 : t = 3.060 , p = 0.006 ) , while an adjacent left dorsal prefrontal region responded sensitively to eye contact cues as indexed by a significantly increased oxyhb concentration when eye contact is compared to averted gaze ( channel 6 : t = 2.291 , p = 0.034 ) . these results were confirmed by non - parametric tests ( wilcoxon 's z = 2.053 , p = 0.040 for the visual modality [ channel 6 , 13 out of 20 infants showed the effect ] and wilcoxon 's z = 2.539 , p = 0.011 , for the auditory modality [ channel 9 , 14 out of 20 infants showed the effect ] ) . five - month - old infants prefrontal cortex responses to eye contact ( channel 6 ) and own name ( channel 9 ) . time courses ( with 0 indicating the stimulus onset ) of the hemodynamic response for these two channels are shown in the top row . the gray windows in the time courses represent the time windows during which significant differences between conditions were observed . the bottom row shows bar charts with the average oxyhb change ( se ) observed during the time windows marked in the top row . furthermore , the prefrontal cortex responses to eye contact ( mutual gaze averted gaze ) and name ( own name control name ) were significantly positively correlated ( r = 0.446 , p = 0.049 ) , indicating that infants who showed a sensitive prefrontal cortex response to eye contact were more likely to also show a sensitive prefrontal cortex response to their own name ( see figure 2 ) . there were no significant correlations between channels 6 and 9 when calculating the overall association of the responses for the four individual conditions ( mutual gaze , averted gaze , own name , and other name ) , suggesting that the correlation reported above was specific to the contrasts calculated ( mutual gaze averted gaze and own name control name ) and not due to a general association of hemodynamic responses between channels . this also renders it unlikely that the nirs signals measured from channels 6 and 9 were sampled from a common vascular supply . our analysis revealed no significant effects of the factors gender and presentation order ( auditory first versus visual first ) for channels 6 and 9 . an additional analysis performed after close inspection of the time courses at these channels showed that there was an earlier effect ( 13 s after stimulus onset ) of eye contact at channel 6 ( increased oxyhb concentration when eye contact was compared to averted gaze , t = 2.176 , p = 0.042 ( wilcoxon 's z = 2.613 , p = 0.009 ; 14 out of 20 infants showed the effect ) , whereas no such early effect of name was observed during auditory stimulation ( see figure 1 ) . correlation between prefrontal cortex responses to eye contact ( channel 6 : difference in oxyhb [ mutual averted gaze ] in mm mm ) and own name ( channel 9 : difference in oxyhb [ own other name ] in mm mm ) . we examined whether 5-month - old infants are sensitive to eye contact and their own name as ostensive signals of communicative intent regardless of modality by measuring prefrontal cortex responses using nirs . the results revealed two adjacent regions in the left prefrontal cortex that are either sensitive to the communicative signals conveyed to the infant through face or voice . specifically , a left dorsal prefrontal region showed sensitivity to infants own name , while an adjacent left dorsal prefrontal region responded sensitively to eye contact cues . thus , these nirs results suggest that 5-month - old infants selectively process and attend to communicative signals directed at them as reflected in their specific prefrontal cortex responses within each modality . interestingly , unlike adults , 5-month - old infants do not seem to recruit a common right prefrontal region when processing communicative signals across modalities . however , infants showed a correlation in the activation of the two prefrontal regions involved . infants who responded sensitively to eye contact in one of the left prefrontal regions were more likely to respond sensitively to their own name in the adjacent region as revealed in a correlation analysis , suggesting that responding to ostensive communicative signals in these two regions might be functionally correlated . this is perhaps an important developmental precursor for a later integration into a more abstract and modality - independent representation of communicative signals directed at the self . it is important to further discuss the question of how these findings using nirs with infants relate to the findings using fmri with adults in order to understand the development of processing communicative signals . while infants in the current study showed sensitive responding to eye contact and own name in the left prefrontal cortex , the effect in adults for the same contrast revealed activation in the right prefrontal cortex ( kampe , et al . , 2003 ) . the difference in the lateralization of the activation patterns between infants and adults suggests different underlying processes that might be due to development , differences in the stimulus material used , or both . in contrast to the adult work , we used smiling faces and infant - directed voices that are both characterized by positive affect . the presentation of positive affect in face and voice directed at the infant may have induced the motivation to approach , which , according to prior infant and adult work , is reflected in left prefrontal activation ( davidson and fox , 1982 ; fox , 1991 ; harmon - jones , 2003 ) . this raises the possibility that infants responded to the positive affective signals directed at them with the motivation to approach rather than with mentalizing like adults do in response to communicative signals . this account does not undermine infants sensitivity to eye contact and own name but it may point to a developmental difference between infants and adults in the interpretation of these communicative signals . however , it should be noted that this is only one potential account for the observed developmental difference . future work should more directly address the question of what kind of psychological process can best account for young infants left prefrontal response patterns during these kinds of communicative scenarios . for example , according to the motivational account put forward above it would be predicted that by using faces and voices expressing anger toward the infant one should observe a different pattern of lateralization in their prefrontal cortex responses since prior work suggests that perceiving aversive emotions results in right prefrontal activation associated with a motivation to withdraw ( davidson and fox , 1982 ; fox , 1991 ; harmon - jones , 2003 ) . another issue that deserves attention when discussing the current findings is the selection of stimuli used . specifically , in order to make it comparable to prior adult work ( kampe et al . , 2003 ) we used static faces in the visual part of the study . from previous infant work it is known that using dynamic rather than static representation of faces has been shown to increase infants attention and cortical responses ( grossmann et al . although we obtained significant effects in both the face and name context , the effect ( as indicated by the t - values ) appeared to be somewhat weaker in the face than in the name context . therefore , presenting infants with a dynamic face that shifts gaze and engages them in eye contact rather than a static face in future studies may be a clearer and more ecologically valid indicator of the intention to communicate with the infant . our nirs results further revealed that , while eye contact cues and name cues resulted in non - overlapping prefrontal cortex responses between 5 and 7 s after stimulus onset , only eye contact evoked an early increase in the hemodynamic response between 1 and 3 s after stimulus onset . this finding suggests that infants recruit particularly fast prefrontal brain processes for the detection of eye contact . this rapid prefrontal response may be related to the fact that eye contact is an especially powerful social cue that infants are sensitive to from birth , and it supports the hypothesis of specialized brain mechanisms that guarantee a fast detection of eye contact ( senju and johnson , 2009 ) . in particular , a fast subcortical pathway involving the superior colliculus and the pulvinar has been hypothesized to convey information to the cerebral cortex including prefrontal areas ( johnson , 2005 ) . processes of eye contact detection stand in contrast to the detection of the own name , which is an arbitrary signal to the self that has to be learned by the infant . our data show that infants as young as 5 months of age are sensitive to this acquired ostensive signal and selectively recruit a specific region of the left prefrontal cortex . thus , the prefrontal cortex also plays a role in integrating learned information into the infants communication comprehension repertoire . in summary , the current data have provided new insights into young infants processing and understanding of communicative signals directed at them . the sensitivity to eye contact and own name demonstrated in selective prefrontal cortex responses may serve as a pivotal building block that helps infants to enter the world of human communication . we have identified developmental precursors of the brain processes that are involved in initiating communication . however , the exact nature of the representation reflected in infants selective prefrontal cortex responses remains unclear . in particular , the present data leave open whether infants already engage adult - like mentalizing processes . this question has been of great interest to developmental and cognitive psychologists in recent years ( see woodward , 2009 ; baillargeon et al . , the current study has shown that a neuroscience perspective can provide additional insights into preverbal infants developing social - cognitive and communicative abilities . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

a precondition for successful communication between people is the detection of signals indicating the intention to communicate , such as eye contact or calling a person 's name . in adults , establishing communication by eye contact or calling a person 's name results in overlapping activity in right prefrontal cortex , suggesting that , regardless of modality , the intention to communicate is detected by the same brain region . we measured prefrontal cortex responses in 5-month - olds using near - infrared spectroscopy ( nirs ) to examine the neural basis of detecting communicative signals across modalities in early development . infants watched human faces that either signaled eye contact or directed their gaze away from the infant , and they also listened to voices that addressed them with their own name or another name . the results revealed that infants recruit adjacent but non - overlapping regions in the left dorsal prefrontal cortex when they process eye contact and own name . moreover , infants that responded sensitively to eye contact in the one prefrontal region were also more likely to respond sensitively to their own name in the adjacent prefrontal region as revealed in a correlation analysis , suggesting that responding to communicative signals in these two regions might be functionally related . these nirs results suggest that infants selectively process and attend to communicative signals directed at them . however , unlike adults , infants do not seem to recruit a common prefrontal region when processing communicative signals of different modalities . the implications of these findings for our understanding of infants developing communicative abilities are discussed .

Introduction Materials and Methods Participants Stimuli and procedure Data acquisition and analysis Results Discussion Conflict of Interest Statement

the ability to detect whether one is being addressed is of critical importance to successfully communicate with others . before decoding the message that is conveyed by another person , one must pick up on signals that indicate that the message is in fact directed at the self ( i.e. , self - referential ) . the most common way to initiate intentional communication is by using so - called ostensive signals such as calling someone 's name and looking directly at someone to establish eye contact . although these two ostensive signals differ in modality and most low - level perceptual features , they both serve to make the intention to communicate manifest to the receiver ( sperber and wilson , 1995 ; csibra , 2010 ) . adults are highly sensitive to eye contact and name cues , which powerfully grab their attention even when they are engaged in cognitively demanding tasks ( moray , 1959 ; von grnau and anston , 1995 ) . critically , it has been argued that mentalizing , the ability to attribute mental states such as intentions to others ( frith and frith , 1999 , 2006 ) , is involved in understanding communication already at the stage at which a sender initiates communication through eye contact or calling someone 's name . accordingly , kampe et al . ( 2003 ) predicted that brain regions implicated in mentalizing would be engaged when detecting communicative signals as referring to the self . ( 2003 ) have shown that , in adults , calling a person 's name or making eye contact activate common brain regions that are implicated in attributing mental states to others ( frith and frith , 1999 , 2006 ) . more specifically , the brain regions adults employ in processing these communicative signals are localized in the right medial prefrontal cortex and the left temporal pole ( kampe et al . these findings suggest that , in adults , the communicative intention of another person directed toward oneself is detected by the same brain region , regardless of modality . this process occurs without deliberate attention ( participants were asked to detect faces with eyes closed rather than direct gaze or to detect last names rather than their own name ) and it also does not depend on subsequent interaction between participants . more support for the view that mentalizing is critical for understanding ostensive communication cues comes from individuals with autism who have been shown to be impaired in their mentalizing skills and also have severe difficulties in recognizing when they are being addressed ( baron - cohen , 2000 ) . indeed , lack of orienting to name and eye contact is perhaps one of the earliest signs of autism identifiable in childhood ( osterling et al . taken together , this poses the important ontogenetic question of whether and when typically developing infants are sensitive to eye contact and own name as ostensive signals of communicative intent and more specifically , whether infants would engage brain regions ( and thus cognitive processes ) similar to adults when they respond to ostensive signals across modalities . findings as to whether young infants already engage in adult - like brain processes associated with mentalizing when they interpret communicative signals can inform theories of early social - cognitive development and may add to recent work that has shown that infants understand certain kinds of intentions and other mental states surprisingly early in development ( woodward , 2009 ; baillargeon et al . it is known that infants are sensitive to eye contact from birth ( farroni et al . , 2002 , 2005 ) , suggesting that eye contact is a potentially hard - wired and pre - specified cue detected without or at least with only very little prior experience . first names on the other hand are arbitrary auditory signals chosen by the parents that must be learned by the infant . sensitivity to own name emerges around 4.5 months of age when infants have been shown to first orient to their name ( mandel et al . , , we measured prefrontal cortex responses in 5-month - old infants using near - infrared spectroscopy ( nirs ) permitting spatial localization of brain activation by measuring hemodynamic responses to examine the neural basis and developmental origins of detecting ostensive communicative signals across modalities ( see lloyd - fox et al . , 2010 , for a detailed review of nirs and its use with infants ) . other neuroimaging techniques that are well established in adults are limited in their use with infants because of methodological concerns . for example , positron emission tomography ( pet ) exposes participants to radioisotopes , and fmri requires the participant to remain very still and exposes them to a noisy environment . although both pet and fmri have been used with infants , this work is restricted to the study of sleeping , sedated or very young infants . nirs is better suited for infant research because it can accommodate a good degree of movement from the infants , enabling them to sit upright on their parent 's lap and behave relatively freely while watching or listening to certain stimuli . in addition , unlike pet and fmri , nirs systems are portable . , 2002 , for evidence of a strong correlation between hemodynamic responses measured with fmri and nirs ) . we studied 5-month - old infants because , as mentioned above , at this age they have been shown to be sensitive to eye contact as well as name cues . moreover , we focused on prefrontal cortical responses since this is one of the parts of the brain that showed common activation to eye contact and name cues in adults ( kampe , et al . , 2003 ) , and previous infant nirs studies have successfully obtained specialized prefrontal cortex responses in social tasks ( grossmann , et al . infants watched human faces that either signaled eye contact or directed their gaze away from the infant , and they also listened to voices that addressed them with their own name or with the name of another infant . twenty 5-month - old infants were included in the final sample ( 11 girls , range 139166 days , m = 151.8 days ) . nine additional infants were tested but not included in the final sample because of fussiness ( n = 4 ) , too many motion artifacts ( n = 3 ) , technical problems or experimenter errors ( n = 2 ) . all infants contributed a total of four trials for each experimental condition . the required minimum number of artifact - free trials per condition was 2 ( see section data acquisition and analysis for artifact treatment ) . please note that an attrition rate at this level is within the normal range for an infant nirs study ( lloyd - fox et al . all infants were born full - term ( 3742 weeks gestation ) and with normal birthweight ( > 2500 g ) . the faces subtended a visual angle of 22 17 at a viewing distance of 60 cm . each infant heard her own name and a stranger 's name , matched for syllable number and always differing from the infant 's name in its first phoneme . infants sat on their parent 's lap while watching the stimuli on a computer monitor within an acoustically shielded , dimly lit room . the experiment consisted of two sessions balanced across participants : a visual and an auditory session . a star appeared on the center of the screen for 300 ms before and after each stimulus . the two experimental conditions within each session were randomly distributed , with no more than two trials of the same condition occurring in a row . the inter - trial interval varied randomly between 10 and 12 s. non - social moving visual stimuli were presented during the inter - trial interval to keep infants attention . two custom - built arrays consisting of nine optodes ( five sources , four detectors ) in a 12-channel ( source detector pairs ) arrangement with an inter - optode separation of 25 mm were placed over the frontal lobe on each hemisphere using an easycap ( www.easycap.de ) . , 2007 , for nirs methodology that allows for the measurement of depth - dependent hemodynamic responses in infants ) . after calculation of the oxyhb concentration changes , pulse - related signal changes and overall trends were eliminated by low - pass filtering ( butterworth , fifth order , lower cutoff 0.5 hz ) . movement artifacts were corrected by an established procedure ( see grossmann et al . , 2010 ) , which allows marking of artifacts and then padding the contaminated data segments by linear interpolation . after visual inspection of the time course of the concentration changes a time window around the peak of the hemodynamic response ( between 5 and 7 s after stimulus onset ) was chosen for statistical analysis . paired t - test were used in order to compare between conditions within each modality : eye contact versus averted gaze in the visual modality and infant 's own name versus stranger 's name in the auditory modality . please note that similar to previous nirs studies ( see lloyd - fox et al . , 2010 ) p - values twenty 5-month - old infants were included in the final sample ( 11 girls , range 139166 days , m = 151.8 days ) . nine additional infants were tested but not included in the final sample because of fussiness ( n = 4 ) , too many motion artifacts ( n = 3 ) , technical problems or experimenter errors ( n = 2 ) . all infants contributed a total of four trials for each experimental condition . the required minimum number of artifact - free trials per condition was 2 ( see section data acquisition and analysis for artifact treatment ) . each infant heard her own name and a stranger 's name , matched for syllable number and always differing from the infant 's name in its first phoneme . the two experimental conditions within each session were randomly distributed , with no more than two trials of the same condition occurring in a row . the inter - trial interval varied randomly between 10 and 12 s. non - social moving visual stimuli were presented during the inter - trial interval to keep infants attention . two custom - built arrays consisting of nine optodes ( five sources , four detectors ) in a 12-channel ( source detector pairs ) arrangement with an inter - optode separation of 25 mm were placed over the frontal lobe on each hemisphere using an easycap ( www.easycap.de ) . , 2007 , for nirs methodology that allows for the measurement of depth - dependent hemodynamic responses in infants ) . after calculation of the oxyhb concentration changes , pulse - related signal changes and overall trends were eliminated by low - pass filtering ( butterworth , fifth order , lower cutoff 0.5 hz ) . paired t - test were used in order to compare between conditions within each modality : eye contact versus averted gaze in the visual modality and infant 's own name versus stranger 's name in the auditory modality . please note that similar to previous nirs studies ( see lloyd - fox et al . , 2010 ) our analysis of 5-month - old infants prefrontal brain responses revealed two adjacent regions in the left hemisphere sensitive to the communicative signals conveyed to the infant ( see figure 1 ) . specifically , a left dorsal prefrontal region showed sensitivity to infants own name as indexed by a significantly increased oxyhb concentration ( average of concentration change between 5 and 7 s after stimulus onset ) when own name was compared to the control name ( channel 9 : t = 3.060 , p = 0.006 ) , while an adjacent left dorsal prefrontal region responded sensitively to eye contact cues as indexed by a significantly increased oxyhb concentration when eye contact is compared to averted gaze ( channel 6 : t = 2.291 , p = 0.034 ) . these results were confirmed by non - parametric tests ( wilcoxon 's z = 2.053 , p = 0.040 for the visual modality [ channel 6 , 13 out of 20 infants showed the effect ] and wilcoxon 's z = 2.539 , p = 0.011 , for the auditory modality [ channel 9 , 14 out of 20 infants showed the effect ] ) . five - month - old infants prefrontal cortex responses to eye contact ( channel 6 ) and own name ( channel 9 ) . time courses ( with 0 indicating the stimulus onset ) of the hemodynamic response for these two channels are shown in the top row . the gray windows in the time courses represent the time windows during which significant differences between conditions were observed . the bottom row shows bar charts with the average oxyhb change ( se ) observed during the time windows marked in the top row . furthermore , the prefrontal cortex responses to eye contact ( mutual gaze averted gaze ) and name ( own name control name ) were significantly positively correlated ( r = 0.446 , p = 0.049 ) , indicating that infants who showed a sensitive prefrontal cortex response to eye contact were more likely to also show a sensitive prefrontal cortex response to their own name ( see figure 2 ) . there were no significant correlations between channels 6 and 9 when calculating the overall association of the responses for the four individual conditions ( mutual gaze , averted gaze , own name , and other name ) , suggesting that the correlation reported above was specific to the contrasts calculated ( mutual gaze averted gaze and own name control name ) and not due to a general association of hemodynamic responses between channels . this also renders it unlikely that the nirs signals measured from channels 6 and 9 were sampled from a common vascular supply . an additional analysis performed after close inspection of the time courses at these channels showed that there was an earlier effect ( 13 s after stimulus onset ) of eye contact at channel 6 ( increased oxyhb concentration when eye contact was compared to averted gaze , t = 2.176 , p = 0.042 ( wilcoxon 's z = 2.613 , p = 0.009 ; 14 out of 20 infants showed the effect ) , whereas no such early effect of name was observed during auditory stimulation ( see figure 1 ) . correlation between prefrontal cortex responses to eye contact ( channel 6 : difference in oxyhb [ mutual averted gaze ] in mm mm ) and own name ( channel 9 : difference in oxyhb [ own other name ] in mm mm ) . we examined whether 5-month - old infants are sensitive to eye contact and their own name as ostensive signals of communicative intent regardless of modality by measuring prefrontal cortex responses using nirs . the results revealed two adjacent regions in the left prefrontal cortex that are either sensitive to the communicative signals conveyed to the infant through face or voice . specifically , a left dorsal prefrontal region showed sensitivity to infants own name , while an adjacent left dorsal prefrontal region responded sensitively to eye contact cues . thus , these nirs results suggest that 5-month - old infants selectively process and attend to communicative signals directed at them as reflected in their specific prefrontal cortex responses within each modality . interestingly , unlike adults , 5-month - old infants do not seem to recruit a common right prefrontal region when processing communicative signals across modalities . however , infants showed a correlation in the activation of the two prefrontal regions involved . infants who responded sensitively to eye contact in one of the left prefrontal regions were more likely to respond sensitively to their own name in the adjacent region as revealed in a correlation analysis , suggesting that responding to ostensive communicative signals in these two regions might be functionally correlated . this is perhaps an important developmental precursor for a later integration into a more abstract and modality - independent representation of communicative signals directed at the self . it is important to further discuss the question of how these findings using nirs with infants relate to the findings using fmri with adults in order to understand the development of processing communicative signals . while infants in the current study showed sensitive responding to eye contact and own name in the left prefrontal cortex , the effect in adults for the same contrast revealed activation in the right prefrontal cortex ( kampe , et al . , 2003 ) . the difference in the lateralization of the activation patterns between infants and adults suggests different underlying processes that might be due to development , differences in the stimulus material used , or both . in contrast to the adult work , we used smiling faces and infant - directed voices that are both characterized by positive affect . the presentation of positive affect in face and voice directed at the infant may have induced the motivation to approach , which , according to prior infant and adult work , is reflected in left prefrontal activation ( davidson and fox , 1982 ; fox , 1991 ; harmon - jones , 2003 ) . this raises the possibility that infants responded to the positive affective signals directed at them with the motivation to approach rather than with mentalizing like adults do in response to communicative signals . this account does not undermine infants sensitivity to eye contact and own name but it may point to a developmental difference between infants and adults in the interpretation of these communicative signals . however , it should be noted that this is only one potential account for the observed developmental difference . future work should more directly address the question of what kind of psychological process can best account for young infants left prefrontal response patterns during these kinds of communicative scenarios . for example , according to the motivational account put forward above it would be predicted that by using faces and voices expressing anger toward the infant one should observe a different pattern of lateralization in their prefrontal cortex responses since prior work suggests that perceiving aversive emotions results in right prefrontal activation associated with a motivation to withdraw ( davidson and fox , 1982 ; fox , 1991 ; harmon - jones , 2003 ) . another issue that deserves attention when discussing the current findings is the selection of stimuli used . , 2003 ) we used static faces in the visual part of the study . from previous infant work it is known that using dynamic rather than static representation of faces has been shown to increase infants attention and cortical responses ( grossmann et al . although we obtained significant effects in both the face and name context , the effect ( as indicated by the t - values ) appeared to be somewhat weaker in the face than in the name context . therefore , presenting infants with a dynamic face that shifts gaze and engages them in eye contact rather than a static face in future studies may be a clearer and more ecologically valid indicator of the intention to communicate with the infant . our nirs results further revealed that , while eye contact cues and name cues resulted in non - overlapping prefrontal cortex responses between 5 and 7 s after stimulus onset , only eye contact evoked an early increase in the hemodynamic response between 1 and 3 s after stimulus onset . this finding suggests that infants recruit particularly fast prefrontal brain processes for the detection of eye contact . this rapid prefrontal response may be related to the fact that eye contact is an especially powerful social cue that infants are sensitive to from birth , and it supports the hypothesis of specialized brain mechanisms that guarantee a fast detection of eye contact ( senju and johnson , 2009 ) . processes of eye contact detection stand in contrast to the detection of the own name , which is an arbitrary signal to the self that has to be learned by the infant . our data show that infants as young as 5 months of age are sensitive to this acquired ostensive signal and selectively recruit a specific region of the left prefrontal cortex . thus , the prefrontal cortex also plays a role in integrating learned information into the infants communication comprehension repertoire . in summary , the current data have provided new insights into young infants processing and understanding of communicative signals directed at them . the sensitivity to eye contact and own name demonstrated in selective prefrontal cortex responses may serve as a pivotal building block that helps infants to enter the world of human communication . we have identified developmental precursors of the brain processes that are involved in initiating communication . however , the exact nature of the representation reflected in infants selective prefrontal cortex responses remains unclear . in particular , the present data leave open whether infants already engage adult - like mentalizing processes . , the current study has shown that a neuroscience perspective can provide additional insights into preverbal infants developing social - cognitive and communicative abilities . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

this is a medical challenge as progressive course from simple steatosis to steatohepatitis and possible cirrhosis with development of liver cancer is known.13 therapy of nafld is currently mainly directed at treating components of the metabolic syndrome . diet and physical exercise are recommended as a basic universal approach . some pharmacological agents show promising results , although on the basis of recent clinical trials no firm conclusions can yet be drawn.4 the situation is comparable with ald.3 the nontoxic essential phospholipids ( epl ) have been already widely accepted to be effective in various liver diseases.5,6 however , as a clinical overview of available results specifically in flds of different origin has not yet been published since 1988 , it is the purpose of this article to evaluate the clinical efficacy and safety of epl in these frequent liver diseases . the term epl with its polyenylphosphatidylcholine ( also called polyene phosphatidylcholine [ ppc ] ) molecules indicates a well - defined , highly purified extract of the semen of soybeans ( glycine max ) with standardized contents of 72%96% ( 3-sn - phosphatidyl ) choline . the quantitatively and qualitatively dominating molecule in epl is 1,2-dilinoleoylphosphatidylcholine ( dlpc ) ( figure 1 ) , with up to 52% of the administered pcs.6 this high level of dlpc is the main difference between epl / ppc and the typical phospholipids as well as pc consumed through diet and synthesized within the body . with a share of 1.3% , highly unsaturated pcs with an additional , unsaturated fatty acid in the first position of the pc molecule are rare.7 by administering epl , the amount of hepatic dlpc significantly increased.8 phospholipids are known to form the double layer of cellular and subcellular membranes and precondition their fluidity and biological activity . the efficacy of epl in the therapy of liver diseases is confirmed not only by the ability of dlpc to be incorporated into damaged sections of membranes , which improves hepatic regeneration and replaces endogenous , less unsaturated pc molecules but also by its ability to increase membrane fluidity and functioning . with regard to in vitro and animal investigations , epl influences membrane - dependent cellular functions and shows antioxidant , anti - inflammatory , antifibrotic , apoptosis - modulating , regenerative , membrane - repairing and -protective , cell - signaling and receptor - influencing , and lipid - regulating effects.6 database research was carried out on medline , embase , cochrane library , country - specific journals , and follow - up literature citations for relevant hepatogastroenterological articles published between 1988 and 2014 . in a two - step process , we first used combinations of keywords including nafld , ald , essentiale , esls , polyenylphosphatidylcholine , polyene phosphatidylcholine , dilinoleoylphosphatidylcholine , phosphatidylcholine , and therapy . in the second step , we reviewed those papers that indicated minimum extraction amount of 72% pc from soybean as being necessary to treat patients with a considerable amount of dlpc as a key component in epl ( for details see later ) , or that were based on the trade name essentiale . studies with less purified pc from soybean or with pc from other provenience were excluded . all papers that were reported in different languages other than english were completely translated into english . of the 83 clinical studies in total , 25 representative ones regarding the causes of fld , study design , and investigated variables were selected , including one case report study and seven double - blind studies ( table 1 ) . fifty - three clinical studies about epl and nafld have been published since 1988 , of which 45 were published since 2000 . twenty - eight , mostly randomized studies , were open controlled and three double - blind . dosage of epl and treatment duration varied as a rule between 1.05 g / d and 1.8 g / d per os and lasted from 4 weeks to > 24 months , in most studies 36 months with 1.8 g of epl / d . therapy started in some studies with 5001,000 mg epl iv over 10 days to 4 weeks , before continuing with oral epl administration ; one study was done with 500 mg epl iv for 30 days only . nafld and its more severe form , nonalcoholic steatohepatitis ( nash ) , were mainly induced by obesity and type ii diabetes mellitus but partly also associated with hyperlipidemia , arterial hypertension , coronary heart disease / atherosclerotic cardiosclerosis , chronic viral hepatitis , cholecystitis / cholelithiasis , hypothyroidism , and pregnancy , or with mixed forms of these diseases / disorders . according to the available studies , analysis of subjective symptoms , clinical findings , biochemical and imaging data , and histology was possible . in the first clinical study from japan , six nonalcoholic and nondiabetic patients with fatty liver due to obesity were successfully treated with a low - calorie diet , physical exercise , and 1.5 g epl / d for 8 weeks and compared to six patients under the same diet , physical exercise , and a nicotinic acid derivative prodrug.9 it was found that computed tomography ( ct ) determined liver fat accumulation significantly improved with epl ( 2718 hu to 548 hu indicating the decrease in intensity of steatosis ) . cairella et al10 from italy evaluated the efficacy of epl in therapy of hepatobiliary dysfunction when administering 1.8 g of epl / d for 3 months . the study group and control comprised 20 cases each with a body mass index of 35.344.89 kg / m . the excretory parameters , total bilirubin , alkaline phosphatase , and y - gt , were normalized in all patients of the epl group but remained increased in 15%35% of the control patients . in the epl group , the ultrasonographic picture clearly improved in 14 and normalized in six cases , while it only slightly changed in three cases of the control group . koga et al11 confirmed the continuous , epl - induced improvement or normalization of the ultrasonic picture in 39 patients with fatty liver due to obesity . bright liver was reduced , and decrement of backward echocardiogram and the obscurity of the intrahepatic venogram were decreased . in 2000 , a randomized double - blind study by li et al12 reported about the positive influence of 1.8 g of epl / d on total cholesterol ( tc ) , triglycerides ( tg ) , and transaminases , supported by significant improvement of the fatty liver ( ct scan ) . experience with epl in patients suffering from maturity- onset diabetes mellitus and fatty liver started with a randomized , placebo - controlled double - blind study.13 thirty patients were instructed to reduce diet to 1,200 kcal / d containing 1 g / kg protein . there were five patients in the placebo and seven patients in the epl group who were treated with 1.01.5 g tolbutamide per day . half of the patients were additionally given 1.8 g of epl / d , the second half a corresponding placebo . during the 6-month treatment phase , liver size hardly changed in the patients of the placebo group , but there was a significant reduction in liver size in the epl group . the histological examination of the liver biopsy yielded corresponding results : marked improvement was observed in four patients treated with epl and only in one patient of the placebo group . although no fatty infiltration was seen in the posttreatment liver biopsy of this patient , he developed the feature of progression to cirrhosis . also in one patient treated with epl , whose fatty infiltration was more pronounced at the second biopsy , morphological features of developing discrete fibrosis in portal tract were observed . altogether , there were seven patients in the epl group and four under placebo including the patient with developing cirrhosis , whose steatosis improved histologically during the study . additionally , y - gt was significantly lowered after 1 month , 3 months , and 6 months of epl therapy , whereas in the control group , the deviations were not statistically significant . since this double - blind trial , seven further clinical studies confirmed the influence of epl on diabetes mellitus - associated fatty liver.1420 yin and kong14 treated 185 patients with standard diet , oral antidiabetics ( not specified ) plus physical exercise , and 125 of these patients were treated additionally with 1.8 g of epl / d for 3 months . alanine aminotransferase ( alt ) , serum tc , and tg , low - density lipoprotein- and high - density lipoprotein - cholesterol had significantly improved or normalized within the epl group , the lipid variables also between the groups . the improvements were seen in 90.2% of the epl cases and in 51% of the controls . this difference was statistically significant ( p<0.05 ) . fasting blood sugar values improved in both groups , but the difference between the groups was not significant . twenty - two patients from the study of poongothai et al15 received , besides a standard diet , antidiabetics depending on the severity of the glycemic level : 22.7% were on metformin therapy , 22.7% on sulfonylurea , 45.5% on combination of both , and 9% on insulin therapy . epl 2.1 g / d per os was additionally given for 6 months . ultrasonographic improvement was seen in 54.5% of patients , 40.9% did not show any change , and one case worsened . hepatic echotexture improved in 80% of subjects with severe diffusion fine echos in hepatic parenchyma , and in 50% of those with slight changes . ohbayashi et al16 treated a female patient with nash with nateglinide as insulin secretagogue for 7 months , followed by an additional 1.5 g of epl / d for a total of 2 years . the homeostasis model assessment , ( an index of insulin resistance ) normalized , same as hepatorenal echo contrast , transaminases , y - gt , and ferritin . liver biopsy , evaluated using brunt s criteria , improved from stage two to zero after 9 months of epl administration ; steatosis decreased , and ballooning , intra - acinus inflammation , and portal tract inflammation disappeared . during the last 8 years , three studies investigated the combination of metformin and epl on patients with diabetes , and the authors observed the same positive effects on aspartate aminotransferase , y - gt , blood lipids , and ultrasonic appearance.1719 according to the latest study , hepatic fibrogenesis slowed down in the epl group versus control ( p=0.03 ) , and steatosis decreased under epl and increased in the control group ( difference between groups p=0.02).19 besides the abovementioned study , ohbayashi20 and ohbayashi et al21 confirmed with further 18 and eight patients with nash that long - term administration of epl continues to improve hepatic function until completion of the study . first , significantly decreased transaminase levels were seen within 4-week treatment and continued over a 12-month therapy.20 of the seven patients of the second study with a biopsy before and after 6-month treatment , one had a significantly improved histological picture , four others had some improvements of steatosis , ballooning , inflammation of the liver lobe , and/or of the periportal area , one had no histological change , and one had a slight aggravation of the picture.21 during the last years , epl was compared to other compounds , discussed to be effective in nafld . the randomized , open controlled studies compared epl to gynostemma pentaphyllum gypenosides , to an extract from red yeast rice ( xuezhikang ; each capsule contains 2.5 mg of lovastatin ) , or to diammonium glycyrrhizinate.2225 compared to the gypenosides of g. pentaphyllum , epl showed better relief of the clinical symptoms , improvement in serum tc and tg , ultrasound picture , and liver function . epl showed obvious effects in 93.3% of the cases.22 the randomized , open controlled studies from fan et al23 and guo et al24 showed no differences between the extracts of the control group and epl in the significant recovery of liver function . however , epl was more effective on serum lipid levels and fatty liver severity than diammonium glycyrrhizinate . especially , the proportion of serious fatty liver changed significantly in the epl group from 23 to nine patients versus 15 to 12 patients in the control group.24 comparison of liver ultrasonography for the number of markedly effective and effective improvements showed significant differences in favor of epl versus diammonium glycyrrhizinate.25 the abovementioned randomized , open controlled studies2225 are added by a double - blind one , which compared 3-month therapy of 1.05 g of epl / d with 710 mg / kg ursodeoxycholic acid ( udca ) per day.26 ten patients of each group were diabetics with nafld , and the remaining were obese individuals ( body mass index > 30 kg / m ) with nafld . patients with cardiovascular disease , liver disease other than hepatosteatosis , complications of diabetes including ketoacidosis , pregnant and lactating women , and patients with a history of alcohol consumption were excluded from the study . forty - five percent of patients on epl expressed satisfaction with therapy in terms of a significant reduction in symptoms ( nausea , malaise , and abdominal distension ) at the end of therapy as opposed to 30% of patients on udca . no noteworthy ultrasonic changes were seen after 4 weeks , but after 12 weeks , 20% of patients on epl and 10% of patients on udca displayed some degree of ultrasonic improvement . in contrast , only alt fell significantly in subjects on udca but not aspartate aminotransferase or ap . the clinical picture of epl and nafld is completed by studies from the beginning of this decade , which investigated a combination therapy of epl with other drugs versus epl therapy alone.27,28 in these two randomized , open controlled studies , the combination of epl with udca in patients with nash and the combination of epl with silybin , glucuronolactone , and vitamin b complex in patients with fatty liver were more effective than epl alone . thirty clinical studies about epl and ald have been published since 1988 , and 17 since 2000 . twelve , partly randomized studies were open controlled , one single - blind , and two double - blind . indications were either not further specified or included different types of ald ( number of studies [ n ] = 10 ) , alcoholic fatty liver ( n=11 ) , acute ( n=1 ) or chronic ( n=6 ) alcoholic hepatitis , chronic active alcoholic hepatitis or liver cirrhosis ( n=1 ) , or patients with alcoholic perivenular fibrosis to incomplete cirrhosis ( n=1 ) were investigated . in most studies , patients were advised to stop alcohol intake during the observation period . with the exception of two clinical trials ( for more details see later ) , dosage of epl was 1.8 g / d per os , and treatment duration varied from 4 weeks to 6 months . in some of the studies ( with higher activity of the inflammatory process in the liver ) , therapy started with 5001,000 mg epl / d iv , followed by 1,800 mg epl / d per os . three studies were done with 5001,000 mg epl / d iv over 4 weeks without peroral epl administration . hu et al29 published a systematic review of six randomized double - blind studies in the treatment of ald and fld.29 according to two independently evaluating persons , the methodological quality of these studies was high enough to be included for evaluation . two studies were in the frame of our database research,30,31 two were older,32,33 and in two studies patients suffered from nafld.12,13 only one study reported on mortality rate , which was 22.6% in the treatment group and 39.2% in the control group.30 the criteria of efficacy of four trials of the systematic review with 146 patients were remission of clinical symptoms , signs , and biochemical variables ( overall clinical efficacy).12,13,32,33 the response rate was 21 of 24 in the treatment group and three of 12 in the control group . a meta - analysis of the four studies showed an efficacy rate of 83.5% in the treatment group and 41.7% in the control group ( p=0.03).29 this suggests that in every 100 patients with ald / fld treated with epl , 41 will benefit and respond to the treatment . additionally , a total of 923 patients in three trials could be included for analysis of histology.13,30,31 according to the meta - analysis , epl did not improve the patients histology ( p=0.07 ) , but histological worsening was prevented ( p=0.02 ) . serious adverse events were not reported in the six studies . the randomized , prospective double - blind trials from panos et al30 and lieber et al31 were both performed according to good clinical practice and differed from the older double - blind studies32,33 by the attempt to increase the efficacy of epl by increasing dosage and duration of treatment . in the study by panos et al,30 53 patients received 6 g epl daily and 51 patients a corresponding placebo over a period of 2 years . a total of 46 patients completed the trial ( epl : n=27 ; placebo : n=19 ) . among the dropouts , 12 patients of the epl group and 20 of the control group ( 31% and 51% of those patients who entered the trial and did not default ) died . the survival curves , constructed by the kaplan meier method , showed a notable trend toward improved survival by epl as compared to placebo , although not being significant ( p=0.086 ) . patients with child pugh b score usually carry a higher death risk than those in classification a. taking into account classification b only , the difference was more pronounced : two of the 12 epl - treated patients died ( 17% of those who entered , 25% of those who did not default ) in comparison with seven of the 16 control patients ( 44% of those who entered , 64% of those who did not default ) ( not significant ) . the mean survival in this subgroup was 83.9 weeks ( epl ) and 56.6 weeks ( placebo ) , respectively . in the study by lieber et al,31 789 heavy drinkers of 20 veterans affairs medical centers / usa with a history of daily average alcohol consumption of 16 drinks per day ( one drink = 14 g of alcohol ) for 19 years and with the evidence of perivenular or septal fibrosis or incomplete cirrhosis were randomly assigned to receive daily either 4.5 g epl or placebo for up to 46 years . epl did not significantly differ from placebo in its effect on the stage of fibrosis as the main outcome as alcohol intake was unexpectedly reduced in both groups to 2.5 drinks per day as a result of a new brief intervention accordingly , there was no more progression of the fibrosis and therefore no way to test whether epl could oppose such a progression except in a subgroup of patients who were still consuming six or more drinks a day and showed beneficial effects against fibrosis . ascites , an important secondary clinical measure of liver disease , was also less frequently observed during follow - up of epl - treated patients . furthermore , improvements in aminotransferases and bilirubin favoring epl were seen at some time points in the subgroups of drinkers who were positive for hepatitis c virus and in nondrinkers.34 in a randomized single - blind clinical study from sas et al,35 86 patients with uncomplicated ald were adequately controlled by diet and abstinent from alcohol and followed a basic treatment scheme , including diet , physical regimen , and abstinence from alcohol . fifty - six of these patients were additionally treated with 1.8 g of epl / d for 6 months , and 30 patients of the control group with 400 mg vitamin e per day . basic treatment plus vitamin e led to less reduction of transaminase levels than basic treatment plus epl . moreover , the mean value of disease activity evaluated by metavir scale was after treatment a1 ( mild activity ) in the epl group and a3 ( severe activity ) in the control group . ultrasound studies revealed significant improvement in the hepatic echo - structure in 49 of the 56 patients of the investigational group . liver biopsy and fibromax test showed that the additional epl administration significantly slowed the progress of hepatic fibrosis to f1 ( portal fibrosis without septa ) versus f3 ( numerous septa without cirrhosis ) in the control group . additionally , the authors reported about significant reduction of glucose , insulin levels , and insulin resistance index . according to the results of a meta - analysis of nine randomized , placebo - controlled double - blind studies on the clinical efficacy of epl in 409 hospitalized patients , who suffered from chronic liver disease ( chronic active hepatitis or fatty degeneration of the liver ) , the overall effect was in favor of epl ( p<0.0001 ) , with the mean difference of the responder rates being 26.6% better for epl.36 fifty - three clinical studies about epl and nafld have been published since 1988 , of which 45 were published since 2000 . twenty - eight , mostly randomized studies , were open controlled and three double - blind . dosage of epl and treatment duration varied as a rule between 1.05 g / d and 1.8 g / d per os and lasted from 4 weeks to > 24 months , in most studies 36 months with 1.8 g of epl / d . therapy started in some studies with 5001,000 mg epl iv over 10 days to 4 weeks , before continuing with oral epl administration ; one study was done with 500 mg epl iv for 30 days only . nafld and its more severe form , nonalcoholic steatohepatitis ( nash ) , were mainly induced by obesity and type ii diabetes mellitus but partly also associated with hyperlipidemia , arterial hypertension , coronary heart disease / atherosclerotic cardiosclerosis , chronic viral hepatitis , cholecystitis / cholelithiasis , hypothyroidism , and pregnancy , or with mixed forms of these diseases / disorders . according to the available studies , analysis of subjective symptoms , clinical findings , biochemical and imaging data , and histology was possible . in the first clinical study from japan , six nonalcoholic and nondiabetic patients with fatty liver due to obesity were successfully treated with a low - calorie diet , physical exercise , and 1.5 g epl / d for 8 weeks and compared to six patients under the same diet , physical exercise , and a nicotinic acid derivative prodrug.9 it was found that computed tomography ( ct ) determined liver fat accumulation significantly improved with epl ( 2718 hu to 548 hu indicating the decrease in intensity of steatosis ) . cairella et al10 from italy evaluated the efficacy of epl in therapy of hepatobiliary dysfunction when administering 1.8 g of epl / d for 3 months . the study group and control comprised 20 cases each with a body mass index of 35.344.89 kg / m . the excretory parameters , total bilirubin , alkaline phosphatase , and y - gt , were normalized in all patients of the epl group but remained increased in 15%35% of the control patients . in the epl group , the ultrasonographic picture clearly improved in 14 and normalized in six cases , while it only slightly changed in three cases of the control group . koga et al11 confirmed the continuous , epl - induced improvement or normalization of the ultrasonic picture in 39 patients with fatty liver due to obesity . bright liver was reduced , and decrement of backward echocardiogram and the obscurity of the intrahepatic venogram were decreased . in 2000 , a randomized double - blind study by li et al12 reported about the positive influence of 1.8 g of epl / d on total cholesterol ( tc ) , triglycerides ( tg ) , and transaminases , supported by significant improvement of the fatty liver ( ct scan ) . experience with epl in patients suffering from maturity- onset diabetes mellitus and fatty liver started with a randomized , placebo - controlled double - blind study.13 thirty patients were instructed to reduce diet to 1,200 kcal / d containing 1 g / kg protein . there were five patients in the placebo and seven patients in the epl group who were treated with 1.01.5 g tolbutamide per day . half of the patients were additionally given 1.8 g of epl / d , the second half a corresponding placebo . during the 6-month treatment phase , liver size hardly changed in the patients of the placebo group , but there was a significant reduction in liver size in the epl group . the histological examination of the liver biopsy yielded corresponding results : marked improvement was observed in four patients treated with epl and only in one patient of the placebo group . although no fatty infiltration was seen in the posttreatment liver biopsy of this patient , he developed the feature of progression to cirrhosis . also in one patient treated with epl , whose fatty infiltration was more pronounced at the second biopsy , morphological features of developing discrete fibrosis in portal tract were observed . altogether , there were seven patients in the epl group and four under placebo including the patient with developing cirrhosis , whose steatosis improved histologically during the study . additionally , y - gt was significantly lowered after 1 month , 3 months , and 6 months of epl therapy , whereas in the control group , the deviations were not statistically significant . since this double - blind trial , seven further clinical studies confirmed the influence of epl on diabetes mellitus - associated fatty liver.1420 yin and kong14 treated 185 patients with standard diet , oral antidiabetics ( not specified ) plus physical exercise , and 125 of these patients were treated additionally with 1.8 g of epl / d for 3 months . alanine aminotransferase ( alt ) , serum tc , and tg , low - density lipoprotein- and high - density lipoprotein - cholesterol had significantly improved or normalized within the epl group , the lipid variables also between the groups . the improvements were seen in 90.2% of the epl cases and in 51% of the controls . this difference was statistically significant ( p<0.05 ) . fasting blood sugar values improved in both groups , but the difference between the groups was not significant . twenty - two patients from the study of poongothai et al15 received , besides a standard diet , antidiabetics depending on the severity of the glycemic level : 22.7% were on metformin therapy , 22.7% on sulfonylurea , 45.5% on combination of both , and 9% on insulin therapy . epl 2.1 g / d per os was additionally given for 6 months . ultrasonographic improvement was seen in 54.5% of patients , 40.9% did not show any change , and one case worsened . hepatic echotexture improved in 80% of subjects with severe diffusion fine echos in hepatic parenchyma , and in 50% of those with slight changes . ohbayashi et al16 treated a female patient with nash with nateglinide as insulin secretagogue for 7 months , followed by an additional 1.5 g of epl / d for a total of 2 years . the homeostasis model assessment , ( an index of insulin resistance ) normalized , same as hepatorenal echo contrast , transaminases , y - gt , and ferritin . liver biopsy , evaluated using brunt s criteria , improved from stage two to zero after 9 months of epl administration ; steatosis decreased , and ballooning , intra - acinus inflammation , and portal tract inflammation disappeared . during the last 8 years , three studies investigated the combination of metformin and epl on patients with diabetes , and the authors observed the same positive effects on aspartate aminotransferase , y - gt , blood lipids , and ultrasonic appearance.1719 according to the latest study , hepatic fibrogenesis slowed down in the epl group versus control ( p=0.03 ) , and steatosis decreased under epl and increased in the control group ( difference between groups p=0.02).19 besides the abovementioned study , ohbayashi20 and ohbayashi et al21 confirmed with further 18 and eight patients with nash that long - term administration of epl continues to improve hepatic function until completion of the study . first , significantly decreased transaminase levels were seen within 4-week treatment and continued over a 12-month therapy.20 of the seven patients of the second study with a biopsy before and after 6-month treatment , one had a significantly improved histological picture , four others had some improvements of steatosis , ballooning , inflammation of the liver lobe , and/or of the periportal area , one had no histological change , and one had a slight aggravation of the picture.21 during the last years , epl was compared to other compounds , discussed to be effective in nafld . the randomized , open controlled studies compared epl to gynostemma pentaphyllum gypenosides , to an extract from red yeast rice ( xuezhikang ; each capsule contains 2.5 mg of lovastatin ) , or to diammonium glycyrrhizinate.2225 compared to the gypenosides of g. pentaphyllum , epl showed better relief of the clinical symptoms , improvement in serum tc and tg , ultrasound picture , and liver function . epl showed obvious effects in 93.3% of the cases.22 the randomized , open controlled studies from fan et al23 and guo et al24 showed no differences between the extracts of the control group and epl in the significant recovery of liver function . however , epl was more effective on serum lipid levels and fatty liver severity than diammonium glycyrrhizinate . especially , the proportion of serious fatty liver changed significantly in the epl group from 23 to nine patients versus 15 to 12 patients in the control group.24 comparison of liver ultrasonography for the number of markedly effective and effective improvements showed significant differences in favor of epl versus diammonium glycyrrhizinate.25 the abovementioned randomized , open controlled studies2225 are added by a double - blind one , which compared 3-month therapy of 1.05 g of epl / d with 710 mg / kg ursodeoxycholic acid ( udca ) per day.26 ten patients of each group were diabetics with nafld , and the remaining were obese individuals ( body mass index > 30 kg / m ) with nafld . patients with cardiovascular disease , liver disease other than hepatosteatosis , complications of diabetes including ketoacidosis , pregnant and lactating women , and patients with a history of alcohol consumption were excluded from the study . forty - five percent of patients on epl expressed satisfaction with therapy in terms of a significant reduction in symptoms ( nausea , malaise , and abdominal distension ) at the end of therapy as opposed to 30% of patients on udca . no noteworthy ultrasonic changes were seen after 4 weeks , but after 12 weeks , 20% of patients on epl and 10% of patients on udca displayed some degree of ultrasonic improvement . in contrast , only alt fell significantly in subjects on udca but not aspartate aminotransferase or ap . the clinical picture of epl and nafld is completed by studies from the beginning of this decade , which investigated a combination therapy of epl with other drugs versus epl therapy alone.27,28 in these two randomized , open controlled studies , the combination of epl with udca in patients with nash and the combination of epl with silybin , glucuronolactone , and vitamin b complex in patients with fatty liver were more effective than epl alone . thirty clinical studies about epl and ald have been published since 1988 , and 17 since 2000 . twelve , partly randomized studies were open controlled , one single - blind , and two double - blind . indications were either not further specified or included different types of ald ( number of studies [ n ] = 10 ) , alcoholic fatty liver ( n=11 ) , acute ( n=1 ) or chronic ( n=6 ) alcoholic hepatitis , chronic active alcoholic hepatitis or liver cirrhosis ( n=1 ) , or patients with alcoholic perivenular fibrosis to incomplete cirrhosis ( n=1 ) were investigated . in most studies , patients were advised to stop alcohol intake during the observation period . with the exception of two clinical trials ( for more details see later ) , dosage of epl was 1.8 g / d per os , and treatment duration varied from 4 weeks to 6 months . in some of the studies ( with higher activity of the inflammatory process in the liver ) , therapy started with 5001,000 mg epl / d iv , followed by 1,800 mg epl / d per os . three studies were done with 5001,000 mg epl / d iv over 4 weeks without peroral epl administration . hu et al29 published a systematic review of six randomized double - blind studies in the treatment of ald and fld.29 according to two independently evaluating persons , the methodological quality of these studies was high enough to be included for evaluation . two studies were in the frame of our database research,30,31 two were older,32,33 and in two studies patients suffered from nafld.12,13 only one study reported on mortality rate , which was 22.6% in the treatment group and 39.2% in the control group.30 the criteria of efficacy of four trials of the systematic review with 146 patients were remission of clinical symptoms , signs , and biochemical variables ( overall clinical efficacy).12,13,32,33 the response rate was 21 of 24 in the treatment group and three of 12 in the control group . a meta - analysis of the four studies showed an efficacy rate of 83.5% in the treatment group and 41.7% in the control group ( p=0.03).29 this suggests that in every 100 patients with ald / fld treated with epl , 41 will benefit and respond to the treatment . additionally , a total of 923 patients in three trials could be included for analysis of histology.13,30,31 according to the meta - analysis , epl did not improve the patients histology ( p=0.07 ) , but histological worsening was prevented ( p=0.02 ) . the randomized , prospective double - blind trials from panos et al30 and lieber et al31 were both performed according to good clinical practice and differed from the older double - blind studies32,33 by the attempt to increase the efficacy of epl by increasing dosage and duration of treatment . in the study by panos et al,30 53 patients received 6 g epl daily and 51 patients a corresponding placebo over a period of 2 years . a total of 46 patients completed the trial ( epl : n=27 ; placebo : n=19 ) . among the dropouts , 12 patients of the epl group and 20 of the control group ( 31% and 51% of those patients who entered the trial and did not default ) died . the survival curves , constructed by the kaplan meier method , showed a notable trend toward improved survival by epl as compared to placebo , although not being significant ( p=0.086 ) . patients with child pugh b score usually carry a higher death risk than those in classification a. taking into account classification b only , the difference was more pronounced : two of the 12 epl - treated patients died ( 17% of those who entered , 25% of those who did not default ) in comparison with seven of the 16 control patients ( 44% of those who entered , 64% of those who did not default ) ( not significant ) . the mean survival in this subgroup was 83.9 weeks ( epl ) and 56.6 weeks ( placebo ) , respectively . in the study by lieber et al,31 789 heavy drinkers of 20 veterans affairs medical centers / usa with a history of daily average alcohol consumption of 16 drinks per day ( one drink = 14 g of alcohol ) for 19 years and with the evidence of perivenular or septal fibrosis or incomplete cirrhosis were randomly assigned to receive daily either 4.5 g epl or placebo for up to 46 years . epl did not significantly differ from placebo in its effect on the stage of fibrosis as the main outcome as alcohol intake was unexpectedly reduced in both groups to 2.5 drinks per day as a result of a new brief intervention accordingly , there was no more progression of the fibrosis and therefore no way to test whether epl could oppose such a progression except in a subgroup of patients who were still consuming six or more drinks a day and showed beneficial effects against fibrosis . ascites , an important secondary clinical measure of liver disease , was also less frequently observed during follow - up of epl - treated patients . furthermore , improvements in aminotransferases and bilirubin favoring epl were seen at some time points in the subgroups of drinkers who were positive for hepatitis c virus and in nondrinkers.34 in a randomized single - blind clinical study from sas et al,35 86 patients with uncomplicated ald were adequately controlled by diet and abstinent from alcohol and followed a basic treatment scheme , including diet , physical regimen , and abstinence from alcohol . fifty - six of these patients were additionally treated with 1.8 g of epl / d for 6 months , and 30 patients of the control group with 400 mg vitamin e per day . basic treatment plus vitamin e led to less reduction of transaminase levels than basic treatment plus epl . moreover , the mean value of disease activity evaluated by metavir scale was after treatment a1 ( mild activity ) in the epl group and a3 ( severe activity ) in the control group . ultrasound studies revealed significant improvement in the hepatic echo - structure in 49 of the 56 patients of the investigational group . liver biopsy and fibromax test showed that the additional epl administration significantly slowed the progress of hepatic fibrosis to f1 ( portal fibrosis without septa ) versus f3 ( numerous septa without cirrhosis ) in the control group . additionally , the authors reported about significant reduction of glucose , insulin levels , and insulin resistance index . according to the results of a meta - analysis of nine randomized , placebo - controlled double - blind studies on the clinical efficacy of epl in 409 hospitalized patients , who suffered from chronic liver disease ( chronic active hepatitis or fatty degeneration of the liver ) , the overall effect was in favor of epl ( p<0.0001 ) , with the mean difference of the responder rates being 26.6% better for epl.36 nafld is the best investigated indication of epl in liver diseases during the last 12 years . not much therapeutic options for nafld are accepted until today besides correction of obesity with hypocaloric diets and physical exercise and controlling hyperglycemia with diet , insulin , or oral hypoglycemic agents . therefore , any therapeutic intervention that targets fat accumulation in the liver and ameliorates hepatic histology would be of great benefit.37 not much has changed therapeutically since this statement was published in 2006 , but epl is one of the drugs under discussion with significant positive effects on nafld ( table 1 ) . because of its membranous , antioxidative , and antifibrotic effects , administration of epl in nafl and nash is pathogenetically justified . an additional important reason for the efficacy of epl in nafld was recently published by ling et al,38 who showed that decreased hepatic pc - to - phosphatidylethanolamine ratio in cellular membrane is a predictor of nafld and survival following partial hepatectomy . epl is able to re - improve this disturbed ratio . as mentioned , besides low - calorie diets and physical loads , therapy of nafld should be directed at treating components of metabolic syndrome , which may be additionally beneficial for the liver . the international diabetes federation consensus worldwide definition of the metabolic syndrome from 2006 is central obesity ( defined as waist circumference with ethnicity - specific values ) and any two of the following criteria : raised tg , reduced high - density lipoprotein cholesterol , raised blood pressure , or raised fasting plasma glucose ( or previously diagnosed type 2 diabetes).39 this definition includes tg and hdl - cholesterol , both criteria , which have been successfully treated by epl , too.40,41 ald is also intensively investigated with epl.5 the investigations from okiyama et al42 exemplarily show that the mode of action of epl in ald is already quite well understood . though the authors found four double - blind and one single - blinded studies for analysis , two are older ones and two were done with significantly higher doses than recommended in the marketed form.30,31 the patients with acute alcoholic hepatitis had a daily intake of 12 capsules with a total of 6 g of epl over 2 years.30 it is questionable whether alcohol - abusing patients with basically well - known low compliance take 12 capsules every day for 2 years . apart from that , it has been shown that the compliance of patients with ald can be increased by a good intervention approach , which leads to alcohol abstinence or at least clear reduction of the alcohol consumption , with corresponding improvements of the hepatic status.43 therefore , the first request is always absolute alcohol abstinence and then to consider drug treatment such as epl , the latter especially to include from the beginning of treatment those cases who have problems to stop drinking . as there was a trend for higher survival of the patients with acute alcoholic hepatitis under epl , especially of those with child pugh b disease severity,30 a further promising double - blind study is recommended with such patients but this time with a lower daily dosage of epl and a shorter duration of treatment , for example , with 1.8 g of epl / d over 1-year treatment . the veterans affairs cooperative study had additional shortcomings besides too high daily epl dose and the long duration of treatment to show epl efficacy.31 planning the study , it was estimated that in the group of long - term heavy drinkers with early stages of fibrosis , the natural progression rate would be 30% over 24 months under conditions of continued drinking . however , the observed natural progression rate based on the placebo group outcome was 20% , with a comparable rate of regression . the slower progression rate was supposed to be related to the reduction in drinking that occurred . therefore , most patients did not meet the protocol entry definition of heavy drinking ( six or more drinks per day ) during the follow - up . even in the subgroup of heavy drinkers , a second drawback was that more than double the rate of patients anticipated in the sample size adjustment withdrew or were lost to follow - up before the 24-month biopsy . on the other hand , most of the analyzed clinical studies on epl in nafld and ald have their shortcomings : too low number of patients , incomplete methodological description including unclear statistical evaluation , varying dosages of epl , data published in secondary papers , or others . therefore , further randomized double - blind clinical studies with higher number of patients and of comparable histology or imaging status are necessary to regard the administration of epl as a standard treatment . until today , only diet and physical exercise in nafld and alcohol abuse in ald are worldwide accepted standard procedures , though even the exact optimum diet and kind and duration of physical exercise are not yet known . to sum it up , most studies from different countries show some advantage of epl versus other investigated drugs or if added to other drugs . this estimation is substantiated by the knowledge that all liver diseases are associated with cellular membrane damages and by the high number of positive pharmacological studies.6 epl accelerates the improvement or normalization of subjective symptoms and pathological findings , such as pain in the right hypochondrium , dyspeptic symptoms , and hepato megaly , in nafld and ald . the findings are supported by imaging procedures , such as ultrasonography , ct , and fibromax ; by biochemical markers of hepatic cytolysis , detoxification , excretion , synthesis , and clearance ; and by liver histology . additional adequately conducted randomized clinical trials on epl versus placebo or other active treatment are recommended to investigate the synergistic effects of epl with other liver drugs .

aimalthough essential phospholipids ( epl ) from soybean are often used in membrane - associated disorders and diseases , their high quality of purification and effects on prevalent liver diseases , especially on fatty liver diseases ( flds ) of different origin , are still widely unknown and a matter of continuous active research . the aim of this article is to review , discuss , and summarize the available results of epl in the treatment of fld.methodsdatabase research was carried out on medline , embase , cochrane library , country - specific journals , and follow - up literature citations for relevant hepatogastroenterological articles published between 1988 and 2014 . we searched for and reviewed only those papers that indicated minimum extraction amount of 72% ( 3-sn - phosphatidyl)choline from soybean as being necessary to treat patients with a considerable amount of 1,2-dilinoleoylphosphatidylcholine as a key component in epl.resultsepl has a well - established mode of action , therapeutic effectiveness , and lack of toxicity , which ensures clinically relevant efficacy - to - safety ratio . it influences membrane- dependent cellular functions and shows anti - inflammatory , antioxidant , antifibrogenic , anti apoptotic , membrane - protective , and lipid - regulating effects . due to its positive effects on membrane composition and functions , it accelerates the improvement or normalization of subjective symptoms ; pathological , clinical , and biochemical findings ; hepatic imaging ; and liver histology . it is justified to administer epl together with other therapeutic measurements in the liver.conclusionpharmacological and clinical results confirm the efficacy of epl in the treatment of fld .

Introduction Materials and methods Results Clinical efficacy of EPL in NAFLD Clinical efficacy of EPL in ALD Discussion Conclusion

some pharmacological agents show promising results , although on the basis of recent clinical trials no firm conclusions can yet be drawn.4 the situation is comparable with ald.3 the nontoxic essential phospholipids ( epl ) have been already widely accepted to be effective in various liver diseases.5,6 however , as a clinical overview of available results specifically in flds of different origin has not yet been published since 1988 , it is the purpose of this article to evaluate the clinical efficacy and safety of epl in these frequent liver diseases . the term epl with its polyenylphosphatidylcholine ( also called polyene phosphatidylcholine [ ppc ] ) molecules indicates a well - defined , highly purified extract of the semen of soybeans ( glycine max ) with standardized contents of 72%96% ( 3-sn - phosphatidyl ) choline . with a share of 1.3% , highly unsaturated pcs with an additional , unsaturated fatty acid in the first position of the pc molecule are rare.7 by administering epl , the amount of hepatic dlpc significantly increased.8 phospholipids are known to form the double layer of cellular and subcellular membranes and precondition their fluidity and biological activity . the efficacy of epl in the therapy of liver diseases is confirmed not only by the ability of dlpc to be incorporated into damaged sections of membranes , which improves hepatic regeneration and replaces endogenous , less unsaturated pc molecules but also by its ability to increase membrane fluidity and functioning . with regard to in vitro and animal investigations , epl influences membrane - dependent cellular functions and shows antioxidant , anti - inflammatory , antifibrotic , apoptosis - modulating , regenerative , membrane - repairing and -protective , cell - signaling and receptor - influencing , and lipid - regulating effects.6 database research was carried out on medline , embase , cochrane library , country - specific journals , and follow - up literature citations for relevant hepatogastroenterological articles published between 1988 and 2014 . in the second step , we reviewed those papers that indicated minimum extraction amount of 72% pc from soybean as being necessary to treat patients with a considerable amount of dlpc as a key component in epl ( for details see later ) , or that were based on the trade name essentiale . according to the available studies , analysis of subjective symptoms , clinical findings , biochemical and imaging data , and histology was possible . in the first clinical study from japan , six nonalcoholic and nondiabetic patients with fatty liver due to obesity were successfully treated with a low - calorie diet , physical exercise , and 1.5 g epl / d for 8 weeks and compared to six patients under the same diet , physical exercise , and a nicotinic acid derivative prodrug.9 it was found that computed tomography ( ct ) determined liver fat accumulation significantly improved with epl ( 2718 hu to 548 hu indicating the decrease in intensity of steatosis ) . cairella et al10 from italy evaluated the efficacy of epl in therapy of hepatobiliary dysfunction when administering 1.8 g of epl / d for 3 months . koga et al11 confirmed the continuous , epl - induced improvement or normalization of the ultrasonic picture in 39 patients with fatty liver due to obesity . in 2000 , a randomized double - blind study by li et al12 reported about the positive influence of 1.8 g of epl / d on total cholesterol ( tc ) , triglycerides ( tg ) , and transaminases , supported by significant improvement of the fatty liver ( ct scan ) . experience with epl in patients suffering from maturity- onset diabetes mellitus and fatty liver started with a randomized , placebo - controlled double - blind study.13 thirty patients were instructed to reduce diet to 1,200 kcal / d containing 1 g / kg protein . additionally , y - gt was significantly lowered after 1 month , 3 months , and 6 months of epl therapy , whereas in the control group , the deviations were not statistically significant . since this double - blind trial , seven further clinical studies confirmed the influence of epl on diabetes mellitus - associated fatty liver.1420 yin and kong14 treated 185 patients with standard diet , oral antidiabetics ( not specified ) plus physical exercise , and 125 of these patients were treated additionally with 1.8 g of epl / d for 3 months . during the last 8 years , three studies investigated the combination of metformin and epl on patients with diabetes , and the authors observed the same positive effects on aspartate aminotransferase , y - gt , blood lipids , and ultrasonic appearance.1719 according to the latest study , hepatic fibrogenesis slowed down in the epl group versus control ( p=0.03 ) , and steatosis decreased under epl and increased in the control group ( difference between groups p=0.02).19 besides the abovementioned study , ohbayashi20 and ohbayashi et al21 confirmed with further 18 and eight patients with nash that long - term administration of epl continues to improve hepatic function until completion of the study . first , significantly decreased transaminase levels were seen within 4-week treatment and continued over a 12-month therapy.20 of the seven patients of the second study with a biopsy before and after 6-month treatment , one had a significantly improved histological picture , four others had some improvements of steatosis , ballooning , inflammation of the liver lobe , and/or of the periportal area , one had no histological change , and one had a slight aggravation of the picture.21 during the last years , epl was compared to other compounds , discussed to be effective in nafld . especially , the proportion of serious fatty liver changed significantly in the epl group from 23 to nine patients versus 15 to 12 patients in the control group.24 comparison of liver ultrasonography for the number of markedly effective and effective improvements showed significant differences in favor of epl versus diammonium glycyrrhizinate.25 the abovementioned randomized , open controlled studies2225 are added by a double - blind one , which compared 3-month therapy of 1.05 g of epl / d with 710 mg / kg ursodeoxycholic acid ( udca ) per day.26 ten patients of each group were diabetics with nafld , and the remaining were obese individuals ( body mass index > 30 kg / m ) with nafld . patients with cardiovascular disease , liver disease other than hepatosteatosis , complications of diabetes including ketoacidosis , pregnant and lactating women , and patients with a history of alcohol consumption were excluded from the study . the clinical picture of epl and nafld is completed by studies from the beginning of this decade , which investigated a combination therapy of epl with other drugs versus epl therapy alone.27,28 in these two randomized , open controlled studies , the combination of epl with udca in patients with nash and the combination of epl with silybin , glucuronolactone , and vitamin b complex in patients with fatty liver were more effective than epl alone . hu et al29 published a systematic review of six randomized double - blind studies in the treatment of ald and fld.29 according to two independently evaluating persons , the methodological quality of these studies was high enough to be included for evaluation . two studies were in the frame of our database research,30,31 two were older,32,33 and in two studies patients suffered from nafld.12,13 only one study reported on mortality rate , which was 22.6% in the treatment group and 39.2% in the control group.30 the criteria of efficacy of four trials of the systematic review with 146 patients were remission of clinical symptoms , signs , and biochemical variables ( overall clinical efficacy).12,13,32,33 the response rate was 21 of 24 in the treatment group and three of 12 in the control group . a meta - analysis of the four studies showed an efficacy rate of 83.5% in the treatment group and 41.7% in the control group ( p=0.03).29 this suggests that in every 100 patients with ald / fld treated with epl , 41 will benefit and respond to the treatment . the randomized , prospective double - blind trials from panos et al30 and lieber et al31 were both performed according to good clinical practice and differed from the older double - blind studies32,33 by the attempt to increase the efficacy of epl by increasing dosage and duration of treatment . ascites , an important secondary clinical measure of liver disease , was also less frequently observed during follow - up of epl - treated patients . furthermore , improvements in aminotransferases and bilirubin favoring epl were seen at some time points in the subgroups of drinkers who were positive for hepatitis c virus and in nondrinkers.34 in a randomized single - blind clinical study from sas et al,35 86 patients with uncomplicated ald were adequately controlled by diet and abstinent from alcohol and followed a basic treatment scheme , including diet , physical regimen , and abstinence from alcohol . according to the results of a meta - analysis of nine randomized , placebo - controlled double - blind studies on the clinical efficacy of epl in 409 hospitalized patients , who suffered from chronic liver disease ( chronic active hepatitis or fatty degeneration of the liver ) , the overall effect was in favor of epl ( p<0.0001 ) , with the mean difference of the responder rates being 26.6% better for epl.36 fifty - three clinical studies about epl and nafld have been published since 1988 , of which 45 were published since 2000 . according to the available studies , analysis of subjective symptoms , clinical findings , biochemical and imaging data , and histology was possible . in the first clinical study from japan , six nonalcoholic and nondiabetic patients with fatty liver due to obesity were successfully treated with a low - calorie diet , physical exercise , and 1.5 g epl / d for 8 weeks and compared to six patients under the same diet , physical exercise , and a nicotinic acid derivative prodrug.9 it was found that computed tomography ( ct ) determined liver fat accumulation significantly improved with epl ( 2718 hu to 548 hu indicating the decrease in intensity of steatosis ) . cairella et al10 from italy evaluated the efficacy of epl in therapy of hepatobiliary dysfunction when administering 1.8 g of epl / d for 3 months . koga et al11 confirmed the continuous , epl - induced improvement or normalization of the ultrasonic picture in 39 patients with fatty liver due to obesity . in 2000 , a randomized double - blind study by li et al12 reported about the positive influence of 1.8 g of epl / d on total cholesterol ( tc ) , triglycerides ( tg ) , and transaminases , supported by significant improvement of the fatty liver ( ct scan ) . additionally , y - gt was significantly lowered after 1 month , 3 months , and 6 months of epl therapy , whereas in the control group , the deviations were not statistically significant . since this double - blind trial , seven further clinical studies confirmed the influence of epl on diabetes mellitus - associated fatty liver.1420 yin and kong14 treated 185 patients with standard diet , oral antidiabetics ( not specified ) plus physical exercise , and 125 of these patients were treated additionally with 1.8 g of epl / d for 3 months . during the last 8 years , three studies investigated the combination of metformin and epl on patients with diabetes , and the authors observed the same positive effects on aspartate aminotransferase , y - gt , blood lipids , and ultrasonic appearance.1719 according to the latest study , hepatic fibrogenesis slowed down in the epl group versus control ( p=0.03 ) , and steatosis decreased under epl and increased in the control group ( difference between groups p=0.02).19 besides the abovementioned study , ohbayashi20 and ohbayashi et al21 confirmed with further 18 and eight patients with nash that long - term administration of epl continues to improve hepatic function until completion of the study . first , significantly decreased transaminase levels were seen within 4-week treatment and continued over a 12-month therapy.20 of the seven patients of the second study with a biopsy before and after 6-month treatment , one had a significantly improved histological picture , four others had some improvements of steatosis , ballooning , inflammation of the liver lobe , and/or of the periportal area , one had no histological change , and one had a slight aggravation of the picture.21 during the last years , epl was compared to other compounds , discussed to be effective in nafld . especially , the proportion of serious fatty liver changed significantly in the epl group from 23 to nine patients versus 15 to 12 patients in the control group.24 comparison of liver ultrasonography for the number of markedly effective and effective improvements showed significant differences in favor of epl versus diammonium glycyrrhizinate.25 the abovementioned randomized , open controlled studies2225 are added by a double - blind one , which compared 3-month therapy of 1.05 g of epl / d with 710 mg / kg ursodeoxycholic acid ( udca ) per day.26 ten patients of each group were diabetics with nafld , and the remaining were obese individuals ( body mass index > 30 kg / m ) with nafld . patients with cardiovascular disease , liver disease other than hepatosteatosis , complications of diabetes including ketoacidosis , pregnant and lactating women , and patients with a history of alcohol consumption were excluded from the study . the clinical picture of epl and nafld is completed by studies from the beginning of this decade , which investigated a combination therapy of epl with other drugs versus epl therapy alone.27,28 in these two randomized , open controlled studies , the combination of epl with udca in patients with nash and the combination of epl with silybin , glucuronolactone , and vitamin b complex in patients with fatty liver were more effective than epl alone . hu et al29 published a systematic review of six randomized double - blind studies in the treatment of ald and fld.29 according to two independently evaluating persons , the methodological quality of these studies was high enough to be included for evaluation . two studies were in the frame of our database research,30,31 two were older,32,33 and in two studies patients suffered from nafld.12,13 only one study reported on mortality rate , which was 22.6% in the treatment group and 39.2% in the control group.30 the criteria of efficacy of four trials of the systematic review with 146 patients were remission of clinical symptoms , signs , and biochemical variables ( overall clinical efficacy).12,13,32,33 the response rate was 21 of 24 in the treatment group and three of 12 in the control group . a meta - analysis of the four studies showed an efficacy rate of 83.5% in the treatment group and 41.7% in the control group ( p=0.03).29 this suggests that in every 100 patients with ald / fld treated with epl , 41 will benefit and respond to the treatment . the randomized , prospective double - blind trials from panos et al30 and lieber et al31 were both performed according to good clinical practice and differed from the older double - blind studies32,33 by the attempt to increase the efficacy of epl by increasing dosage and duration of treatment . furthermore , improvements in aminotransferases and bilirubin favoring epl were seen at some time points in the subgroups of drinkers who were positive for hepatitis c virus and in nondrinkers.34 in a randomized single - blind clinical study from sas et al,35 86 patients with uncomplicated ald were adequately controlled by diet and abstinent from alcohol and followed a basic treatment scheme , including diet , physical regimen , and abstinence from alcohol . according to the results of a meta - analysis of nine randomized , placebo - controlled double - blind studies on the clinical efficacy of epl in 409 hospitalized patients , who suffered from chronic liver disease ( chronic active hepatitis or fatty degeneration of the liver ) , the overall effect was in favor of epl ( p<0.0001 ) , with the mean difference of the responder rates being 26.6% better for epl.36 nafld is the best investigated indication of epl in liver diseases during the last 12 years . therefore , any therapeutic intervention that targets fat accumulation in the liver and ameliorates hepatic histology would be of great benefit.37 not much has changed therapeutically since this statement was published in 2006 , but epl is one of the drugs under discussion with significant positive effects on nafld ( table 1 ) . because of its membranous , antioxidative , and antifibrotic effects , administration of epl in nafl and nash is pathogenetically justified . an additional important reason for the efficacy of epl in nafld was recently published by ling et al,38 who showed that decreased hepatic pc - to - phosphatidylethanolamine ratio in cellular membrane is a predictor of nafld and survival following partial hepatectomy . the international diabetes federation consensus worldwide definition of the metabolic syndrome from 2006 is central obesity ( defined as waist circumference with ethnicity - specific values ) and any two of the following criteria : raised tg , reduced high - density lipoprotein cholesterol , raised blood pressure , or raised fasting plasma glucose ( or previously diagnosed type 2 diabetes).39 this definition includes tg and hdl - cholesterol , both criteria , which have been successfully treated by epl , too.40,41 ald is also intensively investigated with epl.5 the investigations from okiyama et al42 exemplarily show that the mode of action of epl in ald is already quite well understood . though the authors found four double - blind and one single - blinded studies for analysis , two are older ones and two were done with significantly higher doses than recommended in the marketed form.30,31 the patients with acute alcoholic hepatitis had a daily intake of 12 capsules with a total of 6 g of epl over 2 years.30 it is questionable whether alcohol - abusing patients with basically well - known low compliance take 12 capsules every day for 2 years . as there was a trend for higher survival of the patients with acute alcoholic hepatitis under epl , especially of those with child pugh b disease severity,30 a further promising double - blind study is recommended with such patients but this time with a lower daily dosage of epl and a shorter duration of treatment , for example , with 1.8 g of epl / d over 1-year treatment . even in the subgroup of heavy drinkers , a second drawback was that more than double the rate of patients anticipated in the sample size adjustment withdrew or were lost to follow - up before the 24-month biopsy . therefore , further randomized double - blind clinical studies with higher number of patients and of comparable histology or imaging status are necessary to regard the administration of epl as a standard treatment . this estimation is substantiated by the knowledge that all liver diseases are associated with cellular membrane damages and by the high number of positive pharmacological studies.6 epl accelerates the improvement or normalization of subjective symptoms and pathological findings , such as pain in the right hypochondrium , dyspeptic symptoms , and hepato megaly , in nafld and ald .

we retrospectively reviewed our institutional database for liver mris conducted between january 2004 and september 2006 on patients suspected of having liver malignancy based on clinical and prior ct findings . we identified 391 patients with hccs and 55 patients with liver metastases who underwent a double contrast mri that consisted of a sequentially acquired gadolinium - enhanced and ferucarbotran - enhanced liver mri . no institutional review board approval was required for retrospective review of medical records and images . of the 446 potential study patients , only those who met the following criteria were included in the study : ( a ) nodular hcc 2 cm or smaller in size that had been proven on histology or showed typical imaging findings on hepatic angiography , lipiodol ct , and dynamic ct , ( b ) liver metastases 2 cm or smaller in size that had been proven on histology or showed typical imaging findings on three - phase dynamic ct and follow - up ct or mri ( rapid progression of disease ) , ( c ) follow - up contrast - enhanced ct or mri performed for at least six months ( range : 6 - 30 months ) . consequently , the study population included 151 patients ( 114 men and 37 women ; age range : 40 - 74 years ) : 119 patients with 157 hccs and 32 patients with 98 metastases . of 51 hccs ( from 38 patients ) that were confirmed by pathologic analysis of surgical specimens , 45 lesions were grade i ( n = 15 ) or ii ( n = 30 ) according to edmondson 's classification of hcc ( 11 ) , and the remaining six were grade iii . for the 81 patients with 106 hccs who underwent transarterial chemoembolization , the final diagnosis was based on a combined interpretation of findings from all diagnostic procedures by two experienced radiologists in consensus . diagnostic procedures included the following : image - guided biopsy ( n = 12 ) , characteristic imaging findings on three - phase multidetector ct ( mdct ) and mri ( arterial hypervascularization and early washout on dynamic imaging and arterial hypervascularization plus hyperintensity on spio - enhanced mri ) ( n = 106 ) , and persistent dense compact nodular lipiodol uptake of the detected lesions on mdct and mri after tace ( n = 106 ) ( 12 ) , as well as aggravation ( local recurrence or new growing hccs ) on follow - up ct or mri ( n = 19 ) . of all the patients with hccs , four underwent ablation therapy , and all patients with hccs had liver cirrhosis ( n = 116 ) or chronic hepatitis ( n = 3 ) associated with viral hepatitis b. seven of them also had viral hepatitis c. during the inclusion period for this retrospective study , there was no patient with small hcc ( 2.0 cm ) who was without underlying liver parenchymal disease . the diagnosis of chronic hepatitis or cirrhosis was made by means of histology in 50 patients and by a combination of clinical course , blood chemistry tests ( aspartate aminotransferase , alanine aminotransferase , alkaline phosphatase , bilirubin , albumin , and globulin ) , and typical ct and mri findings in the remaining 69 patients . based on the child - pugh classification , 98 patients were classified as child class a , and the remaining 18 patients were classified as class b. liver metastases arose from the following primary tumors : colorectal carcinoma ( n = 25 ) , gastric carcinoma ( n = 5 ) , and pancreatic carcinoma ( n = 2 ) . no patients with liver metastases had cirrhosis , and all had normal hepatic function test results , including prothrombin time and blood chemistry ( serum albumin , total bilirubin level , and choline esterase ) . all mri examinations were performed using a 1.5 t unit ( magnetom symphony ; siemens , enlargen , germany ) with a combination of a phased array body coil for signal reception . baseline mr images included a respiratory - triggered t2w - tse sequence and a breath - hold t2w - gre sequence . respiratory - triggered t2w - tse imaging was obtained using the following parameters : tr / te of 3300 - 4200/76 , echo train length of 13 , 150 flip angle , matrix of 202 384 , signal averages of two , and average scan time 3 minutes . breath - hold t2w - gre imaging was obtained using the following parameters : tr / te of 180/12 , 30 flip angle , matrix of 144 256 , signal average of one , two sequential acquisitions , 20 slices , and average scan time 35 seconds ( two stacks with 10 slices each in a 15 - 17-second breath - hold ) . a relatively long te ( 12 ms ) and lower flip angle ( 30 ) were used for increasing the sensitivity to the field inhomogeneity induced by spio administration and for decreasing the t1 contrast , respectively ( 7 , 13 ) . for all sequences , a 6 - 7 mm slice thickness was used with a 10% intersection gap and a field of view of 35 - 40 cm , depending on the size of the liver . in the first session , dynamic mris ( volumetric interpolated breath - hold examination , vibe : siemens , erlangen , germany ) with msense were performed using the following parameters : tr / te of 4.3/2.0 , flip angle of 12 , bandwidth of 450 hz / px , matrix of 256 ( read)135 ( phase)40 - 46 ( partition ) , effective slice thickness of 3.5 - 4 mm , and field of view of 32 - 35 cm . the determination of scan delay for the image acquisition timing was achieved using the test bolus technique , in which 1 ml of contrast media was injected , followed by a 20 ml saline flush , and the vessel of interest ( the abdominal aorta ) was then scanned approximately once per second . the mean delay time to peak aortic enhancement was 21 seconds ( range : 17.0 - 23.0 sec ) . thus , for a sequential mode of k - space acquisition ( center lines of k - space are acquired during the middle of the acquisition time ) , the mean delay times ( time interval between bolus administration and the start of image acquisition ) for the early arterial , late arterial , portal , and equilibrium phase were 20 , 30 , 60 , and 180 seconds , respectively . gadopentetate dimeglumine ( magnevist , bayer healthcare ) was injected at a dosage of 0.1 mmol / kg body weight at a rate of 2 ml / sec . the contrast was injected into the antecubital vein using an automated injector ( spectris mr ; medrad europe , maastricht , the netherlands ) , and a 20 ml saline flush followed the contrast injection . in the second session , ferucarbotran ( shu-555-a ; resovist , bayer healthcare)-enhanced imaging was performed immediately after completion of the dynamic mr examination . ferucarbotran - enhanced imaging was comprised of the respiratory - triggered t2-weighted tse sequence and the breath - hold t2-weighted gre sequence for the baseline mri . patients up to 60 kg body weight received 0.9 ml of ferucarbotran , and patients above 60 kg body weight received 1.4 ml of ferucarbotran ( 8 - 12 mol of iron per kilogram of body weight ) . ferucarbotran was rapidly injected intravenously through a 5 m filter and was followed by a 20 ml saline flush ; imaging then commenced approximately 10 minutes after the intravenous injection of contrast agent . two gastrointestinal radiologists ( one on - site reviewer and one off - site reviewer ) , who had at least six years experience interpreting liver imaging in their daily clinical practice , reviewed the images independently and separately . all images were reviewed on a 20002000 picture archiving and communication systems ( pacs ; marotech , seoul , korea ) monitor . the radiologists were aware of the overall goal of the study before the reading session , and they knew that the patients were at risk for hcc or metastasis , but were unaware of the presence or location of any liver lesions or of the results of other imaging studies . they independently reviewed three sets of mr images in two reading sessions : ( a ) unenhanced t1- and t2-weighted imaging , gadolinium - enhanced dynamic imaging , and ferucarbotran - enhanced t2w - gre imaging ( the t2w - gre set ) , ( b ) unenhanced t1- and t2-weighted imaging , gadolinium - enhanced dynamic imaging , and ferucarbotran - enhanced t2w - tse imaging ( the t2w - tse set ) , and ( c ) a combination of the t2w - gre set and t2w - tse set ( the combined set ) . in the first session , either the t2w - gre set or the t2w - tse set was randomly presented with no specific patient order . in the second session , the remaining t2w - gre set or t2w - tse set was randomly presented , and the t2w - gre or t2w - tse reviewed in the first reading recession was added for a combined review . to minimize any learning bias , there was at least a two - week interval between the two reading sessions . the criteria for diagnosing hcc or metastasis on the ferucarbotran - enhanced mri were defined as a focal , discrete , nodular high signal intensity area relative to adjacent liver parenchyma ( lower than the signal intensity of csf or gallbladder on a t2w - tse sequence to exclude the cyst ) on both t2w - gre and t2w - tse images . on gadolinium - enhanced mri , early enhancement with rapid washout ( typical of hcc ) and irregular peripheral enhancement with a peripheral washout on delayed phase images ( typical of metastases ) were used as the criteria for diagnosing malignancy . for each eligible lesion , one of four confidence levels was assigned to each decision as follows : " 1 " as ' probably not present ' ; " 2 " as ' possibly present ' ; " 3 " as ' probably present ' ; and " 4 " as ' definitely present ' . lesions that showed only arterial hypervascularization were regarded as category 2 if they showed no delayed capsular enhancement , mosaic pattern , or moderate hyperintensity on t2-weighted imaging ( category 3 ) . during gadolinium - enhanced mri , 115 of the total 157 hccs showed early enhancement with rapid washout , and the remaining 42 appeared as hyperintense nodules during arterial phase imaging and were occult during portal and equilibrium phase imaging . to achieve an accurate correlation between the findings of the scored lesions and the findings of the reference standard , each observer made markings over all lesions with arrows on the pacs monitor . after the two observers conducted the two review sessions , the study coordinator , who was not involved in image interpretation , compared the scoring results of each of the observers to a gold standard , and then a possible explanation was formulated for any false - positive or false - negative findings . based on the reviews submitted by the two observers , an alternative - free response receiver operating characteristic ( roc ) curve analysis was performed on a lesion - by - lesion basis ( 14 ) . for each imaging set ( t2w - gre images , t2w - tse images , and combined ) , an alternative - free response roc curve was fitted to each observer 's confidence rating data using a maximum likelihood estimation program ( rockit 0.9b ; courtesy of metz ce , university of chicago , il , 1998 ) ( 15 ) . the area under the alternative - free response roc curve ( az ) was calculated to determine the diagnostic accuracy of each image , each observer , and lesion type . the differences between the imaging sets with regard to the area under the alternative - free response roc curves were compared using a two - tailed student 's t test for paired data . the sensitivities for each image and for each observer , as well as according to the lesion type , were then calculated . the sensitivity was defined as the number of true positive diagnoses , using a confidence level of 3 or 4 . the sensitivities and positive predictive values for each image set we also calculated the 95% confidence interval ( ci ) to determine a range of plausible sensitivity differences . in order to assess interobserver agreement for evaluating the two images and the combined approach , we calculated the kappa statistic for multiple observers ( 16 ) . kappa values of less than 0.20 indicated positive , but poor , agreement , those from 0.21 - 0.40 indicated fair agreement , those from 0.41 - 0.60 indicated moderate agreement , those from 0.61 - 0.80 indicated good agreement , and those greater than 0.81 indicated excellent agreement . we retrospectively reviewed our institutional database for liver mris conducted between january 2004 and september 2006 on patients suspected of having liver malignancy based on clinical and prior ct findings . we identified 391 patients with hccs and 55 patients with liver metastases who underwent a double contrast mri that consisted of a sequentially acquired gadolinium - enhanced and ferucarbotran - enhanced liver mri . no institutional review board approval was required for retrospective review of medical records and images . of the 446 potential study patients , only those who met the following criteria were included in the study : ( a ) nodular hcc 2 cm or smaller in size that had been proven on histology or showed typical imaging findings on hepatic angiography , lipiodol ct , and dynamic ct , ( b ) liver metastases 2 cm or smaller in size that had been proven on histology or showed typical imaging findings on three - phase dynamic ct and follow - up ct or mri ( rapid progression of disease ) , ( c ) follow - up contrast - enhanced ct or mri performed for at least six months ( range : 6 - 30 months ) . consequently , the study population included 151 patients ( 114 men and 37 women ; age range : 40 - 74 years ) : 119 patients with 157 hccs and 32 patients with 98 metastases . of 51 hccs ( from 38 patients ) that were confirmed by pathologic analysis of surgical specimens , 45 lesions were grade i ( n = 15 ) or ii ( n = 30 ) according to edmondson 's classification of hcc ( 11 ) , and the remaining six were grade iii . for the 81 patients with 106 hccs who underwent transarterial chemoembolization , the final diagnosis was based on a combined interpretation of findings from all diagnostic procedures by two experienced radiologists in consensus . diagnostic procedures included the following : image - guided biopsy ( n = 12 ) , characteristic imaging findings on three - phase multidetector ct ( mdct ) and mri ( arterial hypervascularization and early washout on dynamic imaging and arterial hypervascularization plus hyperintensity on spio - enhanced mri ) ( n = 106 ) , and persistent dense compact nodular lipiodol uptake of the detected lesions on mdct and mri after tace ( n = 106 ) ( 12 ) , as well as aggravation ( local recurrence or new growing hccs ) on follow - up ct or mri ( n = 19 ) . of all the patients with hccs , four underwent ablation therapy , and all patients with hccs had liver cirrhosis ( n = 116 ) or chronic hepatitis ( n = 3 ) associated with viral hepatitis b. seven of them also had viral hepatitis c. during the inclusion period for this retrospective study , there was no patient with small hcc ( 2.0 cm ) who was without underlying liver parenchymal disease . the diagnosis of chronic hepatitis or cirrhosis was made by means of histology in 50 patients and by a combination of clinical course , blood chemistry tests ( aspartate aminotransferase , alanine aminotransferase , alkaline phosphatase , bilirubin , albumin , and globulin ) , and typical ct and mri findings in the remaining 69 patients . based on the child - pugh classification , 98 patients were classified as child class a , and the remaining 18 patients were classified as class b. liver metastases arose from the following primary tumors : colorectal carcinoma ( n = 25 ) , gastric carcinoma ( n = 5 ) , and pancreatic carcinoma ( n = 2 ) . no patients with liver metastases had cirrhosis , and all had normal hepatic function test results , including prothrombin time and blood chemistry ( serum albumin , total bilirubin level , and choline esterase ) . all mri examinations were performed using a 1.5 t unit ( magnetom symphony ; siemens , enlargen , germany ) with a combination of a phased array body coil for signal reception . baseline mr images included a respiratory - triggered t2w - tse sequence and a breath - hold t2w - gre sequence . respiratory - triggered t2w - tse imaging was obtained using the following parameters : tr / te of 3300 - 4200/76 , echo train length of 13 , 150 flip angle , matrix of 202 384 , signal averages of two , and average scan time 3 minutes . breath - hold t2w - gre imaging was obtained using the following parameters : tr / te of 180/12 , 30 flip angle , matrix of 144 256 , signal average of one , two sequential acquisitions , 20 slices , and average scan time 35 seconds ( two stacks with 10 slices each in a 15 - 17-second breath - hold ) . a relatively long te ( 12 ms ) and lower flip angle ( 30 ) were used for increasing the sensitivity to the field inhomogeneity induced by spio administration and for decreasing the t1 contrast , respectively ( 7 , 13 ) . for all sequences , a 6 - 7 mm slice thickness was used with a 10% intersection gap and a field of view of 35 - 40 cm , depending on the size of the liver . in the first session , dynamic mris ( volumetric interpolated breath - hold examination , vibe : siemens , erlangen , germany ) with msense were performed using the following parameters : tr / te of 4.3/2.0 , flip angle of 12 , bandwidth of 450 hz / px , matrix of 256 ( read)135 ( phase)40 - 46 ( partition ) , effective slice thickness of 3.5 - 4 mm , and field of view of 32 - 35 cm . the determination of scan delay for the image acquisition timing was achieved using the test bolus technique , in which 1 ml of contrast media was injected , followed by a 20 ml saline flush , and the vessel of interest ( the abdominal aorta ) was then scanned approximately once per second . the mean delay time to peak aortic enhancement was 21 seconds ( range : 17.0 - 23.0 sec ) . thus , for a sequential mode of k - space acquisition ( center lines of k - space are acquired during the middle of the acquisition time ) , the mean delay times ( time interval between bolus administration and the start of image acquisition ) for the early arterial , late arterial , portal , and equilibrium phase were 20 , 30 , 60 , and 180 seconds , respectively . gadopentetate dimeglumine ( magnevist , bayer healthcare ) was injected at a dosage of 0.1 mmol / kg body weight at a rate of 2 ml / sec . the contrast was injected into the antecubital vein using an automated injector ( spectris mr ; medrad europe , maastricht , the netherlands ) , and a 20 ml saline flush followed the contrast injection . in the second session , ferucarbotran ( shu-555-a ; resovist , bayer healthcare)-enhanced imaging was performed immediately after completion of the dynamic mr examination . ferucarbotran - enhanced imaging was comprised of the respiratory - triggered t2-weighted tse sequence and the breath - hold t2-weighted gre sequence for the baseline mri . patients up to 60 kg body weight received 0.9 ml of ferucarbotran , and patients above 60 kg body weight received 1.4 ml of ferucarbotran ( 8 - 12 mol of iron per kilogram of body weight ) . ferucarbotran was rapidly injected intravenously through a 5 m filter and was followed by a 20 ml saline flush ; imaging then commenced approximately 10 minutes after the intravenous injection of contrast agent . two gastrointestinal radiologists ( one on - site reviewer and one off - site reviewer ) , who had at least six years experience interpreting liver imaging in their daily clinical practice , reviewed the images independently and separately . all images were reviewed on a 20002000 picture archiving and communication systems ( pacs ; marotech , seoul , korea ) monitor . the radiologists were aware of the overall goal of the study before the reading session , and they knew that the patients were at risk for hcc or metastasis , but were unaware of the presence or location of any liver lesions or of the results of other imaging studies . they independently reviewed three sets of mr images in two reading sessions : ( a ) unenhanced t1- and t2-weighted imaging , gadolinium - enhanced dynamic imaging , and ferucarbotran - enhanced t2w - gre imaging ( the t2w - gre set ) , ( b ) unenhanced t1- and t2-weighted imaging , gadolinium - enhanced dynamic imaging , and ferucarbotran - enhanced t2w - tse imaging ( the t2w - tse set ) , and ( c ) a combination of the t2w - gre set and t2w - tse set ( the combined set ) . in the first session , either the t2w - gre set or the t2w - tse set was randomly presented with no specific patient order . in the second session , the remaining t2w - gre set or t2w - tse set was randomly presented , and the t2w - gre or t2w - tse reviewed in the first reading recession was added for a combined review . to minimize any learning bias , there was at least a two - week interval between the two reading sessions . the criteria for diagnosing hcc or metastasis on the ferucarbotran - enhanced mri were defined as a focal , discrete , nodular high signal intensity area relative to adjacent liver parenchyma ( lower than the signal intensity of csf or gallbladder on a t2w - tse sequence to exclude the cyst ) on both t2w - gre and t2w - tse images . on gadolinium - enhanced mri , early enhancement with rapid washout ( typical of hcc ) and irregular peripheral enhancement with a peripheral washout on delayed phase images ( typical of metastases ) were used as the criteria for diagnosing malignancy . for each eligible lesion , one of four confidence levels was assigned to each decision as follows : " 1 " as ' probably not present ' ; " 2 " as ' possibly present ' ; " 3 " as ' probably present ' ; and " 4 " as ' definitely present ' . lesions that showed only arterial hypervascularization were regarded as category 2 if they showed no delayed capsular enhancement , mosaic pattern , or moderate hyperintensity on t2-weighted imaging ( category 3 ) . during gadolinium - enhanced mri , 115 of the total 157 hccs showed early enhancement with rapid washout , and the remaining 42 appeared as hyperintense nodules during arterial phase imaging and were occult during portal and equilibrium phase imaging . to achieve an accurate correlation between the findings of the scored lesions and the findings of the reference standard , each observer made markings over all lesions with arrows on the pacs monitor . after the two observers conducted the two review sessions , the study coordinator , who was not involved in image interpretation , compared the scoring results of each of the observers to a gold standard , and then a possible explanation was formulated for any false - positive or false - negative findings . based on the reviews submitted by the two observers , an alternative - free response receiver operating characteristic ( roc ) curve analysis was performed on a lesion - by - lesion basis ( 14 ) . for each imaging set ( t2w - gre images , t2w - tse images , and combined ) , an alternative - free response roc curve was fitted to each observer 's confidence rating data using a maximum likelihood estimation program ( rockit 0.9b ; courtesy of metz ce , university of chicago , il , 1998 ) ( 15 ) . the area under the alternative - free response roc curve ( az ) was calculated to determine the diagnostic accuracy of each image , each observer , and lesion type . the differences between the imaging sets with regard to the area under the alternative - free response roc curves were compared using a two - tailed student 's t test for paired data . the sensitivities for each image and for each observer , as well as according to the lesion type , were then calculated . the sensitivity was defined as the number of true positive diagnoses , using a confidence level of 3 or 4 . the sensitivities and positive predictive values for each image set we also calculated the 95% confidence interval ( ci ) to determine a range of plausible sensitivity differences . in order to assess interobserver agreement for evaluating the two images and the combined approach , we calculated the kappa statistic for multiple observers ( 16 ) . kappa values of less than 0.20 indicated positive , but poor , agreement , those from 0.21 - 0.40 indicated fair agreement , those from 0.41 - 0.60 indicated moderate agreement , those from 0.61 - 0.80 indicated good agreement , and those greater than 0.81 indicated excellent agreement . for all 255 lesions , including the 157 hccs and 98 metastases , there was a trend toward increased diagnostic accuracy ( az values ) for the combined set ( mean , 0.966 ) when compared with each set alone ( mean , 0.892 for t2w - gre set ; 0.910 for t2w - tse set ) , to a statistically significant degree ( p = 0.004 [ observer 1 ] , 0.003 [ observer 2 ] for the t2w - gre set ; p = 0.033 [ observer 1 ] , 0.025 [ observer 2 ] for the t2w - tse set ) ( table 2 ) . furthermore , considering the sensitivity for all lesions , the combined set ( mean , 94.7% ) was significantly more sensitive than each set alone ( mean , 86.1% for t2w - gre set ; 87.7% for t2w - tse set , p = 0.0001 ) ( table 3 ) . for detection of hccs , the az value of the combined set was significantly higher than that of each set alone , for both observers ( p = 0.016 [ observer 1 ] , 0.013 [ observer 2 ] for the t2w - gre set ; p = 0.042 for the t2w - tse set ) ( table 2 ) . although the az values of the t2w - gre set were slightly better than those of the t2w - tse set for both observers ( fig . 1 ) , no significant difference was noted for either observer ( p = 0.635 for observer 1 ; p = 0.801 for observer 2 ) . among the 157 hccs , the t2w - tse set allowed for the depiction of 127 lesions ( sensitivity , 80.9% ; 95% ci : 73.9% , 86.7% ) by observer 1 and 129 lesions ( sensitivity , 82.2% ; 95% ci : 75.35% , 87.8% ) by observer 2 ; the t2w - gre set allowed the depiction of 135 lesions ( sensitivity , 86.0% ; 95% ci : 79.6% , 91.0% ) by observer 1 and 137 lesions ( sensitivity , 87.3% ; 95% ci : 81.0% , 92.0% ) by observer 2 . there was a significant difference in sensitivities between the two image sets for both observers ( p = 0.01 ) . the combined approach allowed for the depiction of 145 lesions ( sensitivity , 92.4% ; 95% ci : 87.05% , 96.0% ) by observer 1 and 147 lesions ( sensitivity , 93.6% ; 95% ci : 88.6% , 96.9% ) by observer 2 ( mean , 93.0% ) , which was significantly better than the values obtained using each set alone ( mean , 81.6% for t2w - tse set , 86.7% for t2w - gre set ) ( p = 0.0001 for t2w - tse set , p = 0.002 for t2w - gre set ) . there were nine and eight hccs ( size range : 0.6 - 1.2 cm , mean size : 0.9 cm ) for each observer , respectively , which were not detected on the t2w - gre set , but which were detected on the t2w - tse set ( fig . 2 ) . there were 17 hccs ( size range : 0.6 - 1.9 cm , mean size : 1.2 cm ) for both observers that were not detected on the t2w - tse set , but which were detected on the t2w - gre set . of the 51 hccs with surgical confirmation , four well - differentiated hccs were not detected by either observer on the t2w - tse set , but were detected on the t2w - gre set by both observers . for the remaining lesions , the two image sets showed similar results . for the detection of liver metastases , the az values of the combined set and the t2w - tse set were better than those of the t2w - gre set for both observers , although the difference was not statistically significant ( p = 0.134 for observer 1 ; p = 0.162 , 0.772 for observer 2 ) . the sensitivity of the combined set was the same as that of the t2w - tse set ( 98.0% ; 95% ci : 92.8% , 99.7% , for observer 1 ; 96.9% ; 95% ci : 91.3% , 99.3% , for observer 2 ; mean 97.5% ) , which was significantly better than that of the t2w - gre set for both observers ( 84.7% ; 95% ci : 76.0% , 91.2% , for observer 1 ; 85.7% ; 95% ci : 77.2% , 92.0% , for observer 2 ; mean 85.2% ) ( p = 0.0001 ) ( fig . 3 ) . there were 11 metastases ( 0.4 - 0.8 cm ) that were not detected by either observer on the t2w - gre set , but which were clearly revealed on the t2w - tse set ( fig . 4 ) . however , there was no lesion detected on the t2w - gre set that could not be verified on the t2w - tse set . each image set showed a similar positive predictive value for each observer ( table 3 ) . both observers had six false - positive findings on the t2w - gre set , four false - positive findings on the t2w - tse set , and two false - positive findings on the combined set . all false - positive findings were attributed to the end - on of vessels or fibrosis in the case of cirrhotic patients . the kappa values for the two observers were 0.746 for the t2w - gre set , 0.762 for the t2w - tse set , and 0.685 for the combined set , indicating good inter - observer agreement with regard to the presence of lesions . t2w - gre and t2w - tse are techniques mainly used with spio - enhancement ( 7 - 10 ) . t2w - gre in spio enhancement provides the highest lesion to liver contrast because of its strong magnetic susceptibility resulting from local field inhomogeneity ( 7 , 8) , but it suffers from low signal - to - noise ratio ( snr ) and has poor pre - contrast image quality . furthermore , t2w - gre might be limited in advanced cirrhotic livers because the decrease in signal intensity of cirrhotic livers on t2w - gre sequences is less pronounced than on t2w - tse sequences ( 17 ) . meanwhile , the respiratory - triggered t2w - tse sequence offers higher spatial resolution because of its large image matrices , less blooming effect because of the robustness of the t2 effect , and preferable t2-weighted image contrast ; small rounded areas of hyperintensity caused by hepatic cysts or hepatic vessels are efficiently differentiated from small malignant tumors ( 18 ) . however , tse is less sensitive to spio , probably because of increased magnetization transfer saturation and decreased susceptibility effects due to the use of multiple 180 refocusing pulses ( 19 ) . furthermore , image degradation from motion artifact could be severe on tse . in the present study , we hypothesized that combining spio - enhanced t2w - gre and t2w - tse sequences had additive value compared to each sequence alone for the detection of small liver malignancies , including hccs and metastases , due to these theoretical relative advantages and disadvantages of t2w - gre and t2w - tse sequences . our study showed that there was a trend toward increased az values for the combined approach compared to each image set alone . regarding the sensitivities for the detection of hccs , the combined approach was significantly better than each image set alone ( p < 0.05 ) , and the t2w - gre set was better than the t2w - tse set for both observers ( p < 0.05 ) . the higher detectability of t2w - gre for hcc could be explained by its higher inherent lesion to liver contrast and possible inclusion of well differentiated hccs in study cases ; most lesions included in this study were 2 cm or smaller ( 17 , 20 ) . ( 17 , 20 ) showed that relative post - spio - enhanced snr changes in the liver parenchyma between cirrhotic and non - cirrhotic patients were remarkable on the t2w - gre sequence , but not on the t2w - tse sequence . they claimed that small intracellular spio clusters caused by decreased kupffer cell function in cirrhosis caused little snr drop in the liver parenchyma on the t2w - gre sequence compared to the t2w - tse sequence , because snr loss on the magnetic susceptibility - insensitive t2w - tse sequence is related to the greater free water interaction made possible by the relatively larger surface area of the small spio clusters ( 20 ) . however , given that kupffer cells density in well - differentiated hccs would be maintained , but kupffer cells function would be reduced compared to the surrounding liver , small intracellular spio clusters in well - differentiated hccs also produce little snr decrease on the t2w - gre sequence , which could result in better lesion conspicuity than on the t2w - tse sequence ( 21 ) . of the 51 hccs in our study with surgical confirmation , four lesions were not detected by either observer on the t2w - tse set , but were detected on the t2w - gre set by both observers . all four lesions proved to be well - differentiated hccs . with regard to the detection of metastases that do not contain kupffer cells , the sensitivity of the combined approach was the same as that of the t2w - tse set , which was significantly better than that of the t2w - gre set for both observers . despite the higher inherent lesion to liver contrast on the t2w - gre , the better detection capability of the t2w - tse for metastases could be explained by signal loss caused by a magnetization transfer effect , with the tse sequence being lower in metastases than in hcc because tumor necrosis and abundant interstitial space in metastases generally contain more abundant free water compared with hcc ( 22 , 23 ) . there were 11 metastases ( 0.4 - 0.8 cm ) that were not detected by either observer on the t2w - gre set , but which were clearly revealed on the t2w - tse set . when we retrospectively reviewed these lesions on the t2w - gre image , all were depicted as tiny high signal intensities with irregular margins , and some small lesions were abutted on intrahepatic venous structures . these lesions were difficult to differentiate from the end - on of the vessel because vessels are depicted with bright hyperintensity , as is true malignancy on the t2w - gre image ( 8 , 10 ) . we assumed that this was also attributable to an exaggerated t2-shortening effect on the t2w - gre sequence , which has a relatively long te ( 12 ms ) , that may have partially obscured the margins of the smaller liver lesions , especially those located at the dome abutting the lung or the left hepatic lobe abutting the gastric lumen ( 13 ) . moreover , the relatively lower spatial resolution of the t2w - gre sequence ( 144 256 ) compared to the t2w - tse sequence ( 202 384 ) and the inherent blurring of high signal intensity structures on the t2w - gre sequence may also be behind this phenomenon . in addition to the shortcomings of the t2w - gre sequence in advanced cirrhotic livers , these could also explain the nine and eight hccs detected only on the t2w - tse set , but not on the t2w - gre set , by observers 1 and 2 , respectively . first , not all lesions were surgically confirmed , which could have resulted in overestimation of the sensitivity of each image by reducing false - negative lesions . however , our study was not intended to determine the absolute diagnostic accuracy and sensitivity of the mr sequence , but to determine the relative diagnostic efficacy of combined mr sequences compared to single sequences for the detection of liver malignancy . furthermore , the relatively thick slice thickness ( 0.6 or 0.7 cm ) of ferucarbotran - enhanced mr sequences is inherently limiting in the detection of small hepatic tumors . a second limitation is that a quantitative analysis to determine the conspicuity of hcc according to the histologic differentiation or the number of kupffer cells was not made . in addition , quantitative analysis was not done regarding the tumor - to - liver contrast . however , optimal contrast - to - noise ratio does not necessarily translate into better detecting capability ( 24 ) . in this regard , the 12 ms te for the t2w - gre sequence used in our protocol might be slightly long to obscure the margin of small hyperintense nodules ( 13 ) . hccs only detected on the t2w - tse set ( mean size , 0.9 cm ) tended to be smaller than those only detected on the t2w - gre set ( mean size , 1.2 cm ) . additionally , since the image quality of the liver was higher with the respiratory - triggered tse sequence owing to its large image matrices , exact direct comparison between the two sequences in this study could not be made . in our study , the diagnostic accuracy of the combined set for metastases was slightly better than that for each image set for observer 2 . although diagnostic confidence is a subjective parameter that can not be directly translated into diagnostic performance , such as sensitivity , an increase in physician confidence might contribute significantly to patient management . therefore , considering the higher inherent lesion to liver contrast of t2w - gre in spio enhancement , we believe that the t2w - gre sequence has the potential to provide an additional role for t2w - tse in the detection of metastases . third , the results of this study do not necessarily reflect the pure diagnostic capabilities of spio - enhanced images for the detection of hcc , because we included an analysis of all sequences combined . lastly , differences in the mr imager , mr parameters , and methods for image review used may result in different results . therefore , the results of this study might not be able to be extrapolated to other facilities with different mr imagers or sequences . additionally , we did not evaluate other commercially used sequences for spio - enhanced mri in this study ( 19 ) . in conclusion , a combination of ferucarbotran - enhanced t2w - gre and t2w - tse images in combining gadolinium and ferucarbotran - enhanced mri had better diagnostic capability compared to each sequence alone for the detection of hcc in cirrhotic patients . no difference in sensitivity for the detection of metastases was noted between the t2w - tse set and the combined t2w - gre / t2w - tse set , suggesting that the respiratory triggered t2w - tse sequence in ferucarbotran - enhanced mri is preferred for the detection of small liver metastases in non - cirrhotic patients .

objectiveto determine if a combination of ferucarbotran - enhanced t2*weighted - gradient echo ( t2*w - gre ) and t2-weighted turbo spin echo ( t2w - tse ) images in gadolinium- and ferucarbotran - enhanced mri has additive efficacy compared to each image alone for detecting small ( 2.0 cm ) hepatocellular carcinoma ( hcc ) lesions in a group of cirrhotic patients and metastases in a group of non - cirrhotic patients.materials and methodstwo readers retrospectively analyzed gadolinium- and ferucarbotran - enhanced t2*w - gre , t2w - tse , and combined t2*w - gre / t2w - tse images of 119 patients with 157 hccs and 32 patients with 98 metastases . the diagnostic accuracy and sensitivity for each image set and the combined set were evaluated using the alternative - free response receiver operating characteristic method.resultsthe mean area under the curve value of the combined set ( 0.966 ) tended to be better than that for each individual image set ( t2w - tse [ 0.910 ] , t2*w - gre [ 0.892 ] ) . sensitivities in the combined set were higher than those in each individual image set for detecting hcc ( mean , 93.0% versus 81.6% and 86.7% , respectively , p < 0.01 ) . sensitivities in the combined set and the t2w - tse set were the same for detecting metastases , and both were higher than the sensitivity seen in the t2*w - gre set ( mean , 97.5% versus 85.2% , p < 0.01).conclusioncombining ferucarbotran - enhanced t2*w - gre and t2w - tse has additive efficacy for detecting hcc in cirrhotic patients , but t2w - tse is preferred for detecting metastases in non - cirrhotic patients .

MATERIALS AND METHODS Patients MR Examination Imaging Analysis Statistical Analysis RESULTS DISCUSSION

of the 446 potential study patients , only those who met the following criteria were included in the study : ( a ) nodular hcc 2 cm or smaller in size that had been proven on histology or showed typical imaging findings on hepatic angiography , lipiodol ct , and dynamic ct , ( b ) liver metastases 2 cm or smaller in size that had been proven on histology or showed typical imaging findings on three - phase dynamic ct and follow - up ct or mri ( rapid progression of disease ) , ( c ) follow - up contrast - enhanced ct or mri performed for at least six months ( range : 6 - 30 months ) . consequently , the study population included 151 patients ( 114 men and 37 women ; age range : 40 - 74 years ) : 119 patients with 157 hccs and 32 patients with 98 metastases . of all the patients with hccs , four underwent ablation therapy , and all patients with hccs had liver cirrhosis ( n = 116 ) or chronic hepatitis ( n = 3 ) associated with viral hepatitis b. seven of them also had viral hepatitis c. during the inclusion period for this retrospective study , there was no patient with small hcc ( 2.0 cm ) who was without underlying liver parenchymal disease . the diagnosis of chronic hepatitis or cirrhosis was made by means of histology in 50 patients and by a combination of clinical course , blood chemistry tests ( aspartate aminotransferase , alanine aminotransferase , alkaline phosphatase , bilirubin , albumin , and globulin ) , and typical ct and mri findings in the remaining 69 patients . breath - hold t2w - gre imaging was obtained using the following parameters : tr / te of 180/12 , 30 flip angle , matrix of 144 256 , signal average of one , two sequential acquisitions , 20 slices , and average scan time 35 seconds ( two stacks with 10 slices each in a 15 - 17-second breath - hold ) . they independently reviewed three sets of mr images in two reading sessions : ( a ) unenhanced t1- and t2-weighted imaging , gadolinium - enhanced dynamic imaging , and ferucarbotran - enhanced t2w - gre imaging ( the t2w - gre set ) , ( b ) unenhanced t1- and t2-weighted imaging , gadolinium - enhanced dynamic imaging , and ferucarbotran - enhanced t2w - tse imaging ( the t2w - tse set ) , and ( c ) a combination of the t2w - gre set and t2w - tse set ( the combined set ) . in the first session , either the t2w - gre set or the t2w - tse set was randomly presented with no specific patient order . in the second session , the remaining t2w - gre set or t2w - tse set was randomly presented , and the t2w - gre or t2w - tse reviewed in the first reading recession was added for a combined review . the criteria for diagnosing hcc or metastasis on the ferucarbotran - enhanced mri were defined as a focal , discrete , nodular high signal intensity area relative to adjacent liver parenchyma ( lower than the signal intensity of csf or gallbladder on a t2w - tse sequence to exclude the cyst ) on both t2w - gre and t2w - tse images . during gadolinium - enhanced mri , 115 of the total 157 hccs showed early enhancement with rapid washout , and the remaining 42 appeared as hyperintense nodules during arterial phase imaging and were occult during portal and equilibrium phase imaging . based on the reviews submitted by the two observers , an alternative - free response receiver operating characteristic ( roc ) curve analysis was performed on a lesion - by - lesion basis ( 14 ) . for each imaging set ( t2w - gre images , t2w - tse images , and combined ) , an alternative - free response roc curve was fitted to each observer 's confidence rating data using a maximum likelihood estimation program ( rockit 0.9b ; courtesy of metz ce , university of chicago , il , 1998 ) ( 15 ) . the area under the alternative - free response roc curve ( az ) was calculated to determine the diagnostic accuracy of each image , each observer , and lesion type . the differences between the imaging sets with regard to the area under the alternative - free response roc curves were compared using a two - tailed student 's t test for paired data . of the 446 potential study patients , only those who met the following criteria were included in the study : ( a ) nodular hcc 2 cm or smaller in size that had been proven on histology or showed typical imaging findings on hepatic angiography , lipiodol ct , and dynamic ct , ( b ) liver metastases 2 cm or smaller in size that had been proven on histology or showed typical imaging findings on three - phase dynamic ct and follow - up ct or mri ( rapid progression of disease ) , ( c ) follow - up contrast - enhanced ct or mri performed for at least six months ( range : 6 - 30 months ) . consequently , the study population included 151 patients ( 114 men and 37 women ; age range : 40 - 74 years ) : 119 patients with 157 hccs and 32 patients with 98 metastases . of all the patients with hccs , four underwent ablation therapy , and all patients with hccs had liver cirrhosis ( n = 116 ) or chronic hepatitis ( n = 3 ) associated with viral hepatitis b. seven of them also had viral hepatitis c. during the inclusion period for this retrospective study , there was no patient with small hcc ( 2.0 cm ) who was without underlying liver parenchymal disease . breath - hold t2w - gre imaging was obtained using the following parameters : tr / te of 180/12 , 30 flip angle , matrix of 144 256 , signal average of one , two sequential acquisitions , 20 slices , and average scan time 35 seconds ( two stacks with 10 slices each in a 15 - 17-second breath - hold ) . they independently reviewed three sets of mr images in two reading sessions : ( a ) unenhanced t1- and t2-weighted imaging , gadolinium - enhanced dynamic imaging , and ferucarbotran - enhanced t2w - gre imaging ( the t2w - gre set ) , ( b ) unenhanced t1- and t2-weighted imaging , gadolinium - enhanced dynamic imaging , and ferucarbotran - enhanced t2w - tse imaging ( the t2w - tse set ) , and ( c ) a combination of the t2w - gre set and t2w - tse set ( the combined set ) . in the first session , either the t2w - gre set or the t2w - tse set was randomly presented with no specific patient order . in the second session , the remaining t2w - gre set or t2w - tse set was randomly presented , and the t2w - gre or t2w - tse reviewed in the first reading recession was added for a combined review . the criteria for diagnosing hcc or metastasis on the ferucarbotran - enhanced mri were defined as a focal , discrete , nodular high signal intensity area relative to adjacent liver parenchyma ( lower than the signal intensity of csf or gallbladder on a t2w - tse sequence to exclude the cyst ) on both t2w - gre and t2w - tse images . during gadolinium - enhanced mri , 115 of the total 157 hccs showed early enhancement with rapid washout , and the remaining 42 appeared as hyperintense nodules during arterial phase imaging and were occult during portal and equilibrium phase imaging . based on the reviews submitted by the two observers , an alternative - free response receiver operating characteristic ( roc ) curve analysis was performed on a lesion - by - lesion basis ( 14 ) . for each imaging set ( t2w - gre images , t2w - tse images , and combined ) , an alternative - free response roc curve was fitted to each observer 's confidence rating data using a maximum likelihood estimation program ( rockit 0.9b ; courtesy of metz ce , university of chicago , il , 1998 ) ( 15 ) . the area under the alternative - free response roc curve ( az ) was calculated to determine the diagnostic accuracy of each image , each observer , and lesion type . the differences between the imaging sets with regard to the area under the alternative - free response roc curves were compared using a two - tailed student 's t test for paired data . for all 255 lesions , including the 157 hccs and 98 metastases , there was a trend toward increased diagnostic accuracy ( az values ) for the combined set ( mean , 0.966 ) when compared with each set alone ( mean , 0.892 for t2w - gre set ; 0.910 for t2w - tse set ) , to a statistically significant degree ( p = 0.004 [ observer 1 ] , 0.003 [ observer 2 ] for the t2w - gre set ; p = 0.033 [ observer 1 ] , 0.025 [ observer 2 ] for the t2w - tse set ) ( table 2 ) . furthermore , considering the sensitivity for all lesions , the combined set ( mean , 94.7% ) was significantly more sensitive than each set alone ( mean , 86.1% for t2w - gre set ; 87.7% for t2w - tse set , p = 0.0001 ) ( table 3 ) . for detection of hccs , the az value of the combined set was significantly higher than that of each set alone , for both observers ( p = 0.016 [ observer 1 ] , 0.013 [ observer 2 ] for the t2w - gre set ; p = 0.042 for the t2w - tse set ) ( table 2 ) . although the az values of the t2w - gre set were slightly better than those of the t2w - tse set for both observers ( fig . among the 157 hccs , the t2w - tse set allowed for the depiction of 127 lesions ( sensitivity , 80.9% ; 95% ci : 73.9% , 86.7% ) by observer 1 and 129 lesions ( sensitivity , 82.2% ; 95% ci : 75.35% , 87.8% ) by observer 2 ; the t2w - gre set allowed the depiction of 135 lesions ( sensitivity , 86.0% ; 95% ci : 79.6% , 91.0% ) by observer 1 and 137 lesions ( sensitivity , 87.3% ; 95% ci : 81.0% , 92.0% ) by observer 2 . the combined approach allowed for the depiction of 145 lesions ( sensitivity , 92.4% ; 95% ci : 87.05% , 96.0% ) by observer 1 and 147 lesions ( sensitivity , 93.6% ; 95% ci : 88.6% , 96.9% ) by observer 2 ( mean , 93.0% ) , which was significantly better than the values obtained using each set alone ( mean , 81.6% for t2w - tse set , 86.7% for t2w - gre set ) ( p = 0.0001 for t2w - tse set , p = 0.002 for t2w - gre set ) . there were nine and eight hccs ( size range : 0.6 - 1.2 cm , mean size : 0.9 cm ) for each observer , respectively , which were not detected on the t2w - gre set , but which were detected on the t2w - tse set ( fig . there were 17 hccs ( size range : 0.6 - 1.9 cm , mean size : 1.2 cm ) for both observers that were not detected on the t2w - tse set , but which were detected on the t2w - gre set . of the 51 hccs with surgical confirmation , four well - differentiated hccs were not detected by either observer on the t2w - tse set , but were detected on the t2w - gre set by both observers . for the detection of liver metastases , the az values of the combined set and the t2w - tse set were better than those of the t2w - gre set for both observers , although the difference was not statistically significant ( p = 0.134 for observer 1 ; p = 0.162 , 0.772 for observer 2 ) . the sensitivity of the combined set was the same as that of the t2w - tse set ( 98.0% ; 95% ci : 92.8% , 99.7% , for observer 1 ; 96.9% ; 95% ci : 91.3% , 99.3% , for observer 2 ; mean 97.5% ) , which was significantly better than that of the t2w - gre set for both observers ( 84.7% ; 95% ci : 76.0% , 91.2% , for observer 1 ; 85.7% ; 95% ci : 77.2% , 92.0% , for observer 2 ; mean 85.2% ) ( p = 0.0001 ) ( fig . there were 11 metastases ( 0.4 - 0.8 cm ) that were not detected by either observer on the t2w - gre set , but which were clearly revealed on the t2w - tse set ( fig . however , there was no lesion detected on the t2w - gre set that could not be verified on the t2w - tse set . both observers had six false - positive findings on the t2w - gre set , four false - positive findings on the t2w - tse set , and two false - positive findings on the combined set . the kappa values for the two observers were 0.746 for the t2w - gre set , 0.762 for the t2w - tse set , and 0.685 for the combined set , indicating good inter - observer agreement with regard to the presence of lesions . t2w - gre and t2w - tse are techniques mainly used with spio - enhancement ( 7 - 10 ) . furthermore , t2w - gre might be limited in advanced cirrhotic livers because the decrease in signal intensity of cirrhotic livers on t2w - gre sequences is less pronounced than on t2w - tse sequences ( 17 ) . in the present study , we hypothesized that combining spio - enhanced t2w - gre and t2w - tse sequences had additive value compared to each sequence alone for the detection of small liver malignancies , including hccs and metastases , due to these theoretical relative advantages and disadvantages of t2w - gre and t2w - tse sequences . regarding the sensitivities for the detection of hccs , the combined approach was significantly better than each image set alone ( p < 0.05 ) , and the t2w - gre set was better than the t2w - tse set for both observers ( p < 0.05 ) . ( 17 , 20 ) showed that relative post - spio - enhanced snr changes in the liver parenchyma between cirrhotic and non - cirrhotic patients were remarkable on the t2w - gre sequence , but not on the t2w - tse sequence . they claimed that small intracellular spio clusters caused by decreased kupffer cell function in cirrhosis caused little snr drop in the liver parenchyma on the t2w - gre sequence compared to the t2w - tse sequence , because snr loss on the magnetic susceptibility - insensitive t2w - tse sequence is related to the greater free water interaction made possible by the relatively larger surface area of the small spio clusters ( 20 ) . however , given that kupffer cells density in well - differentiated hccs would be maintained , but kupffer cells function would be reduced compared to the surrounding liver , small intracellular spio clusters in well - differentiated hccs also produce little snr decrease on the t2w - gre sequence , which could result in better lesion conspicuity than on the t2w - tse sequence ( 21 ) . of the 51 hccs in our study with surgical confirmation , four lesions were not detected by either observer on the t2w - tse set , but were detected on the t2w - gre set by both observers . with regard to the detection of metastases that do not contain kupffer cells , the sensitivity of the combined approach was the same as that of the t2w - tse set , which was significantly better than that of the t2w - gre set for both observers . despite the higher inherent lesion to liver contrast on the t2w - gre , the better detection capability of the t2w - tse for metastases could be explained by signal loss caused by a magnetization transfer effect , with the tse sequence being lower in metastases than in hcc because tumor necrosis and abundant interstitial space in metastases generally contain more abundant free water compared with hcc ( 22 , 23 ) . there were 11 metastases ( 0.4 - 0.8 cm ) that were not detected by either observer on the t2w - gre set , but which were clearly revealed on the t2w - tse set . moreover , the relatively lower spatial resolution of the t2w - gre sequence ( 144 256 ) compared to the t2w - tse sequence ( 202 384 ) and the inherent blurring of high signal intensity structures on the t2w - gre sequence may also be behind this phenomenon . in addition to the shortcomings of the t2w - gre sequence in advanced cirrhotic livers , these could also explain the nine and eight hccs detected only on the t2w - tse set , but not on the t2w - gre set , by observers 1 and 2 , respectively . however , our study was not intended to determine the absolute diagnostic accuracy and sensitivity of the mr sequence , but to determine the relative diagnostic efficacy of combined mr sequences compared to single sequences for the detection of liver malignancy . furthermore , the relatively thick slice thickness ( 0.6 or 0.7 cm ) of ferucarbotran - enhanced mr sequences is inherently limiting in the detection of small hepatic tumors . hccs only detected on the t2w - tse set ( mean size , 0.9 cm ) tended to be smaller than those only detected on the t2w - gre set ( mean size , 1.2 cm ) . in our study , the diagnostic accuracy of the combined set for metastases was slightly better than that for each image set for observer 2 . in conclusion , a combination of ferucarbotran - enhanced t2w - gre and t2w - tse images in combining gadolinium and ferucarbotran - enhanced mri had better diagnostic capability compared to each sequence alone for the detection of hcc in cirrhotic patients . no difference in sensitivity for the detection of metastases was noted between the t2w - tse set and the combined t2w - gre / t2w - tse set , suggesting that the respiratory triggered t2w - tse sequence in ferucarbotran - enhanced mri is preferred for the detection of small liver metastases in non - cirrhotic patients .

cancer is a major health problem in the united states and other parts of the world ( 1 ) . cancer has been identified as a growing problem in the middle east countries ( 2 ) . cancer is not a disease with a single cause , but rather a collection of different causes with various symptoms , treatments and prognoses ( 3 ) . several aspects of the daily lives of cancer patients are affected which include financial situation , ability to work and family life , mood and temper , relationships with others , sleep and life quality ( 4 ) . cancer patients deal with various problems in different individual , family and social areas and also with the reduced life quality ( 5 ) . pain in patients with cancer is a stressful event which can affect patients life style as well as their feeling of satisfaction and comfort and also cause pain and discomfort , loss of control , fatigue , impaired quality of life and sexual activity , loss of interpersonal relationships and the concept of life , reduced performance , sleep and daily activities in them ( 6 ) . moreover , patients with cancer may also experience a feeling of anxiousness caused by the illness , treatment methods , etc . , which has a negative impact on their treatment and recovery process , therefore , it is essential to be controlled and mitigated ( 7 ) . the main treatments for cancers include surgery , chemotherapy , hormone therapy , radiation therapy , and immunotherapy or biological therapy . depending on the type of cancer , specialists use one or a combination of these treatments ( 8) . numerous studies have shown that a variety of complementary medicine can be influential in solving or reducing the problems caused by the disease in cancer patients ( 9 ) . complementary and alternative medicine ( cam ) is a group of medical and health care systems , practices , and products that are not currently considered to be part of conventional medicine ( 10 ) . the aim of this approach is to improve the health and quality of life as well as increased longevity by using natural approaches and different methods of alternative medicine based on clinical and research experiences ( 11 ) . in a similar vein , the studies around the world indicate an increasing desire to control health through using cam ( 12 ) . according to the american cancer society ( 2011 ) , complementary medicine for cancer includes methods that lead to the prevention , diagnosis and treatment of cancer . some types of complementary therapies can help to relieve from some certain symptoms of cancer and side effects caused by the treatment , such as fatigue , anxiety and pain , or lead to an increased sense of well - being in a person ( 10 ) . in the classification of complementary medicine , energy healing class includes treatments in which the energy emanates from the human body ( biofield ) or is originated from an external source such as therapeutic touch , reiki , qigong , polarity therapy , etc . ( 10 ) the simplest definition of therapeutic touch ( tt ) is the use of hands on or near the body to help the treatment . tt is the interpretation of an old healing experience in the modern era ( 11 ) . special theories underlying tt include dora kunz s model of human energy fields , electromagnetism and quantum physics , interpersonal psychology , martha roger s theory of human unity , ( relying on quantum and whole system theory , and eastern science and philosophy ) ( 13 , 14 ) . rogers in the subject of nursing : knowledge of human unity , ( 1990 ) , considers people as the continuous multi - dimensional energy fields that interact with the energy field of the environment . since the energy of therapists is not stopped in recipients , energy can be exchanged without any physical contact or with a very slight one ( 15 ) . from the rogers perspective , tt is an example of how professionals work to strengthen the integrity and perfection of humans and their environmental fields so that patients can reach an optimal health status ( 16 ) . tt is a non - invasive nursing intervention which takes place by the hands and in the form of energy transfer ( 17 ) . jackson et al . by investigating twelve studies found that tt can be an acceptable method for reducing physical and psychological symptoms of patients with cancer ( 18 ) . given the importance of tt in nursing and its potential positive outcomes in cancer patients , and also by taking into account the clinical evidence and the growing publication of articles in this field , this review study was conducted comprehensively to evaluate the results of tt clinical trials in patients with cancer . this review study was carried out by searching scientific databases including scopus , scholar google , science direct , pubmed using key words of healing touch , therapeutic touch , touch therapy , cancer , neoplasm within the year 1990 to november , 2015 . the mesh terms and keywords were exploded in the databases to the extent possible . moreover , the thematic relevance of studies was assessed by examining the titles and abstracts . in this study , the papers were selected on the basis of the availability of full texts of clinical trial articles in english , with the focus on the impact of tt in patients with cancer which had developed designs and methodologies . moreover , any trials with tt as part of their complex intervention , aimed at the development of methodologies for tt procedures without having any clinical outcomes , and those in which no data or statistical comparisons were reported , and healthy participants were also assessed , were excluded from the study . furthermore , papers presented at seminars ( due to the lack of having full texts and failure to complete the revision process for reviewing ) were not also evaluated . the number of 334 articles was found on the basis of the key words , of which 317 articles were excluded after carrying out the investigation process and 17 articles related to the clinical trial were examined in accordance with the objectives of the study . moreover , a total of 6 articles fully related to the field that had proper design and reliable data were ultimately selected and analyzed for the process of conducting the present study ( figure 1 ) . the intervention , results and the methodology ( table 1 ) as well as the setting and participants ( age , sex , stage of cancer , and site of cancer ) were examined in each study for the process of extracting the data . literature search and retrieval flow diagram summary of the basic features of the research articles included in the review . bfi - brief fatigue inventory ; bpi - brief pain index ; ct - chemotherapy ; dbp - diastolic blood pressure ; fact - f - functional assessment of cancer therapy fatigue ; fsi - fatigue symptom inventory ; hr - heart rate ; mt - massage therapy ; nsaids - nonsteroidal anti - inflammatory drugs ; poms - profile of mood states ; qol quality of life ; rr - respiratory rate ; rt - radiotherapy ; sbp - systolic blood pressure ; tt - therapeutic touch ; studies obtained by searching databases were organized and selected . the results indicated that most studies expressed a positive and significant association between therapeutic touch and dependent variables ( nausea , pain , quality of life , fatigue and temper ) . the studies were undertaken in the united states ( n= 3 ) , the uk ( n= 1 ) and iran ( n= 2 ) and were dated between 2003 and 2015 . the participants were adults with an age range of 18 to 83 who had not a wide range of cancer diagnoses except in the study of weze ( breast , gastrointestinal , gynecological , lung , undisclosed ) and in the study of post - white with two breast and gynecology cancers . the total number of participants in this current review was 601 , of which , 96% were females and 4% were males . all the participants in the studies varied in degree from the stage 1 up to the advanced , metastatic cancer . all the studies had used control groups in order for comparison with the intervention and only the study by weze was a single group study ( 19 ) . the number of therapeutic touch sessions ranged from one session ( 20 ) to six sessions ( 21 ) . a variety of measurement instruments were used to assess fatigue , pain , and nausea across the studies . to measure the fatigue , aghabati ( 22 ) and lutgendorf ( 23 ) respectively used rhoten fatigue scale ( rfs ) and fatigue symptom inventory ( fsi ) . to measure the pain interference and pain intensity , post - white ( 24 ) used the brief pain inventory ( bpi ) . other measurement instruments used for measuring pain included the visual analogue scale ( vas ) used by aghabati and weze and 36 -hrqol short used by cook . moreover , postwhite used the brief nausea index ( bni ) to measure the nausea . however , matourpour only made use of the check list . in a single - blind clinical trial study conducted by matourpour et al . the results showed that the nausea was significantly shorter for the intervention group compared to the placebo and the control groups . however , the tt intervention did not result in the reduced the severity of nausea in the intervention group . moreover , the number of incidence of nausea in the acute phase was also significant in the intervention group in comparison with the other two groups ( 20 ) . investigated the effects of massage therapy ( mt ) and tt compared to the standard care on inducing relaxation and reducing symptoms in 203 patients . the results demonstrated a greater relaxed feeling and short - term pain reduction , along with less disturbed mood and fatigue in the patients in the intervention group with the touch and massage therapies compared to the patients in the control group . the researchers concluded that the use of various methods of alternative medicine to relieve pain in patients with chronic pain may be more beneficial than relying on a single approach ( 24 ) . moreover , cook et al . in the study reported the reduction in pain and improvement in the physical function . in this study , the intervention group had a greater improvement than the control group in terms of quality of life . additionally , the improvement in physical function , pain and vitality was observed in the intervention group compared to the control group ( 21 ) . the results of this study indicate that therapeutic touch can cause the pain reduction in the breast and oncology cancers . . indicated positive effects of therapeutic touch on reducing pain in cancer patients ( 19 ) . the studies were undertaken in the united states ( n= 3 ) , the uk ( n= 1 ) and iran ( n= 2 ) and were dated between 2003 and 2015 . the participants were adults with an age range of 18 to 83 who had not a wide range of cancer diagnoses except in the study of weze ( breast , gastrointestinal , gynecological , lung , undisclosed ) and in the study of post - white with two breast and gynecology cancers . the total number of participants in this current review was 601 , of which , 96% were females and 4% were males . all the participants in the studies varied in degree from the stage 1 up to the advanced , metastatic cancer . all the studies had used control groups in order for comparison with the intervention and only the study by weze was a single group study ( 19 ) . the number of therapeutic touch sessions ranged from one session ( 20 ) to six sessions ( 21 ) . a variety of measurement instruments were used to assess fatigue , pain , and nausea across the studies . to measure the fatigue , aghabati ( 22 ) and lutgendorf ( 23 ) respectively used rhoten fatigue scale ( rfs ) and fatigue symptom inventory ( fsi ) . to measure the pain interference and pain intensity , post - white ( 24 ) used the brief pain inventory ( bpi ) . other measurement instruments used for measuring pain included the visual analogue scale ( vas ) used by aghabati and weze and 36 -hrqol short used by cook . moreover , postwhite used the brief nausea index ( bni ) to measure the nausea . however , matourpour only made use of the check list . in a single - blind clinical trial study conducted by matourpour et al . the results showed that the nausea was significantly shorter for the intervention group compared to the placebo and the control groups . however , the tt intervention did not result in the reduced the severity of nausea in the intervention group . moreover , the number of incidence of nausea in the acute phase was also significant in the intervention group in comparison with the other two groups ( 20 ) . post - white et al . in their study investigated the effects of massage therapy ( mt ) and tt compared to the standard care on inducing relaxation and reducing symptoms in 203 patients . the results demonstrated a greater relaxed feeling and short - term pain reduction , along with less disturbed mood and fatigue in the patients in the intervention group with the touch and massage therapies compared to the patients in the control group . the researchers concluded that the use of various methods of alternative medicine to relieve pain in patients with chronic pain may be more beneficial than relying on a single approach ( 24 ) . moreover , cook et al . in the study reported the reduction in pain and improvement in the physical function . in this study , the intervention group had a greater improvement than the control group in terms of quality of life . additionally , the improvement in physical function , pain and vitality was observed in the intervention group compared to the control group ( 21 ) . the results of this study indicate that therapeutic touch can cause the pain reduction in the breast and oncology cancers . furthermore , the results of a study by weze et al . indicated positive effects of therapeutic touch on reducing pain in cancer patients ( 19 ) . the studies were undertaken in the united states ( n= 3 ) , the uk ( n= 1 ) and iran ( n= 2 ) and were dated between 2003 and 2015 . the participants were adults with an age range of 18 to 83 who had not a wide range of cancer diagnoses except in the study of weze ( breast , gastrointestinal , gynecological , lung , undisclosed ) and in the study of post - white with two breast and gynecology cancers . the total number of participants in this current review was 601 , of which , 96% were females and 4% were males . all the participants in the studies varied in degree from the stage 1 up to the advanced , metastatic cancer . all the studies had used control groups in order for comparison with the intervention and only the study by weze was a single group study ( 19 ) . the number of therapeutic touch sessions ranged from one session ( 20 ) to six sessions ( 21 ) . a variety of measurement instruments were used to assess fatigue , pain , and nausea across the studies . to measure the fatigue , aghabati ( 22 ) and lutgendorf ( 23 ) respectively used rhoten fatigue scale ( rfs ) and fatigue symptom inventory ( fsi ) . to measure the pain interference and pain intensity , post - white ( 24 ) used the brief pain inventory ( bpi ) . other measurement instruments used for measuring pain included the visual analogue scale ( vas ) used by aghabati and weze and 36 -hrqol short used by cook . moreover , postwhite used the brief nausea index ( bni ) to measure the nausea . however , matourpour only made use of the check list . in a single - blind clinical trial study conducted by matourpour et al . the results showed that the nausea was significantly shorter for the intervention group compared to the placebo and the control groups . however , the tt intervention did not result in the reduced the severity of nausea in the intervention group . moreover , the number of incidence of nausea in the acute phase was also significant in the intervention group in comparison with the other two groups ( 20 ) . post - white et al . in their study investigated the effects of massage therapy ( mt ) and tt compared to the standard care on inducing relaxation and reducing symptoms in 203 patients . the results demonstrated a greater relaxed feeling and short - term pain reduction , along with less disturbed mood and fatigue in the patients in the intervention group with the touch and massage therapies compared to the patients in the control group . the researchers concluded that the use of various methods of alternative medicine to relieve pain in patients with chronic pain may be more beneficial than relying on a single approach ( 24 ) . moreover , cook et al . in the study reported the reduction in pain and improvement in the physical function . in this study , the intervention group had a greater improvement than the control group in terms of quality of life . additionally , the improvement in physical function , pain and vitality was observed in the intervention group compared to the control group ( 21 ) . the results of this study indicate that therapeutic touch can cause the pain reduction in the breast and oncology cancers . . indicated positive effects of therapeutic touch on reducing pain in cancer patients ( 19 ) . in the studies on cancer patients , significant findings have been reported . even in the absence of significant results in outcomes studied , therapeutic touch recipients often reported subjective benefits , including improved mood , well - being , and interpersonal relationships , reduction in pain , nausea , anxiety , and fatigue , increase in vitality , and satisfaction with the touch therapy . in a descriptive study on patients with fibromyalgia , diener et al . moreover , the participants reported relaxation , spiritual uplifting , and greater mobility ( 25 ) . in addition , meehan et al . in their study also indicated that therapeutic touch can be used as a supplement in relieving pain in patients after the surgery ( 26 ) . evanoff by investigating the effect of therapeutic touch on the degree of pain in patients with osteoarthritis of the knee came to the conclusion that therapeutic touch can reduce pain and improve function in patients ( 27 ) . the results obtained from four studies on elderly people proved to be positive regardless of the diagnosis . physical status such as pain , appetite , and sleep were enhanced ; behavioral markers such as worry , outbursts , and restlessness were decreased ; and behaviors that made the jobs of the staff easier , such as compliance with daily routine , decreased medications , and increased functional ability , all supported the use of tt for this population ( 28 ) . significant decreases in pain , nausea , and anxiety were immediately observed following the intervention on post - operative days one and two , and in pain and anxiety on the post - operative day three compared with the pre - intervention levels . these findings indicated that the tt intervention was feasible and acceptable to patients undergoing bariatric surgery , and significantly improved pain , nausea , and anxiety in them ( 29 ) . coakley believes that the energy therapy can be used to improve various symptoms in patients with cancer . in a similar vein , studies have also shown that such holistic interferences are greatly demanded by the patients ( 16 ) . the goal of tt is to restore balance , harmony , and a sense of well - being . tt is based on a compassionate intention directed through light touch or placement of the hands just off the body which is often performed by nurses ( 30 ) . physiologically , bio - field therapies such as tt appear to affect the autonomic nervous system , altering the high frequency to low frequency ratio of heart rate variability , reflecting a greater parasympathetic tone and decreasing the sympathetic activation ( 31 ) . some studies suggested that biofield healing may decrease stress and enhance immune function ( 30 , 32 ) . it is also possible for the relaxation response to help explain the effects of tt ( 33 ) . the underlying assumption of tt is that human beings are systems of energy and that the energy field extends a few inches beyond the skin s surface . there exist three distinct phases of intervention including ( a ) nurses becoming aware of the helpfulness of tt for their patients , ( b ) the assessment phase , where a nurse uses slow , gentle , sweeping movement of their hands starting from the patient s head and proceeding to the patient s feet to assess the presence of any signs of energy dissymmetry , and ( c ) the un - ruffling phase , where a nurse uses symmetric movement of their hands over the energy field of the patient with the goal of smoothing out or relieving energy congestion ( 34 ) . therapeutic touch is a standardized biofield therapy that uses gentle touch and movements in the patient s energy field with the goal of restoring balance in the patient s energy system and strengthening the patient s healing capacity(35 ) . tt has been shown to increase well - being in cervical and breast cancer patients during radiation ( 21 , 23 ) and to reduce distress and fatigue during chemotherapy ( 24 ) . tt has also a series of benefits in reducing anxiety , increasing relaxation , decreasing pain , diminishing depression , and increasing a sense of well - being ( 36 , 37 ) . although treatment for cancer is recognized as stressful and impairing to quality of life , few tt interventions have been shown to be efficacious in lessening treatment - related symptoms ( 38 ) . this method allows instructors both to provide technical skills and to build a relationship with the patient so that professionals gratification and motivation will be improved . the ease of training and feasibility in multiple settings without the need for special tools also make touch therapy a good option for many patients and caregivers . none of the studies included in this review provided a clear rationale for the treatment specificity or duration . moreover , the studies showed substantial differences in frequency of interventions which were performed every day , once a week , and twice a week . the length of treatment sessions ranged from 1040 minutes ; however , the average time was usually from 1045 minutes ( 34 ) . in the most reviewed studies , the role of some intervening variables such as physical activity , psychological problems and gender was overlooked , or not mentioned . moreover , methodology differences are observable in the reviewed studies , the most important of which is the failure to provide any credible references to indicate the number of tt sessions . the length of the study ranged from one day to six weeks ; however , the average length was 4 - 6 weeks ( 11 , 34 ) most samples selected in the studies were diagnosed with breast cancer which was probably due to their greater prevalence and better uniformity in the designs of the studies . moreover , given the high incidence of cancer , it is necessary to investigate the tt function and impacts on individuals and men with various types of cancer . participants generally reported the improved quality of life physically , emotionally , relationally , and spiritually . studying the different research in the field of tt revealed that none of the studies had pointed to any considerable side effects . however , some patients with chronic diseases were reported to have light - headedness , dizziness , irritability following the bio - field therapy ( 39 ) . a search strategy was created with the help of an information specialist . to reduce subjective selection bias , the inclusion process and the quality of the articles were carefully assessed by four independent researchers . however , there are some limitations to this study . first , the searches were limited to the period between 1990 and early 2015 . second , there was a risk of language bias as the papers in any language other than english were excluded . third , there was a possibility for selecting only positive research due to the publication bias . a search strategy was created with the help of an information specialist . to reduce subjective selection bias , the inclusion process and the quality of the articles were carefully assessed by four independent researchers . however , there are some limitations to this study . second , there was a risk of language bias as the papers in any language other than english were excluded . third , there was a possibility for selecting only positive research due to the publication bias . based on the results of this review , an affirmation can be made regarding the use of therapeutic touch as a non - invasive intervention for improving the health status in patients with cancer . it also seems that this method can be used as a safe method in the management of physical function , pain , anxiety , and nausea in cancer patients . training tt to those interested to the field can possibly be of great help in caring for cancer patients and reducing complications of the disease . however , further studies are needed to explore the impact of tt on additional clinically relevant measures .

background : the use of complementary and alternative medicine ( cam ) techniques has been growing . the national center for complementary and alternative medicine places therapeutic touch ( tt ) into the category of bio field energy . this literature review is aimed at critically evaluating the data from clinical trials examining the clinical efficacy of therapeutic touch as a supportive care modality in adult patients with cancer.methods:electronic databases ( pubmed , scopus , scholar google , and science direct ) were searched from the year 1990 to 2015 to locate potentially relevant peer - reviewed articles using the key words therapeutic touch , touch therapy , neoplasm , cancer , and cam . additionally , relevant journals and references of all the located articles were manually searched for other potentially relevant studies.results:the number of 334 articles was found on the basis of the key words , of which 17 articles related to the clinical trial were examined in accordance with the objectives of the study . a total of 6 articles were in the final dataset in which several examples of the positive effects of healing touch on pain , nausea , anxiety and fatigue , and life quality and also on biochemical parameters were observed.conclusion:based on the results of this study , an affirmation can be made regarding the use of tt , as a non - invasive intervention for improving the health status in patients with cancer . moreover , therapeutic touch was proved to be a useful strategy for adult patients with cancer .

1. INTRODUCTION 2. METHODS 3. FINDINGS None Participants and setting Outcome measures 4. DISCUSSION Strengths and limitations of this review 5. CONCLUSION

cancer is a major health problem in the united states and other parts of the world ( 1 ) . cancer has been identified as a growing problem in the middle east countries ( 2 ) . several aspects of the daily lives of cancer patients are affected which include financial situation , ability to work and family life , mood and temper , relationships with others , sleep and life quality ( 4 ) . cancer patients deal with various problems in different individual , family and social areas and also with the reduced life quality ( 5 ) . pain in patients with cancer is a stressful event which can affect patients life style as well as their feeling of satisfaction and comfort and also cause pain and discomfort , loss of control , fatigue , impaired quality of life and sexual activity , loss of interpersonal relationships and the concept of life , reduced performance , sleep and daily activities in them ( 6 ) . moreover , patients with cancer may also experience a feeling of anxiousness caused by the illness , treatment methods , etc . the main treatments for cancers include surgery , chemotherapy , hormone therapy , radiation therapy , and immunotherapy or biological therapy . depending on the type of cancer , specialists use one or a combination of these treatments ( 8) . numerous studies have shown that a variety of complementary medicine can be influential in solving or reducing the problems caused by the disease in cancer patients ( 9 ) . complementary and alternative medicine ( cam ) is a group of medical and health care systems , practices , and products that are not currently considered to be part of conventional medicine ( 10 ) . the aim of this approach is to improve the health and quality of life as well as increased longevity by using natural approaches and different methods of alternative medicine based on clinical and research experiences ( 11 ) . according to the american cancer society ( 2011 ) , complementary medicine for cancer includes methods that lead to the prevention , diagnosis and treatment of cancer . some types of complementary therapies can help to relieve from some certain symptoms of cancer and side effects caused by the treatment , such as fatigue , anxiety and pain , or lead to an increased sense of well - being in a person ( 10 ) . in the classification of complementary medicine , energy healing class includes treatments in which the energy emanates from the human body ( biofield ) or is originated from an external source such as therapeutic touch , reiki , qigong , polarity therapy , etc . ( 10 ) the simplest definition of therapeutic touch ( tt ) is the use of hands on or near the body to help the treatment . tt is the interpretation of an old healing experience in the modern era ( 11 ) . rogers in the subject of nursing : knowledge of human unity , ( 1990 ) , considers people as the continuous multi - dimensional energy fields that interact with the energy field of the environment . from the rogers perspective , tt is an example of how professionals work to strengthen the integrity and perfection of humans and their environmental fields so that patients can reach an optimal health status ( 16 ) . tt is a non - invasive nursing intervention which takes place by the hands and in the form of energy transfer ( 17 ) . by investigating twelve studies found that tt can be an acceptable method for reducing physical and psychological symptoms of patients with cancer ( 18 ) . given the importance of tt in nursing and its potential positive outcomes in cancer patients , and also by taking into account the clinical evidence and the growing publication of articles in this field , this review study was conducted comprehensively to evaluate the results of tt clinical trials in patients with cancer . this review study was carried out by searching scientific databases including scopus , scholar google , science direct , pubmed using key words of healing touch , therapeutic touch , touch therapy , cancer , neoplasm within the year 1990 to november , 2015 . the mesh terms and keywords were exploded in the databases to the extent possible . moreover , the thematic relevance of studies was assessed by examining the titles and abstracts . in this study , the papers were selected on the basis of the availability of full texts of clinical trial articles in english , with the focus on the impact of tt in patients with cancer which had developed designs and methodologies . moreover , any trials with tt as part of their complex intervention , aimed at the development of methodologies for tt procedures without having any clinical outcomes , and those in which no data or statistical comparisons were reported , and healthy participants were also assessed , were excluded from the study . furthermore , papers presented at seminars ( due to the lack of having full texts and failure to complete the revision process for reviewing ) were not also evaluated . the number of 334 articles was found on the basis of the key words , of which 317 articles were excluded after carrying out the investigation process and 17 articles related to the clinical trial were examined in accordance with the objectives of the study . moreover , a total of 6 articles fully related to the field that had proper design and reliable data were ultimately selected and analyzed for the process of conducting the present study ( figure 1 ) . the intervention , results and the methodology ( table 1 ) as well as the setting and participants ( age , sex , stage of cancer , and site of cancer ) were examined in each study for the process of extracting the data . literature search and retrieval flow diagram summary of the basic features of the research articles included in the review . bfi - brief fatigue inventory ; bpi - brief pain index ; ct - chemotherapy ; dbp - diastolic blood pressure ; fact - f - functional assessment of cancer therapy fatigue ; fsi - fatigue symptom inventory ; hr - heart rate ; mt - massage therapy ; nsaids - nonsteroidal anti - inflammatory drugs ; poms - profile of mood states ; qol quality of life ; rr - respiratory rate ; rt - radiotherapy ; sbp - systolic blood pressure ; tt - therapeutic touch ; studies obtained by searching databases were organized and selected . the results indicated that most studies expressed a positive and significant association between therapeutic touch and dependent variables ( nausea , pain , quality of life , fatigue and temper ) . the studies were undertaken in the united states ( n= 3 ) , the uk ( n= 1 ) and iran ( n= 2 ) and were dated between 2003 and 2015 . the participants were adults with an age range of 18 to 83 who had not a wide range of cancer diagnoses except in the study of weze ( breast , gastrointestinal , gynecological , lung , undisclosed ) and in the study of post - white with two breast and gynecology cancers . the total number of participants in this current review was 601 , of which , 96% were females and 4% were males . all the participants in the studies varied in degree from the stage 1 up to the advanced , metastatic cancer . all the studies had used control groups in order for comparison with the intervention and only the study by weze was a single group study ( 19 ) . the number of therapeutic touch sessions ranged from one session ( 20 ) to six sessions ( 21 ) . a variety of measurement instruments were used to assess fatigue , pain , and nausea across the studies . to measure the fatigue , aghabati ( 22 ) and lutgendorf ( 23 ) respectively used rhoten fatigue scale ( rfs ) and fatigue symptom inventory ( fsi ) . moreover , postwhite used the brief nausea index ( bni ) to measure the nausea . however , matourpour only made use of the check list . in a single - blind clinical trial study conducted by matourpour et al . the results showed that the nausea was significantly shorter for the intervention group compared to the placebo and the control groups . however , the tt intervention did not result in the reduced the severity of nausea in the intervention group . moreover , the number of incidence of nausea in the acute phase was also significant in the intervention group in comparison with the other two groups ( 20 ) . investigated the effects of massage therapy ( mt ) and tt compared to the standard care on inducing relaxation and reducing symptoms in 203 patients . the results demonstrated a greater relaxed feeling and short - term pain reduction , along with less disturbed mood and fatigue in the patients in the intervention group with the touch and massage therapies compared to the patients in the control group . the researchers concluded that the use of various methods of alternative medicine to relieve pain in patients with chronic pain may be more beneficial than relying on a single approach ( 24 ) . moreover , cook et al . in the study reported the reduction in pain and improvement in the physical function . in this study , the intervention group had a greater improvement than the control group in terms of quality of life . additionally , the improvement in physical function , pain and vitality was observed in the intervention group compared to the control group ( 21 ) . the results of this study indicate that therapeutic touch can cause the pain reduction in the breast and oncology cancers . indicated positive effects of therapeutic touch on reducing pain in cancer patients ( 19 ) . the participants were adults with an age range of 18 to 83 who had not a wide range of cancer diagnoses except in the study of weze ( breast , gastrointestinal , gynecological , lung , undisclosed ) and in the study of post - white with two breast and gynecology cancers . the total number of participants in this current review was 601 , of which , 96% were females and 4% were males . all the participants in the studies varied in degree from the stage 1 up to the advanced , metastatic cancer . all the studies had used control groups in order for comparison with the intervention and only the study by weze was a single group study ( 19 ) . the number of therapeutic touch sessions ranged from one session ( 20 ) to six sessions ( 21 ) . a variety of measurement instruments were used to assess fatigue , pain , and nausea across the studies . to measure the fatigue , aghabati ( 22 ) and lutgendorf ( 23 ) respectively used rhoten fatigue scale ( rfs ) and fatigue symptom inventory ( fsi ) . however , matourpour only made use of the check list . in a single - blind clinical trial study conducted by matourpour et al . the results showed that the nausea was significantly shorter for the intervention group compared to the placebo and the control groups . moreover , the number of incidence of nausea in the acute phase was also significant in the intervention group in comparison with the other two groups ( 20 ) . in their study investigated the effects of massage therapy ( mt ) and tt compared to the standard care on inducing relaxation and reducing symptoms in 203 patients . the results demonstrated a greater relaxed feeling and short - term pain reduction , along with less disturbed mood and fatigue in the patients in the intervention group with the touch and massage therapies compared to the patients in the control group . the researchers concluded that the use of various methods of alternative medicine to relieve pain in patients with chronic pain may be more beneficial than relying on a single approach ( 24 ) . in the study reported the reduction in pain and improvement in the physical function . in this study , the intervention group had a greater improvement than the control group in terms of quality of life . additionally , the improvement in physical function , pain and vitality was observed in the intervention group compared to the control group ( 21 ) . the results of this study indicate that therapeutic touch can cause the pain reduction in the breast and oncology cancers . furthermore , the results of a study by weze et al . indicated positive effects of therapeutic touch on reducing pain in cancer patients ( 19 ) . the studies were undertaken in the united states ( n= 3 ) , the uk ( n= 1 ) and iran ( n= 2 ) and were dated between 2003 and 2015 . the participants were adults with an age range of 18 to 83 who had not a wide range of cancer diagnoses except in the study of weze ( breast , gastrointestinal , gynecological , lung , undisclosed ) and in the study of post - white with two breast and gynecology cancers . the total number of participants in this current review was 601 , of which , 96% were females and 4% were males . all the participants in the studies varied in degree from the stage 1 up to the advanced , metastatic cancer . all the studies had used control groups in order for comparison with the intervention and only the study by weze was a single group study ( 19 ) . the number of therapeutic touch sessions ranged from one session ( 20 ) to six sessions ( 21 ) . a variety of measurement instruments were used to assess fatigue , pain , and nausea across the studies . to measure the fatigue , aghabati ( 22 ) and lutgendorf ( 23 ) respectively used rhoten fatigue scale ( rfs ) and fatigue symptom inventory ( fsi ) . moreover , postwhite used the brief nausea index ( bni ) to measure the nausea . however , matourpour only made use of the check list . in a single - blind clinical trial study conducted by matourpour et al . the results showed that the nausea was significantly shorter for the intervention group compared to the placebo and the control groups . moreover , the number of incidence of nausea in the acute phase was also significant in the intervention group in comparison with the other two groups ( 20 ) . in their study investigated the effects of massage therapy ( mt ) and tt compared to the standard care on inducing relaxation and reducing symptoms in 203 patients . the results demonstrated a greater relaxed feeling and short - term pain reduction , along with less disturbed mood and fatigue in the patients in the intervention group with the touch and massage therapies compared to the patients in the control group . the researchers concluded that the use of various methods of alternative medicine to relieve pain in patients with chronic pain may be more beneficial than relying on a single approach ( 24 ) . moreover , cook et al . in the study reported the reduction in pain and improvement in the physical function . in this study , the intervention group had a greater improvement than the control group in terms of quality of life . additionally , the improvement in physical function , pain and vitality was observed in the intervention group compared to the control group ( 21 ) . the results of this study indicate that therapeutic touch can cause the pain reduction in the breast and oncology cancers . indicated positive effects of therapeutic touch on reducing pain in cancer patients ( 19 ) . even in the absence of significant results in outcomes studied , therapeutic touch recipients often reported subjective benefits , including improved mood , well - being , and interpersonal relationships , reduction in pain , nausea , anxiety , and fatigue , increase in vitality , and satisfaction with the touch therapy . moreover , the participants reported relaxation , spiritual uplifting , and greater mobility ( 25 ) . in their study also indicated that therapeutic touch can be used as a supplement in relieving pain in patients after the surgery ( 26 ) . evanoff by investigating the effect of therapeutic touch on the degree of pain in patients with osteoarthritis of the knee came to the conclusion that therapeutic touch can reduce pain and improve function in patients ( 27 ) . the results obtained from four studies on elderly people proved to be positive regardless of the diagnosis . physical status such as pain , appetite , and sleep were enhanced ; behavioral markers such as worry , outbursts , and restlessness were decreased ; and behaviors that made the jobs of the staff easier , such as compliance with daily routine , decreased medications , and increased functional ability , all supported the use of tt for this population ( 28 ) . significant decreases in pain , nausea , and anxiety were immediately observed following the intervention on post - operative days one and two , and in pain and anxiety on the post - operative day three compared with the pre - intervention levels . these findings indicated that the tt intervention was feasible and acceptable to patients undergoing bariatric surgery , and significantly improved pain , nausea , and anxiety in them ( 29 ) . coakley believes that the energy therapy can be used to improve various symptoms in patients with cancer . the goal of tt is to restore balance , harmony , and a sense of well - being . it is also possible for the relaxation response to help explain the effects of tt ( 33 ) . the underlying assumption of tt is that human beings are systems of energy and that the energy field extends a few inches beyond the skin s surface . there exist three distinct phases of intervention including ( a ) nurses becoming aware of the helpfulness of tt for their patients , ( b ) the assessment phase , where a nurse uses slow , gentle , sweeping movement of their hands starting from the patient s head and proceeding to the patient s feet to assess the presence of any signs of energy dissymmetry , and ( c ) the un - ruffling phase , where a nurse uses symmetric movement of their hands over the energy field of the patient with the goal of smoothing out or relieving energy congestion ( 34 ) . therapeutic touch is a standardized biofield therapy that uses gentle touch and movements in the patient s energy field with the goal of restoring balance in the patient s energy system and strengthening the patient s healing capacity(35 ) . tt has been shown to increase well - being in cervical and breast cancer patients during radiation ( 21 , 23 ) and to reduce distress and fatigue during chemotherapy ( 24 ) . tt has also a series of benefits in reducing anxiety , increasing relaxation , decreasing pain , diminishing depression , and increasing a sense of well - being ( 36 , 37 ) . although treatment for cancer is recognized as stressful and impairing to quality of life , few tt interventions have been shown to be efficacious in lessening treatment - related symptoms ( 38 ) . this method allows instructors both to provide technical skills and to build a relationship with the patient so that professionals gratification and motivation will be improved . the ease of training and feasibility in multiple settings without the need for special tools also make touch therapy a good option for many patients and caregivers . moreover , the studies showed substantial differences in frequency of interventions which were performed every day , once a week , and twice a week . in the most reviewed studies , the role of some intervening variables such as physical activity , psychological problems and gender was overlooked , or not mentioned . moreover , methodology differences are observable in the reviewed studies , the most important of which is the failure to provide any credible references to indicate the number of tt sessions . the length of the study ranged from one day to six weeks ; however , the average length was 4 - 6 weeks ( 11 , 34 ) most samples selected in the studies were diagnosed with breast cancer which was probably due to their greater prevalence and better uniformity in the designs of the studies . moreover , given the high incidence of cancer , it is necessary to investigate the tt function and impacts on individuals and men with various types of cancer . participants generally reported the improved quality of life physically , emotionally , relationally , and spiritually . studying the different research in the field of tt revealed that none of the studies had pointed to any considerable side effects . to reduce subjective selection bias , the inclusion process and the quality of the articles were carefully assessed by four independent researchers . however , there are some limitations to this study . first , the searches were limited to the period between 1990 and early 2015 . third , there was a possibility for selecting only positive research due to the publication bias . to reduce subjective selection bias , the inclusion process and the quality of the articles were carefully assessed by four independent researchers . however , there are some limitations to this study . third , there was a possibility for selecting only positive research due to the publication bias . based on the results of this review , an affirmation can be made regarding the use of therapeutic touch as a non - invasive intervention for improving the health status in patients with cancer . it also seems that this method can be used as a safe method in the management of physical function , pain , anxiety , and nausea in cancer patients . training tt to those interested to the field can possibly be of great help in caring for cancer patients and reducing complications of the disease . however , further studies are needed to explore the impact of tt on additional clinically relevant measures .

tooth bleaching is one of the most noninvasive dental treatments to improve people 's appearance . bleaching is a decolorization or whitening process that can occur in solution or on a surface . the color producing materials in solution or on a surface are typically organic compounds that effect the teeth color . intrinsic tooth color is usually associated with the light scattering and adsorption properties of the enamel and dentine , while extrinsic stains tend to form in areas of the teeth that are less accessible to tooth brushing and is often promoted by smoking , dietary intake , the use of certain cationic agents such as chlorhexidine or metal salts . tooth color can be improved by a number of methods and approaches including internal bleaching of nonvital teeth , external bleaching of vital teeth , whitening toothpastes , micro - abrasion of enamel with abrasives . although the benefits and side effects are still controversial for some of them , increased expectancy for aesthetics stops neither the dentists nor the patients to perform such procedures . there are a number of studies and trials in the literature investigates the methods and procedures , two of the most applied approaches in the tooth bleaching treatment are home ( night - guard ) bleaching and in - office bleaching technique . lower concentrations of both carbamide peroxide ( cp ) and hydrogen peroxide ( hp ) are used for home - bleaching , while higher concentrations are necessary for in - office treatments . in - office bleaching application of light - sensitive , high - concentration bleaching agents associated with a power - unit usually performed in a single appointment , reduces the time required to achieve the expected results and decreases the failure possibility . the home - bleaching technique , with a custom tray , offers a conservative , cost effective method for bleaching teeth . in addition using cp formerly used for topical disinfection as an alternative bleaching agent to hp , provided a slower release of active , oxidizing ions which caused more effective and long lasting bleaching impact . despite the favorable results achieved with both bleaching techniques , some reports in the literature have related adverse side effects of cp as a consequence of the treatment . sensitivity following the treatment has been related to the possible removal of mineral content from enamel and dentin . therefore some of the authors advised that bleaching materials could adversely affect dental hard tissues and should be used with caution . an in - vitro study by efeoglu et al . showed that different concentrations of cp can remove mineral structures from enamel , causing morphological alterations with different forms and intensity and can reach to the subsurface . nevertheless , it has been proposed that the loss of mineral content and increased porosity could explain transitory dental sensitivity during bleaching treatment . since little information exists in the literature regarding the clinical response to bleaching treatment , there is a need for studies that simulate clinical conditions in order to evaluate the real effects of such treatment . the hypothesis to be tested is that in a clinical oral simulate condition ( in situ ) , the effects of bleaching agents are less evident than when seen in in - vitro conditions . the scope of the current study is focused on the evaluation the in - vitro bleaching efficiency of different products in similar concentrations used in home and office applications and validate the results with software and hardware methods to assess the sufficiency of the measurement techniques . our hypothesis was similar cp concentration results in similar bleaching effect and different measurement methods validates each other . a total of 110 freshly extracted for aggressive untreatable periodontitis , caries free human incisors were collected from department for oral and maxillo - facial surgery , ege university to obtain similar enamel and dentin thickness , also start - up shades . ethical approval of the ege university committee of medical ethics ( reg . all of the teeth were examined under a stereomicroscope in order to select those without surface defects . all of the specimens were stored in 1% thymol solution until used . the debris and calculus was removed with periodontal scalers mechanically . the labial surfaces were ground and polished with water - cooled finishing and polishing discs ( sof - lex , 3 m espe , st . paul , mn , usa ) removing approximately 100 m of the outermost enamel layer to obtain flat and smooth enamel surfaces . the teeth were artificially stained with whole blood and hemolysate solution prior embedding in prepared acrylic jaws with acrylic resin ( meliodent , herauskulzer , werheim , germany ) , to achieve an adequate and uniform staining [ figure 1a ] . a total of 110 teeth were embed in acrylic jaws according to the random distribution in order to make the templates for bleaching and color measurement procedures . the labial teeth surfaces were covered with 1 mm thick spacer up to 1 mm of the apical foramen [ figure 1b ] . bleaching agents placed in the custom trays on all study groups , in a 2 mm thick layer in order to get enough material to produce active bleaching [ figure 1d ] . baseline tooth color was recorded using a spectrophotometer ( vita easyshade - vita zahnfabrik , germany ) . from the beginning of the study to the end , all specimens were stored for 48 h at 37c in artificial saliva that was renewed everyday . all of the specimens randomly assigned to , eleven groups ( n = 10 ) which would be applied a different type home - bleaching procedure which contains approximately 15.5% cp . the distribution of study and control groups is described below : example of specimen ( a ) after staining , ( b , c ) embed in acrylic jaws and covered with nail polish , ( d ) prepared custom trays for bleaching application nite white excel ( nwe ) ( discus dental , usa ; 16% cp)pola night ( pn ) ( sdi , australia ; 16% cp)zaris white and brite ( zwb ) ( 3 m espe , usa ; 16% cp)opalescence ( op ) ( ultradent , usa ; 15% cp)bite and white ( bw ) ( cavex , netherland ; 15% cp)whiteness perfect ( wp ) ( fgm dental , brazil : 16% cp)rembrandt rem3 ( r3 ) ( oral - b , usa ; 15% cp)illumine ( dentsply , usa ; 15% cp)yotuel ( yo ) ( biocosmetic laboratories spain ; 16% cp)happy smile ( hs ) ( happysmileuk , uk ; 16% cp)control ( zoom , philips , nederland ; office bleaching system : 25% hp ) . nite white excel ( nwe ) ( discus dental , usa ; 16% cp ) pola night ( pn ) ( sdi , australia ; 16% cp ) zaris white and brite ( zwb ) ( 3 m espe , usa ; 16% cp ) opalescence ( op ) ( ultradent , usa ; 15% cp ) bite and white ( bw ) ( cavex , netherland ; 15% cp ) whiteness perfect ( wp ) ( fgm dental , brazil : 16% cp ) rembrandt rem3 ( r3 ) ( oral - b , usa ; 15% cp ) illumine ( dentsply , usa ; 15% cp ) yotuel ( yo ) ( biocosmetic laboratories spain ; 16% cp ) happy smile ( hs ) ( happysmileuk , uk ; 16% cp ) control ( zoom , philips , nederland ; office bleaching system : 25% hp ) . session arranged in the day time , at 37c and lasted for 1 , 2 and 4 h as recommended by the manufacturer [ table 1 ] . subgroups ( sgs ) arranged by the application time of the products ( sg-1 : 1 h products , sg-2 : 2 h products , sg-3 : 4 h products ) . were left to dry for 3 min and after bleaching the agent remnants on the teeth were carefully removed with a soft toothbrush under tap water for 3 min . in control group an office bleaching system was conducted to compare the bleaching efficacy of home - bleaching systems with the control and the other study groups . in control group approximately a 1 - 2 mm thick layer of 25% hp bleaching gel ( zoom! , philips , nederland ) was applied to the buccal surfaces of the teeth . then the light source was positioned according to the manufacturer 's instructions using the integral bite appliance guide to set the distance between the teeth and the light source ( ~6 cm ) . after each 15 min session , the bleaching gel was rinsed off and reapplied with cotton pellets . evaluation of the bleaching products used in this study digital photos of each tooth were carefully taken with a slr camera ( canon eos 650d with a macro lens 100 mm , canon , tokyo , japan ) prior to the staining , before and after each procedure and transferred to a digital imaging software ( adobe photoshop cs4 , adobe , san jose , ca , usa ) to evaluate the color changes objectively using the histogram processing ability of the software . figure 2 displays the assessment of color changes employing the digital photo processing software ( cs4 ) . the range of lightness ( l ) and hue ( h ) values are different when compared to the commission internationale de ieclairage ( cie ) l * and h * values . in photoshop , the range of the mean l * c * h * values , respectively , is 0 - 255 . the cie l * value ranges from 0 to 100 , and the cie c * and h * value ranges from 80 to + 80 . assessment of color changes employing the digital imaging analysis software as a second colorimetric measurement method , shades of the teeth were determined in the l*c*h ( lightness , chroma , and hue ) color space using spectrophotometer ( easyshade ) , which allowed images not affected by a visual determination , such as visual perception , office lighting or time of day . spectrophotometer can express the color in various values ( l * c * h * ) , can be displayed by the software of the system and compared the data with standard shade guides . total color differences or distances between two colors ( e ) are calculated automatically by the software according to the following formulas : cie color space l ( 0 - 100 ) c and h ( 80 to + 80 ) ; e = [ ( l ) + ( c ) + ( h ) ] , e = 247.4 and rgb color space l - c - h ( 0 - 255 ) ; e = 441.7 . tooth color assessments were performed with one evaluator who measured the shades at two different evaluation sessions prior to the staining , before and after bleaching . the final measurement decision was recorded only if it was an exact at both sessions . custom templates were arranged with holes on labial surfaces suitable for the tip of the spectrophotometer and to obtain a definite measurement process , a standardizing jig was used to ensure the positioning of the device is consistent . after the fully insertion of these templates double check have been performed to control the measurement areas left unpolished and clear prior to the spectrophotometric measurements [ figure 3a and b ] . spectrophotometric measurement of bleaching efficacy the results for both experimental and control groups were submitted to statistical analysis software spss 17 , ( ibm , endicott , ny , usa ) . differences in l*c*h values before and after application were tested with a repeated - measures analysis of variance ( anova ) followed by a multiple - comparison scheffe test . prior to the study , a repeatability test of the photograph shooting and the resulting color measurements was performed with posthoc tukey test . digital photos of each tooth were carefully taken with a slr camera ( canon eos 650d with a macro lens 100 mm , canon , tokyo , japan ) prior to the staining , before and after each procedure and transferred to a digital imaging software ( adobe photoshop cs4 , adobe , san jose , ca , usa ) to evaluate the color changes objectively using the histogram processing ability of the software . figure 2 displays the assessment of color changes employing the digital photo processing software ( cs4 ) . the range of lightness ( l ) and hue ( h ) values are different when compared to the commission internationale de ieclairage ( cie ) l * and h * values . in photoshop , the range of the mean l * c * h * values , respectively , is 0 - 255 . the cie l * value ranges from 0 to 100 , and the cie c * and h * value ranges from 80 to + 80 assessment of color changes employing the digital imaging analysis software as a second colorimetric measurement method , shades of the teeth were determined in the l*c*h ( lightness , chroma , and hue ) color space using spectrophotometer ( easyshade ) , which allowed images not affected by a visual determination , such as visual perception , office lighting or time of day . spectrophotometer can express the color in various values ( l * c * h * ) , can be displayed by the software of the system and compared the data with standard shade guides . total color differences or distances between two colors ( e ) are calculated automatically by the software according to the following formulas : cie color space l ( 0 - 100 ) c and h ( 80 to + 80 ) ; e = [ ( l ) + ( c ) + ( h ) ] , e = 247.4 and rgb color space l - c - h ( 0 - 255 ) ; e = 441.7 . tooth color assessments were performed with one evaluator who measured the shades at two different evaluation sessions prior to the staining , before and after bleaching . the final measurement decision was recorded only if it was an exact at both sessions . custom templates were arranged with holes on labial surfaces suitable for the tip of the spectrophotometer and to obtain a definite measurement process , a standardizing jig was used to ensure the positioning of the device is consistent . after the fully insertion of these templates double check have been performed to control the measurement areas left unpolished and clear prior to the spectrophotometric measurements [ figure 3a and b ] . the results for both experimental and control groups were submitted to statistical analysis software spss 17 , ( ibm , endicott , ny , usa ) . differences in l*c*h values before and after application were tested with a repeated - measures analysis of variance ( anova ) followed by a multiple - comparison scheffe test . prior to the study , a repeatability test of the photograph shooting and the resulting color measurements was performed with posthoc tukey test . the degree of repeatability of the photograph shooting was found to be highly reliable , as confirmed by the anova , since no significant difference ( p > 0.05 ) was observed among the values of for each specimen in each groups . l*c*h * values for each study groups , before and after coloration , as well as after each treatment phase , are presented in table 2 . table 3 presents the color differences for each study groups as well as the control group represented by e values together with statistical differences . both spectrophotometric and histogram evaluation showed , zwb had a significant increase of lightness ( ls : 24.6 , lh : 46.3 ) decrease of chroma ( cs : 2.9 , ch : 79 ) and increase in hue ( hs : 6.8 , hh : 74.8 ) that proves zwb has the highest bleaching efficacy compared to the other products ( p < 0.05 ) . pn achieved similar results as increase in lightness ( ls : 23.8 , lh : 51.1 - hs : 8.1 , hh : 68.5 ) and hue , decrease in chroma ( cs : 2.3 , ch : 67.6 ) [ tables 2 - 4 ] . therefore statistical difference between zwb group and pn group was insignificant ( p > 0.05 ) . the increase in lightness ( ls : 6.9 , lh : 10.4 - hs : 4.9 , hh : 19.7 ) and hue , decrease in chroma ( cs : 0.4 , ch : 19.1 ) and change in e values ( es : 8.5 , eh : 29.3 ) for the hs group found relatively low compared to the other groups ( p < 0.05 ) . although initial application increased e values ( e : 19.1 ) for zwb group , significant reduction in increase rates observed after the 3 , 7 and final applications [ table 3 ] . similar to this , in pn group ; the increase rate after the first application , slightly decreased in the next sessions . contrary to these findings , in all other groups except hs group ; slow but stable increase in e values has been monitored ( p > 0.05 ) . in addition , the change in l , c , h , and e values for the remaining groups found similar and differences between these values in comparison to their increase rates were statistically negligible ( p > 0.05 ) . summary of initial , postcoloration , and posttreatment 's l , h- , c , and e values es values of the experimental and control groups histogram values of l*c*h and e values ( rgb ) according to the experimental groups the data acquired from each sg according to time of use has been demonstrated in tables 24 separately . in sg-1 ; pn has the highest and hs has the lowest e value changes amongst the sg-1 after both spectrophotometric and histogram evaluations [ tables 2 - 4 ] . besides , the statistical difference in histogram results between pn and bw is insignificant ( p > 0.05 ) . for sg-2 ; zwb showed the highest e value changes when compared to the other two products ( p < 0.05 ) . likewise , the differences between the values of ih and yo were found statistically insignificant ( p > 0.05 ) . in sg-3 ; although op has higher e values than np and nwe , the statistical analysis showed there were no differences within the group . first of all , this study performed an evaluation about the bleaching effectiveness of different home - bleaching systems with similar cp concentrations . although both short- and long - term clinical efficacy of home bleaching procedures using cp have been well - documented the data about the multiple comparison of this protocol with both software and spectrophotometric analyses are relatively insufficient . the digital imaging analysis reveals the fact that even if a highly standardized photographic procedure is adopted , some factors remain that affect the lightness and color . therefore , a photographic procedure to standardize the measuring process has to be performed and that includes a 200 200 grit pure white circular spot in each picture as a neutral reference point . by this way , color deflections , shadows caused by camera flash and volume of daylight can be eliminated and measurements can be calculated using a standard image - editing software program ( adobe photoshop cs4 , adobe , san jose , ca , usa ) . in this study , software analyses were performed to validate the results and minimize the user - induced errors . because the lack of validation in past studies ; the deviations and differences in measurement process , probably effected the results . although lehmann et al . demonstrated that both traditional and advanced spectrophotometer devices showed excellent repeatability and similar results , validation with an objective software was required because of the substantial deviations of the calculated color coordinates from the spectrophotometric reference system . in this study , although all products under evaluation were home - bleaching systems with similar cp concentrations , it could be observed that there were differences in efficacy between these products , mainly due to the application time and different active ingredients except cp in these products . contrary to expectations , it is shown in this study that the application time of the bleaching gel is not directly related with the bleaching efficacy of the systems . furthermore , the statistical insignificant difference between sg-1 and sg-2 , shows 1 h of application time is enough to achieve desired level of bleaching . in the light of these facts , the continuation of color change is relatively not related with the time of use of bleaching product and it is in agreement with other in - vitro studies . similar to this , sulieman et al . showed that the bleaching efficacy is highly dependent on duration of exposure , if the cp concentration and the catalysts in the composition are identical . in addition most of the studies in scientific literature , investigate the bleaching products either in comparison with combined techniques ( office + home vs. office + home ) or different techniques ( home vs. office vs. over - the - counter ) . such studies generally focused on the assessments about in - vivo / in - vitro bleaching efficiencies of home - bleaching systems , compare the products with negative control groups . few of them tried to compare the home - bleaching products with a positive control group ( active bleaching agent ) to evaluate the effectiveness of a home - bleaching system against an office bleaching system . in this manner , none of these studies explain the bleaching efficiencies of office and home - based products used in combined techniques separately . moreover , in these combined techniques home - bleaching gels were used to be applied after office bleaching technique to enhance the bleaching . as a consequence , the efficacy of home - bleaching gels could not be measured accurately , because the saturation point of enamel was reached . therefore , in this study home - bleaching gels compared with both each other and office bleaching product . this reveals the fact that , home - bleaching gels are as effective as office bleaching products when used alone in bleaching treatments . , zwb proved to bleach faster than other home - bleaching products and relatively similar to office - based control group . although all products contain approximately 16% cp , zaris include a higher level of hp ( 5.6% ) and catalyst which leads a long lasting bleaching effect . because hp is known to penetrate tissues faster than cp , our results seem logical and confirms previous studies . tooth bleaching with home - bleaching products include cp were similarly effective as office bleaching products . it could be suggested the use cp concentrations instead of high level hp for vital tooth bleaching , in accordance with the american dental association guidelines to assure both the safety and the efficiency of bleaching treatments . although application time of the home - bleaching products has a minor influence on bleaching efficacy , concentration of the active ingredients such as carbopol affects the peroxide secretion rate . the bleaching efficacy of home - based products are as effective as professional office - based products if used properly . the results of this trial allow the following conclusions : all bleaching products performed a satisfactory bleaching efficiency while 16% cp gels whiten faster compared with 15% cp gels . the application time did not alter the efficiency of bleaching while 4 h use decrease the e values . both spectrophotometric and digital image analyses presented sufficient and objective evaluation of the bleaching efficiency and validated each other 's findings .

objective : the aim of this in - vitro study was to evaluate the efficacy of bleaching products , determine the applicability and validation of the measurement methods.materials and methods : freshly extracted 110 human incisor teeth were stained with whole blood and hemolysate solution prior to the application of 10 different home - bleaching products . spectrophotometric measurements of the tooth shades were performed for each specimen before and after bleaching at the 1st , 3rd , 7th , and 14 days . differences in lightness ( l ) , chroma ( c ) , hue ( h ) values and shade changes were measured to evaluate process . computerized digital imaging analyses to determine the color changes were performed with photoshop cs4 software ( adobe , san jose , ca , usa ) . statistical analyses were performed with analysis of variance , scheffe and tukey tests.results:in all of the test groups regardless of the material used , a significant increase in lightness and hue , and decrease of chroma were observed , as compared to the control group . after recommended bleaching applications , l and h values respectively increased in group zaris white and brite ( zwb ) and group pola night and c values showed significant decrease in groups zwb and rembrandt rem3 ( p < 0.05 ) . at the end of the procedure both spectrophotometric and digital imaging analysis showed zwb was the most effective product among the others while yotuel and happy smile were the least ( p < 0.05).conclusions : home - bleaching systems showed slower but almost permanent bleaching effect likewise office - based methods . both software and spectrophotometric analyses have advantages such as evaluating the results objectively and numerically , also treatment outcomes could be preserved .

INTRODUCTION MATERIALS AND METHODS Data collection Statistical analyses RESULTS DISCUSSION CLINICAL RELEVANCE CONCLUSIONS

tooth bleaching is one of the most noninvasive dental treatments to improve people 's appearance . the color producing materials in solution or on a surface are typically organic compounds that effect the teeth color . intrinsic tooth color is usually associated with the light scattering and adsorption properties of the enamel and dentine , while extrinsic stains tend to form in areas of the teeth that are less accessible to tooth brushing and is often promoted by smoking , dietary intake , the use of certain cationic agents such as chlorhexidine or metal salts . there are a number of studies and trials in the literature investigates the methods and procedures , two of the most applied approaches in the tooth bleaching treatment are home ( night - guard ) bleaching and in - office bleaching technique . lower concentrations of both carbamide peroxide ( cp ) and hydrogen peroxide ( hp ) are used for home - bleaching , while higher concentrations are necessary for in - office treatments . in - office bleaching application of light - sensitive , high - concentration bleaching agents associated with a power - unit usually performed in a single appointment , reduces the time required to achieve the expected results and decreases the failure possibility . the home - bleaching technique , with a custom tray , offers a conservative , cost effective method for bleaching teeth . an in - vitro study by efeoglu et al . since little information exists in the literature regarding the clinical response to bleaching treatment , there is a need for studies that simulate clinical conditions in order to evaluate the real effects of such treatment . the hypothesis to be tested is that in a clinical oral simulate condition ( in situ ) , the effects of bleaching agents are less evident than when seen in in - vitro conditions . the scope of the current study is focused on the evaluation the in - vitro bleaching efficiency of different products in similar concentrations used in home and office applications and validate the results with software and hardware methods to assess the sufficiency of the measurement techniques . our hypothesis was similar cp concentration results in similar bleaching effect and different measurement methods validates each other . a total of 110 freshly extracted for aggressive untreatable periodontitis , caries free human incisors were collected from department for oral and maxillo - facial surgery , ege university to obtain similar enamel and dentin thickness , also start - up shades . ethical approval of the ege university committee of medical ethics ( reg . all of the teeth were examined under a stereomicroscope in order to select those without surface defects . all of the specimens were stored in 1% thymol solution until used . paul , mn , usa ) removing approximately 100 m of the outermost enamel layer to obtain flat and smooth enamel surfaces . the teeth were artificially stained with whole blood and hemolysate solution prior embedding in prepared acrylic jaws with acrylic resin ( meliodent , herauskulzer , werheim , germany ) , to achieve an adequate and uniform staining [ figure 1a ] . a total of 110 teeth were embed in acrylic jaws according to the random distribution in order to make the templates for bleaching and color measurement procedures . from the beginning of the study to the end , all specimens were stored for 48 h at 37c in artificial saliva that was renewed everyday . all of the specimens randomly assigned to , eleven groups ( n = 10 ) which would be applied a different type home - bleaching procedure which contains approximately 15.5% cp . the distribution of study and control groups is described below : example of specimen ( a ) after staining , ( b , c ) embed in acrylic jaws and covered with nail polish , ( d ) prepared custom trays for bleaching application nite white excel ( nwe ) ( discus dental , usa ; 16% cp)pola night ( pn ) ( sdi , australia ; 16% cp)zaris white and brite ( zwb ) ( 3 m espe , usa ; 16% cp)opalescence ( op ) ( ultradent , usa ; 15% cp)bite and white ( bw ) ( cavex , netherland ; 15% cp)whiteness perfect ( wp ) ( fgm dental , brazil : 16% cp)rembrandt rem3 ( r3 ) ( oral - b , usa ; 15% cp)illumine ( dentsply , usa ; 15% cp)yotuel ( yo ) ( biocosmetic laboratories spain ; 16% cp)happy smile ( hs ) ( happysmileuk , uk ; 16% cp)control ( zoom , philips , nederland ; office bleaching system : 25% hp ) . nite white excel ( nwe ) ( discus dental , usa ; 16% cp ) pola night ( pn ) ( sdi , australia ; 16% cp ) zaris white and brite ( zwb ) ( 3 m espe , usa ; 16% cp ) opalescence ( op ) ( ultradent , usa ; 15% cp ) bite and white ( bw ) ( cavex , netherland ; 15% cp ) whiteness perfect ( wp ) ( fgm dental , brazil : 16% cp ) rembrandt rem3 ( r3 ) ( oral - b , usa ; 15% cp ) illumine ( dentsply , usa ; 15% cp ) yotuel ( yo ) ( biocosmetic laboratories spain ; 16% cp ) happy smile ( hs ) ( happysmileuk , uk ; 16% cp ) control ( zoom , philips , nederland ; office bleaching system : 25% hp ) . subgroups ( sgs ) arranged by the application time of the products ( sg-1 : 1 h products , sg-2 : 2 h products , sg-3 : 4 h products ) . were left to dry for 3 min and after bleaching the agent remnants on the teeth were carefully removed with a soft toothbrush under tap water for 3 min . in control group an office bleaching system was conducted to compare the bleaching efficacy of home - bleaching systems with the control and the other study groups . , philips , nederland ) was applied to the buccal surfaces of the teeth . then the light source was positioned according to the manufacturer 's instructions using the integral bite appliance guide to set the distance between the teeth and the light source ( ~6 cm ) . evaluation of the bleaching products used in this study digital photos of each tooth were carefully taken with a slr camera ( canon eos 650d with a macro lens 100 mm , canon , tokyo , japan ) prior to the staining , before and after each procedure and transferred to a digital imaging software ( adobe photoshop cs4 , adobe , san jose , ca , usa ) to evaluate the color changes objectively using the histogram processing ability of the software . figure 2 displays the assessment of color changes employing the digital photo processing software ( cs4 ) . the range of lightness ( l ) and hue ( h ) values are different when compared to the commission internationale de ieclairage ( cie ) l * and h * values . the cie l * value ranges from 0 to 100 , and the cie c * and h * value ranges from 80 to + 80 . assessment of color changes employing the digital imaging analysis software as a second colorimetric measurement method , shades of the teeth were determined in the l*c*h ( lightness , chroma , and hue ) color space using spectrophotometer ( easyshade ) , which allowed images not affected by a visual determination , such as visual perception , office lighting or time of day . spectrophotometer can express the color in various values ( l * c * h * ) , can be displayed by the software of the system and compared the data with standard shade guides . total color differences or distances between two colors ( e ) are calculated automatically by the software according to the following formulas : cie color space l ( 0 - 100 ) c and h ( 80 to + 80 ) ; e = [ ( l ) + ( c ) + ( h ) ] , e = 247.4 and rgb color space l - c - h ( 0 - 255 ) ; e = 441.7 . tooth color assessments were performed with one evaluator who measured the shades at two different evaluation sessions prior to the staining , before and after bleaching . custom templates were arranged with holes on labial surfaces suitable for the tip of the spectrophotometer and to obtain a definite measurement process , a standardizing jig was used to ensure the positioning of the device is consistent . after the fully insertion of these templates double check have been performed to control the measurement areas left unpolished and clear prior to the spectrophotometric measurements [ figure 3a and b ] . spectrophotometric measurement of bleaching efficacy the results for both experimental and control groups were submitted to statistical analysis software spss 17 , ( ibm , endicott , ny , usa ) . differences in l*c*h values before and after application were tested with a repeated - measures analysis of variance ( anova ) followed by a multiple - comparison scheffe test . prior to the study , a repeatability test of the photograph shooting and the resulting color measurements was performed with posthoc tukey test . digital photos of each tooth were carefully taken with a slr camera ( canon eos 650d with a macro lens 100 mm , canon , tokyo , japan ) prior to the staining , before and after each procedure and transferred to a digital imaging software ( adobe photoshop cs4 , adobe , san jose , ca , usa ) to evaluate the color changes objectively using the histogram processing ability of the software . figure 2 displays the assessment of color changes employing the digital photo processing software ( cs4 ) . the range of lightness ( l ) and hue ( h ) values are different when compared to the commission internationale de ieclairage ( cie ) l * and h * values . in photoshop , the range of the mean l * c * h * values , respectively , is 0 - 255 . the cie l * value ranges from 0 to 100 , and the cie c * and h * value ranges from 80 to + 80 assessment of color changes employing the digital imaging analysis software as a second colorimetric measurement method , shades of the teeth were determined in the l*c*h ( lightness , chroma , and hue ) color space using spectrophotometer ( easyshade ) , which allowed images not affected by a visual determination , such as visual perception , office lighting or time of day . spectrophotometer can express the color in various values ( l * c * h * ) , can be displayed by the software of the system and compared the data with standard shade guides . total color differences or distances between two colors ( e ) are calculated automatically by the software according to the following formulas : cie color space l ( 0 - 100 ) c and h ( 80 to + 80 ) ; e = [ ( l ) + ( c ) + ( h ) ] , e = 247.4 and rgb color space l - c - h ( 0 - 255 ) ; e = 441.7 . tooth color assessments were performed with one evaluator who measured the shades at two different evaluation sessions prior to the staining , before and after bleaching . custom templates were arranged with holes on labial surfaces suitable for the tip of the spectrophotometer and to obtain a definite measurement process , a standardizing jig was used to ensure the positioning of the device is consistent . after the fully insertion of these templates double check have been performed to control the measurement areas left unpolished and clear prior to the spectrophotometric measurements [ figure 3a and b ] . the results for both experimental and control groups were submitted to statistical analysis software spss 17 , ( ibm , endicott , ny , usa ) . differences in l*c*h values before and after application were tested with a repeated - measures analysis of variance ( anova ) followed by a multiple - comparison scheffe test . prior to the study , a repeatability test of the photograph shooting and the resulting color measurements was performed with posthoc tukey test . the degree of repeatability of the photograph shooting was found to be highly reliable , as confirmed by the anova , since no significant difference ( p > 0.05 ) was observed among the values of for each specimen in each groups . l*c*h * values for each study groups , before and after coloration , as well as after each treatment phase , are presented in table 2 . table 3 presents the color differences for each study groups as well as the control group represented by e values together with statistical differences . both spectrophotometric and histogram evaluation showed , zwb had a significant increase of lightness ( ls : 24.6 , lh : 46.3 ) decrease of chroma ( cs : 2.9 , ch : 79 ) and increase in hue ( hs : 6.8 , hh : 74.8 ) that proves zwb has the highest bleaching efficacy compared to the other products ( p < 0.05 ) . pn achieved similar results as increase in lightness ( ls : 23.8 , lh : 51.1 - hs : 8.1 , hh : 68.5 ) and hue , decrease in chroma ( cs : 2.3 , ch : 67.6 ) [ tables 2 - 4 ] . therefore statistical difference between zwb group and pn group was insignificant ( p > 0.05 ) . the increase in lightness ( ls : 6.9 , lh : 10.4 - hs : 4.9 , hh : 19.7 ) and hue , decrease in chroma ( cs : 0.4 , ch : 19.1 ) and change in e values ( es : 8.5 , eh : 29.3 ) for the hs group found relatively low compared to the other groups ( p < 0.05 ) . contrary to these findings , in all other groups except hs group ; slow but stable increase in e values has been monitored ( p > 0.05 ) . in addition , the change in l , c , h , and e values for the remaining groups found similar and differences between these values in comparison to their increase rates were statistically negligible ( p > 0.05 ) . summary of initial , postcoloration , and posttreatment 's l , h- , c , and e values es values of the experimental and control groups histogram values of l*c*h and e values ( rgb ) according to the experimental groups the data acquired from each sg according to time of use has been demonstrated in tables 24 separately . in sg-1 ; pn has the highest and hs has the lowest e value changes amongst the sg-1 after both spectrophotometric and histogram evaluations [ tables 2 - 4 ] . besides , the statistical difference in histogram results between pn and bw is insignificant ( p > 0.05 ) . for sg-2 ; zwb showed the highest e value changes when compared to the other two products ( p < 0.05 ) . likewise , the differences between the values of ih and yo were found statistically insignificant ( p > 0.05 ) . in sg-3 ; although op has higher e values than np and nwe , the statistical analysis showed there were no differences within the group . first of all , this study performed an evaluation about the bleaching effectiveness of different home - bleaching systems with similar cp concentrations . although both short- and long - term clinical efficacy of home bleaching procedures using cp have been well - documented the data about the multiple comparison of this protocol with both software and spectrophotometric analyses are relatively insufficient . the digital imaging analysis reveals the fact that even if a highly standardized photographic procedure is adopted , some factors remain that affect the lightness and color . therefore , a photographic procedure to standardize the measuring process has to be performed and that includes a 200 200 grit pure white circular spot in each picture as a neutral reference point . by this way , color deflections , shadows caused by camera flash and volume of daylight can be eliminated and measurements can be calculated using a standard image - editing software program ( adobe photoshop cs4 , adobe , san jose , ca , usa ) . in this study , software analyses were performed to validate the results and minimize the user - induced errors . because the lack of validation in past studies ; the deviations and differences in measurement process , probably effected the results . in this study , although all products under evaluation were home - bleaching systems with similar cp concentrations , it could be observed that there were differences in efficacy between these products , mainly due to the application time and different active ingredients except cp in these products . contrary to expectations , it is shown in this study that the application time of the bleaching gel is not directly related with the bleaching efficacy of the systems . in the light of these facts , the continuation of color change is relatively not related with the time of use of bleaching product and it is in agreement with other in - vitro studies . in addition most of the studies in scientific literature , investigate the bleaching products either in comparison with combined techniques ( office + home vs. office + home ) or different techniques ( home vs. office vs. over - the - counter ) . such studies generally focused on the assessments about in - vivo / in - vitro bleaching efficiencies of home - bleaching systems , compare the products with negative control groups . few of them tried to compare the home - bleaching products with a positive control group ( active bleaching agent ) to evaluate the effectiveness of a home - bleaching system against an office bleaching system . in this manner , none of these studies explain the bleaching efficiencies of office and home - based products used in combined techniques separately . moreover , in these combined techniques home - bleaching gels were used to be applied after office bleaching technique to enhance the bleaching . as a consequence , the efficacy of home - bleaching gels could not be measured accurately , because the saturation point of enamel was reached . therefore , in this study home - bleaching gels compared with both each other and office bleaching product . this reveals the fact that , home - bleaching gels are as effective as office bleaching products when used alone in bleaching treatments . , zwb proved to bleach faster than other home - bleaching products and relatively similar to office - based control group . although all products contain approximately 16% cp , zaris include a higher level of hp ( 5.6% ) and catalyst which leads a long lasting bleaching effect . tooth bleaching with home - bleaching products include cp were similarly effective as office bleaching products . it could be suggested the use cp concentrations instead of high level hp for vital tooth bleaching , in accordance with the american dental association guidelines to assure both the safety and the efficiency of bleaching treatments . although application time of the home - bleaching products has a minor influence on bleaching efficacy , concentration of the active ingredients such as carbopol affects the peroxide secretion rate . the bleaching efficacy of home - based products are as effective as professional office - based products if used properly . the results of this trial allow the following conclusions : all bleaching products performed a satisfactory bleaching efficiency while 16% cp gels whiten faster compared with 15% cp gels . the application time did not alter the efficiency of bleaching while 4 h use decrease the e values . both spectrophotometric and digital image analyses presented sufficient and objective evaluation of the bleaching efficiency and validated each other 's findings .

microbes and humans are strictly linked in every facet of the society ( evolution , economy , behaviour and lifestyle ) . for instance , malaria ( caused by plasmodium parasites ) is one of the major worldwide health emergences , and this disease represents a strong selective force on human populations . indeed , in different malaria endemic areas , exposed populations developed genetic adaptations that confer resistance to the infection ( shi and su , 2011 ) . moreover , the recent escherichia coli outbreak in germany ( nature editorial , 2011 , vol . 474 ) underlined yet again how microbes can influence our lifedetermining public health emergencies even in developed countries ( fislage , 2011 ) . on the contrary , there are several examples of beneficial interactions of microbes with plants , animals and humans , even in extreme conditions . for instance some bacteria are able to degrade contaminants and clean up polluted ecosystems ( balloi et al . , 2010 ) , plant endophytes or rhizobacteria promote soil fertility and a safe plant growth even under environmental stresses ( hayat et al . , 2010 ) , or animal gut symbionts are positively involved in the stimulation of the host 's immune system and contribute to increase nutrient availability ( kinross et al . , although humans have unconsciously learnt to harness several microbial processes from the dawn of history , for example in the preparation of food ( leavening of dough ) , beverage ( fermentation of wine and beer ) and tissues ( soaking of linen ) , only from the second half of 1800 the development of microbiology slowly built up the awareness that it was possible to exploit the microbial metabolic capabilities for humans ' benefit ( rittmann et al . , willy verstraete theorized this concept and defined the microbial resource management ( mrm ) as the human ability to manage complex microbial systems and their associated metabolic capabilities in order to solve practical problems ( verstraete , 2007 ) . this led to the development of three parameters richness ( rr ) , dynamics ( dy ) and functional organization ( fo ) to describe the complex microbial community and to answer questions like who is there? , who is doing what? , who is with whom? ( marzorati et al . , 2008 ) . this approach , originally designed for the ecological interpretation of raw fingerprinting patterns ( e.g. dgge , lhpcr , trflp ) , has been recently updated to be applied to the new molecular technologies ( i.e. pyrosequencing ) , thus allowing to provide a more accurate picture of the complexity and variability of the microbial communities ( read et al . , 2011 ) . besides proposing a series of parameters to assess the nature of a given microbial community , read and colleagues ( 2011 ) also proposed a practical mindset and a flow sheet based on the economical value of the approach , a clear determination of the endpoints , and an ecological survey to determine the proper microbial weapons , in order to logically identify the correct direction to proceed when implementing the big picture of mrm ( fig . 1 ) . this new approach inaugurated a more conscious phase of the microbial ecology , no longer dominated by the inductive method and based on empirical observations , but by the application of microbial ecology theories , capable to explain and predict the behaviour of a given microbial community . the aim was to establish the base for the control and the steering of microbial resources . a typical example is the change in prospective in the case of probiotics . at the beginning of the 20th century , elia metchinkoff , in his book the prolongation of life , hypothesized that the presence of lactic acid bacteria ( lab ) in human intestinal tract could positively affect health and longevity . he based the hypothesis on the observation of the longevity of populations used to eat high amounts of yogurt ( such as baltic populations ) . following this intuition , the concept of probiotic developed as the use of bacteria that could improve host health . however , the scientific literature presents many studies in which bacteria have been provided to humans with promising but often uncertain effects ( dunne et al . , 1999 ) . just to mention a few examples , the effect of an oral probiotic bacteriotherapy with lactobacillus rhamnosus gg previously shown to be effective in alleviating intestinal inflammation associated with food allergy in small children ( majamaa and isolauri , 1997 ) gave no beneficial effects once administrated to apple and birchpollensensitive teenagers and young adults , who manifested intermittent symptoms of allergy and mild asthma ( helin et al . the same lab was shown to reduce the duration of viral diarrheal illness in european and north african children from 1 month to 3 years of age ( guandalini et al . , 2000 ) , but not in brazilian patients with similar traits ( costaribeiro et al . , 2003 ) . these and similar studies clearly show that the effectiveness of probiotics can be related to the patient traits , dietary habits ( hehemann et al . , 2010 ) and age ( biagi et al . , 2010 ) and that different people may have different needs . these examples show that , even if mrm was initially conceived as a practical approach for the development of an elaborative system that would describe and drive the management of the resources associated to a given microbial community , the practical implementation for many environments is still complex ( read et al . , 2011 ) . this is mainly due to our limited understanding of those key factors that shape the composition and the activity of a microbial community in a complex environment . despite these limitations in fact , the recent literature in the entomological field ( a simplified environment as compared with the human gut ) can provide several examples in which the mrm concept has been used to practically solve real problems . the present work , after briefly discussing the biological role , sometime essential , of microbial simbionts in insects , aims to review these cases classifying them according to the purpose of the microbiota management : ( i ) for the control of insect pest for agriculture ; ( ii ) for the control of insecttransmitted pathogens ; ( iii ) for the protection of beneficial insects from diseases and stresses . moreover , this review will conclude analysing the possibility to develop future studies aimed to verify , improve and apply the mrm concept by using the insect one of the environmental hot topics in mrm is the gastrointestinal tract ( git ) , defined as an outside world inside the living animals ( verstraete , 2007 ) . the microbiota associated to the git is an highly complex community in which microbial cells outnumber , in the case of humans , prokaryotic cells by a factor of 10 , comprising more than 1000 microbial taxa , most of which are unique to each host individual ( dethlefsen et al . , 2007 ; ley et al . , 2008 ; costello et al . , 2009 superorganism , whose components coevolved with the host , and play an essential role for the host 's health and the metabolic regulation . with regards to the invertebrate gut , the microbial communities are generally less complex if compared with those of mammals , with one or two orders of magnitude less in terms of richness . however , remarkable differences could be found among species ( dillon and dillon , 2004 ; dunn and stabb , 2005 ; behar et al . , 2008 ; hongoh , 2010 ; robinson et al . , 2010 ; wong et al . , in fact , the former harbours several hundred species of gut microbes unique to termites , comprising protists , bacteria and archea ( hongoh , 2010 ) , while the fruit fly drosophila melanogaster less than 10 ( wong et al . , 2011 ) . despite these differences microbes exert important and crucial functions for the survival and benefit of the host also in insects . in particular , the interactions established between bacteria and insects , or arthropods in general , have been known since long to go beyond pathogenesis ( dale and moran , 2006 ) . cellular and humoral defences are deployed by insects to defend themselves from pathogens and parasites . inherited protective microbes act as an additional exogenous immune system , highlighting their great relevance in preserving insect health ( hurst and hutchence , 2010 ) . commensal bacteria can modulate the innate immune system and strengthen the epithelial barrier , limiting pathogenic bacterial contact with the epithelium by inducing the secretion of antimicrobial compounds or competing with them ( hamdi et al . , 2011 ) . for instance , in the case of aphids , we can find several examples of symbiontmediated protection . besides the obligate mutualistic symbiont buchnera aphidicola , the aphid acyrthosiphon pisum harbours one or more facultative symbionts , i.e. hamiltonella defensa , regiella insecticola and serratia symbiotica . they explicate a role of protection of the aphid against natural enemies , such as entomopathogenic fungi and parasitoid wasps , or against heat stress ( oliver et al . , 2010 ) . wolbachia infection in the fruit fly results in a strong resistance to rna virus infection ( hedges et al . , 2008 ; teixeira et al . , 2008 ) . to exploit gut microbes in a mrm approach , firstly , the diversity of the gut microbiota is linked to the genotype , diet , developmental stage , sex and physiological conditions of the host ( dethlefsen et al . , 2007 ; , it has been shown that the gut microbiome was constituted by lactobacillus , enterococcus and acetobacter members , in several studies performed on the same species by different authors ( corbyharris et al . , 2007 ; cox and gilmore , 2007 ; ren et al . , 2007 ; ryu et al . , 2008 ; crotti et al . , this is in analogy with the human gut in which recently it has been identified a studies performed on honeybees collected from different geographic regions , such as south africa ( jeyaprakash et al . , 2003 ) , germany ( mohr and tebbe , 2006 ) and switzerland ( babendreier et al . , 2007 ) , gave a similar picture : the presence of a core bacterial microbiota conserved worldwide ( hamdi et al . , 2011 ) . on the other side , in the case of the cabbage white butterfly , the bacterial community shows temporal instability at the species level and conservation at phylum level ( robinson et al . , 2010 ) . these examples show how in different species , nature apparently selected for different mechanisms of adaptation . the essential factor is to maintain the overall functionality of a community rather than to conserve the presence of particular members ( robinson et al . , 2010 ) . cases in which the gut functionality is disrupted by specific changes in the composition of the resident microbial community are known as dysbiosis . host homeostasis and it can be implicated with a pathological state , explicating a role in the occurrence of a disease . an example of insect dysbiosis has been reported by coxfoster and colleagues ( 2007 ) . by the use of a metagenomic survey , it has been demonstrated that in the microbiota of healthy bees there is a predominance of alphaproteobacteria and firmicutes , which are not found when bee specimens affected by colony collapse disorder ( ccd ) are analysed . a phenomenon of dysbiosis occurs in this case and the restoration of a healthy microbiota could counteract the microbial disequilibrium . in humans , such conditions are normally treated by means of therapeutic approaches such as bacteriotherapy ( borody et al . , 2004 ) and bioecological control ( bengmark , 2005 ) which make use of pre and probiotics ( or a combination of the two synbiotics ) in order to modulate the intestinal microbial community and improve the human health . in the next paragraph an elegant example of the manipulation of the insect microbiota is the management of the bacterial community associated to the mediterranean fruit fly , ceratitis capitata ( ben ami et al . , 2010 ; gavriel et al . , one of the strategies , commonly used to control this invasive pest , is the sterile insect technique ( sit ) that foresees , firstly , a mass rearing of overwhelming numbers of male individuals , followed by insect sterilization by gamma irradiation and finally their release in the target area . after releasing , the sterile males compete with the native males for the mating with wild females and , in a successful scenario , the reduction of the next fly generation is expected . however , several studies have emphasized that irradiated males are less competent in attracting and mating with wild females than wild males . as demonstrated by molecular tools by ben ami and colleagues ( 2010 ) , gamma irradiation influences the fly 's gut microbial community leading to a dramatic reduction of klebsiella sp . and to a problematic increase of pseudomonas sp . therefore , a clear case of dysbiosis due to the irradiation process affects phenotypically the sterile male performances . in order to restore the original microbial community , ben ami and colleagues ( 2010 ) fed the insects with the fly symbiont klebsiella oxytoca . the administration of k. oxytoca led to its stable colonization and a decrease of potentially pathogenic pseudomonas spp . , resulting in a higher mating competitiveness as compared with wild males . furthermore , other experiments performed on captured wild medflies had showed that the administration of high levels of a mix of bacteria belonging to the enterobacteriaceae family previously isolated from the fly community and in which one of the members was k. oxytoca , extended the fly 's longevity ( behar et al . , 2008 ) . this approach could be applied in order to extend the life span of sterile male insect and to enhance the success of sit programs . the reported examples show that the manipulation of the insect microbiota by the administration of members of the fly 's community can positively influences several aspects of the insect life . in mrm terms , these experiments showed that within a plan of biological control strategy against a pest , it is of key importance to consider the role of the whole microbiota of the target insect . in the three mentioned studies ( behar et al . , 2008 ; ben ami et al . , 2010 ; gavriel et al . , 2011 ) , the authors were able to reach successful results by applying an mrm approach : they use molecular tools in order to : ( i ) evaluating the microbial community structure , satisfying the question who is there ; ( ii ) defining the key microorganisms , satisfying the question who is doing what ; and ( iii ) planning the strategy to restore the suitable climax community , satisfying the question who is with whom. another strategy proposed for the control of c. capitata foresees the use of cytoplasmic incompatibility ( ci)inducing wolbachia endosymbionts as a novel environmentalfriendly tool ( zabalou et al . , 2004 ) . ceratitis capitata is generally not infected by wolbachia , although a few records referred to the presence of this symbiont in some brazilian medflies ( rocha et al . , 2005 ; coscrato et al . , wolbachia transinfections from a closely related species of the medfly , rhagoletis cerasi , allow obtaining wolbachiatransinfected lines of c. capitata , stably infected with the bacterium with rates of 100% and able to express the ci phenotype . results obtained by zabalou and colleagues ( 2004 ) evidenced that for the suppression of the insect pest a release of wolbachiainfected medflies could be successfully and efficiently used , as demonstrated by laboratory cage trials . this study is an example of a more general application of wolbachia or of other ciinducing agents in strategies defined incompatible insect technique ( iit ) . the introduction of wolbachia into pest and vector species of economic and hygienic relevance could be a powerful tool to suppress or modify natural populations . for a successful implementation of iit it is mandatory to employ an efficient sexing strain of the insect pest , in order to release only the males . thus , a medfly line infected with ciinducing wolbachia and carrying the selectable marker temperature sensitive lethal ( tsl ) for the maleonly production has been developed by zabalou and colleagues ( 2009 ) . , it has been estimated that more than 1.4 billion sterile maleonly pupae were produced per week in different facilities around the world . the sit programs contributed to the eradication of some insect species from specific regions , such as the new world screwworm eradicated from libya or the tsetse fly from zanzibar ( lindquist et al . the sterile insect technique is applied on different insect species and its economic and social benefits have been demonstrated in various cases ( vargastern et al . , 2005 ) . the process of implementing sit requires seven components : suppression of density , mass rearing , sterilization , shipment , release , evaluation , and quality control . the application of this mrm approach for sit or iit could contribute to the implementation of these techniques for the production of males more competitive than wild ones or with wolbachiainduced ci trait for other species of insect . a microbial tool widely used in biocontrol programs of specific insect species is represented by the use of the entomopathogenic bacterium bacillus thuringiensis ( bt ) . bt has been widely studied for its ability to produce parasporal crystalline protein inclusions , usually indicated as crystals , which explicate interesting and exploitable insecticidal activities . bt ability has been used worldwide for the biocontrol of insect pests and for the development of transgenic crops ( van frankenhuyzen , 2009 ) . recently , the b. thuringiensis toxin specificity database has been designed to collect information on the biological specificity of the individual crystal proteins available in literature ( k. van frankenhuyzen and c. nystrom , http://www.glfc.forestry.ca/bacillus , january 2008 ; van frankenhuyzen , 2009 ) . nowadays , bt has become the leading biological insecticide and , along with bacillus sphaericus , it has also been successfully used to control the mosquito vectors of diseases , such as dengue and malaria ( becker , 2000 ) . the use of biopesticides as a component of integrated pest management ( ipm ) have been gaining acceptance over the world . however , in some cases , the lack of proper strategy and effective application methods are among the reasons why the usage of bt is not successful , as it has been recorded for bt ssp . the application of the mrm mindset in this field could enhance the exploitation of this microbial insecticide , which has proven to possess interesting features such as the safety for nontarget organisms , high specificity , easy productivity of the commercial formulates and realistic market positioning . still nowadays infectious diseases pose real and several problems , especially in developing countries , with diseases like malaria , trypanosomiasis , lymphatic filariasis and onchocerciasis , which are vectored by arthropods . in order to eliminate or block the diffusion of a pathogen , one of the recently proposed strategies is based on the exploitation of mutualistic symbiotic bacteria , which are associated to the host vector or to the pathogenic agent and which are essential for the host survival or pathogen reproduction . in this respect , they can be considered as the final target for chemotherapy treatments. an explicative example is again on the alphaproteobacterium wolbachia . generally , wolbachia is not a primary symbiont since it is not essential for the insect survival , though exceptions have been found , like in the case of the drosophila parasitoid , asobara tabida , where wolbachia is necessary for the wasp oogenesis ( dedeine et al . , 2001 ) . on the other hand , in nematodes as brugia malayi , wuchereria bancrofti and oncocherca volvulus ( agents of lymphatic filariasis and river blindness ) wolbachia is a primary obligate symbiont , essential for the host development and survival . the principle of treating filarial diseases through antibiotic treatment exploits this strict association with the host . the therapeutic approach has been attested by multiple studies in which the antifilarial effects of antibiotics such as doxycycline or rifampicin on nematodes have been evaluated in laboratory conditions and by several clinical trials in humans ( bandi et al . ; bazzocchi et al . , 2008 ; hoerauf , 2008 ; supali et al . , 2008 ; coulibaly et al . , 2009 ; mand et al . , 2009 ; specht et al . , 2009 ; nowadays , mass drug administration ( mda ) is used worldwide for the elimination of filariasis , but the employed drugs only temporarily clear the juvenile stage of nematodes without killing all adult specimens ( gyapong et al . , 2005 ) . the antibioticbased treatments against wolbachia are among the top research priorities with new promising insights . the antiwolbachia consortium , awol , was thus established with the aim to discover and develop new antiwolbachia drugs and application , with therapies compatible with mda ( taylor et al . , 2010 ) . this is a clear example of how the manipulation of the host microbiota , with the elimination of an essential primary endosymbiont , results in the impairing of a highly virulent and pathogenic parasite . essential for the transmission of a pathogen is that the pathogen spends a period of extrinsic incubation into the vector , in order to be transmitted . this means that only the vectors from a defined age are able to transmit the pathogen , that is to say that only the oldest part of the vector population transmit the pathogen . wolbachia strain wmelpop , a symbiont of drosophila , is a lifeshortening strain , therefore able to reduce adult life span of its natural host and , as a consequence , to reduce pathogen transmission ( mcmeniman et al . , 2009 ) . a recent strategy proposes to transfer this strain in vectors of medical and agriculture importance . in order to get this achievement in mosquitotransmitted diseases , scientists firstly adapted wmelpop from drosophila in a mosquito cell culture for 3 years and then they microinjected the adapted wmelpop strain into naturally uninfected embryos of the major mosquito vector of dengue aedes aegypti . strain wmelpop halved the life span of the mosquito , inducing ci and maintaining high maternal inheritance , with no differences in fecundity ( mcmeniman et al . , 2009 ) . wolbachia is a powerful tool for the control of vectorborne diseases . in this standpoint different scenario can be pictured : ( i ) wolbachia can be used as a gene driven agent , able to drive refractory genes into the vector population ( rasgon et al . , 2006 ) ; ( ii ) wolbachiainfected males can be released into the insect population and , through wolbachiainduced ci , it could be obtained a reduction of vector population ( see previous paragraph ) ; ( iii ) insect vectors with virulent or pathogenic strains of wolbachia can be released , as the case of the aforementioned wmelpop strain , able to shorter the host life span ( mcmeniman et al . , 2009 ) . moreover , it has been observed that wolbachia is able to exert an interference with transmitted pathogens , being able to inhibit plasmodium falciparum oocysts in mosquito midgut , or the development of the infectious stage of filarial nematodes ( kambris et al . formulations based on entomopathogenic fungi have been proposed as powerful tools in the control of vectorborne diseases . metarhizium anisopliae and beauveria bassiana have been shown to efficiently infect and kill mosquito larvae in laboratory trials ( scholte et al . , 2005 ) . also recombinant strains of m. anisopliae , expressing molecules whose targets were plasmodium sporozoites , in a variation of the so called paratransgenesis approach , resulted in a high inhibition of the malaria protozoan ( fang et al . , 2011 ) . specific formulations have been developed in order to prepare a more useful and persistent product under field conditions for the control of malariatransmitting anophelines ( bukhari et al . , it is not only important to evaluate the effective agent for the foreseen application , but also to consider the best carrier for the delivery of a product and the best delivery way ( where , when and how ) in order to scale up the procedure from the laboratory condition to the open field . paratransgenesis was firstly introduced with the study carried out on the triatomine rhodnius prolixus , the vector of the parasitic protozoan trypanosoma cruzi , the causative agent of the chagas disease ( beard et al . , a member of its microbial community , rodhococcus rhodnii , essential for the growth and development of the host , has been genetically modified ( gm ) to express trypanocidal genes and then it has been a formulation based on gm bacteria , named cruzigard , has been developed , at a laboratory scale , in order to introduce gm symbionts into its host , resulting in a successful application method . similarly , in the tsetse fly glossina morsitans , vector of trypanosoma brucei , the etiological agent of the sleeping sickness , its secondary symbiont sodalis has been proposed as a paratransgenic tool to block the transmission of the disease . sodalis shows a wide tropism in the tsetse body , being mainly localized at the midgut level ( rio et al . , 2004 ) and within the cytoplasm of the secretory cells ( attardo et al . , 2008 ) . promising tools in the control of diseasetransmitting mosquitoes like anopheles are the acetic acid bacterial symbionts of the genus asaia ( favia et al . , 2007 ; crotti et al . , 2010 ) . asaia is tightly associated to different organs and tissues of the anopheles body , including salivary glands and midgut that represent key spots for the development and the transmission of the malarial pathogens . moreover , several features of asaia account for making it a powerful instrument in a applications of mrm applied to the insect microbiome : ( i ) the high prevalence and relative abundance in the mosquito individuals and populations ( favia et al . , 2007 ; chouaia et al . , 2010 ) ; ( ii ) the versatility to be transmitted by horizontal ( via cofeeding or venereal ) and vertical routes ( maternal or paternal ; damiani et al . , 2008 ; crotti et al . 2011 ) ; ( iii ) the ability to efficiently spread through insects populations supported by the capacity of the bacterium to colonize and crosscolonize phylogenetically related or distant hosts ( crotti et al . , 2009 ) ; and ( iv ) the ease to be transformable with exogenous dna ( favia et al . similarly , very recently it has been proposed another symbiont of anopheles , the gammaproteobacterium pantoea agglomerans as a potential carrier of antagonistic factors against plasmodium ( riehle et al . , 2007 ) . by using suitable heterologous secretion signals several antiplasmodium effector proteins could be efficiently secreted by the strain without apparently affecting the growth rate in the mosquito midgut ( bisi and lampe , 2011 ) . another microorganism with a potential for the control of mosquitoborne diseases is the saccharomycetales yeast , wickerhamomyces anomalus , previously known with the name of pichia anomala ( ricci et al . , 2011a , b ) . wickerhamomyces anomalus has been identified in several anopheles and aedes species as a stably associated symbiont in the host midgut and reproductive systems . great attention is placed towards the use of a paratransgenesis approach based on genetically modified yeasts that , as eukaryotic organisms , could allow solving translation and folding biases of eukaryotic recombinant proteins . insecttransmitted plant pathogens are another area in which the mrm approach could be applied with success . more precisely , research has been conducted on phytoplasmas , vectored by leafhoppers , liberibacter pathogens transmitted by psyllids , and the gammaproteobacterium xylella fastidiosa , spread by the glassywinged sharpshooter homalodisca vitripennis . all these microorganisms are responsible of plant diseases that cause devastating yield losses in diverse low and highvalue crops worldwide . disease control is commonly based on the control of the insects , i.e. by spraying various insecticides , and on practices that consist in the removal of symptomatic plants . however , some first steps of mrm applications have been already carried out on the vectors , with the aim of defining the microbial community composition and functionality in the insects ( marzorati et al . , 2006 ; crotti et al . , 2009 ; raddadi et al . , the final aim is to propose a biocontrol approach based on the management of the microbial symbionts associated to the vectors in order to counteract directly the pathogen or to reduce the vector competence . an example is represented by the pierce 's disease of grape caused by the above mentioned x. fastidiosa . a culturable bacterial symbiont of the x. fastidiosa vector h. vitripennis has been isolated from the host foregut . denitrificans , was capable of colonizing the same niche , the foregut , occupied by x. fastidiosa indicating that it has the basic potential of counteracting the pathogen for instance by competitive exclusion during the colonization of the host foregut . by using a variant of the strain transformed with a plasmid for the expression of a fluorescent protein , it was possible to track the behaviour of the symbiont within the host body . a characteristic potentially very useful for the development of an approach of symbiotic control of the pierce 's disease is the versatility of the strain in colonizing different host type . such a feature could be positively exploited to increase the exposure of the transmitted pathogen to antagonistic factors expressed by the bacterial symbiont not only at the level of the insect body but in the target plant species too ( bextine et al . , 2004 ; bextine et al . , 2005 ; miller , 2011 ) . when people think to insects , or arthropods in general , they have the idea of pests or disease vectors. however , most of the insects are useful for human and environmental benefit . some of them ( bees , wasps , butterflies and ants ) are pollinators , others reduce the population of harmful insects , representing a real alternative to chemical application . others produce useful substances for human activities , as honey , wax , lacquer and silk . lastly , in many countries , insects are a part of people 's diets and edible insects , such as caterpillars and grubs , are important sources of protein . nowadays , a serious environmental problem is the decline of pollinators and a number of firms are working in the perspective of producing insect species for pollination management in the field , orchards and greenhouses at the flowering time . honeybees and bumblebees are sold worldwide and guidelines and operative protocols are provided to farmers for an optimal application . however , these beneficial insects are coping with severe stresses , including both abiotic and biotic ones ( e.g. parasites , fungi , bacteria and viruses ) , which are seriously affecting their wellness , activity and productivity . management of microbial symbionts could represent a mean to enhance the defences of beneficial insects from pathogens ' attacks . some microbial groups , as lab or acetic acid bacteria ( aab ) , have been reported as able to enhance innate immune system of bees or fruit flies ( evans and lopez , 2004 ; ryu et al . , 2008 ) . indeed , lab and aab are generating a lot of interest in apiculture , the former for the potential probiotic activity , the latter because it has been shown to be abundant and prevalent symbionts in healthy insects with sugarbased diets ( crotti et al . , 2010 ) . lab and aab own specific features that make them efficient colonizers of the bee midgut in comparison to acidsensitive pathogens . for instance they are able to tolerate low ph , to produce organic acids and to utilize a wide range of sugars , interfering with the potential establishment of pathogenic bacteria . other commensals of the honey bee gut like those of bacillus and related genera have been recently shown to have an antagonistic effect against paenibacillus larvae , the causative agent of american foulbrood disease ( afb , cherif et al . , we can say that this could open the possibility in mrm terms of acting on the microbial structure and functionality of a specific niche in order to reestablish a good balance of the microbiota with a benefit for the host . recently , by using artificial microcosms , it has been proved that microorganisms , once present in a suitable climax community , guarantee a high functionality of the system even during stressing events ( wittebolle et al . , 2009 ) . in the case of the gut microbiota , this functionality contributes to the host protection against pathogen infections ( see the review of hamdi et al . , 2011 ) . in particular in a recent work , it was demonstrated that structural changes in the midgut bacterial communities of cabbage white butterfly ( pieris rapae ) larvae , due to variations in the diet , enhanced the susceptibility to biological invasion . the community of a pool of larvae fed with an artificial diet was compared with other two pools of larvae fed with the same diet , but enriched with brussels sprouts or sinigrin respectively ( both exert an antimicrobial activity ) . in the second trial , larvae were fed with a sterile artificial diet both in the presence and in absence of antibiotics . subsequently , the larvae were exposed to bacteria , commonly present within the larval microbiota , but exogenous to the diet . at the end of the treatment , the microbial community of all the larvae the study revealed that , compared with the microbiota of the larvae reared with the sterile artificial diet , those exposed to antibiotics , brussels sprouts and sinigrin were altered in their structure , resulting to be more susceptible to the invasion ( robinson et al . , 2010 ) . this study , which provides clear evidences on the importance of the native community structure in preventing exogenous invasions , results in particular interest when the mrm parameters are applied to describe the degree of the perturbation of the microbiota organization in the different treatments . of particular utility are the ecological pareto value ( ep ) , which describes the optimal microbial community organization for a specific environment , and the community distortion factor ( cd ) that calculates the degree to which the community organization ( co ) is different from the ecological pareto value ( read et al . , 2011 ) . in both proposed experiments we can consider as the ep value the one referred to the structure of the microbiota of the control community ( sterile diet ) and as co the value of the microbiota subjected to changes in the diet . in both experiments , the cd factors resulted in a value different from the one of ep , indicating that the communities have a low resistance to the applied perturbations ( co values were 24.04 , 24.03 and 33.72 for the communities of the larvae fed with sinigrin , brussels sprouts and antibiotics respectively ) . these results numerically support the observation that perturbations can decrease the resistance of the communities to invasion . in this review , we have evaluated the different possibilities in which the manipulation of the microbial community associated to the insects can be carried out in order to obtain multiple benefits . however , this is just the top of the iceberg and many other possibilities lay in the future . the influence of the microbial partners on the biology and evolution of a eukaryotic host is nowadays well recognized but the main drivers are frequently unknown . this theory considers the holobiont ( the host organism and its symbiotic microbiota ) with its hologenome ( the sum of the genetic information of the host and its microbiota ) acting in a consortium as a dynamic entity and a unit of selection in which some microorganisms multiply and other decrease in number as a function of local condition within the holobiont ( rosenberg and zilberrosenberg , 2011 ) . due to such a close relationship , the possibility of managing the microbial community opens several perspectives in terms of mrm in relation to the comprehensive characterization of the microbiota and the determination of its role in health and disease . the understanding of these principles and the definition of general ecological rules are of key importance to implement mrm to practice . for instance , this is the aim of the human microbiome project that has been initiated by the nih roadmap ( http://nihroadmap.nih.gov/hmp/ ) . however , mammals are far too complex for basic ecological studies . on the contrary , this is not the case for insects that , in comparison to humans , are a more simplified system . this leads to a double opportunity for the insects . on the one side , due to their relatively easy growth under controlled conditions , the possibility to manipulate both hosts and symbionts , the ability to determine precisely the kind of interactions between the partners and the possibility to measure the effects of these interactions , insects can be a more handy holobiont to study specific theories of microbial ecology and develop new aspects of mrm approach . on the other side , extra work has to be conducted to further exploit the mrm approach in the insect world . for example , the already developed mrm parameters ( marzorati et al . , 2008 ; read et al . , 2011 ) do not take in consideration the role of the communication occurring among cells within the microbiota and between cells host and microbiota . the cellular communicative strategies , inter and intrataxa , are quite complex , comprising conjugation systems , secretory systems , systems that use small hormonelike signalling molecules , plasmodesmatas , gap junctions and tunnelling nanotubes and probably other still unknown mechanisms ( dubey and benyehuda , 2011 ) . this ecological aspect can be a promising field of application of mrm to control and manage the ecosystem symbiontinsect .

summarymicroorganisms establish with their animal hosts close interactions . they are involved in many aspects of the host life , physiology and evolution , including nutrition , reproduction , immune homeostasis , defence and speciation . thus , the manipulation and the exploitation the microbiota could result in important practical applications for the development of strategies for the management of insectrelated problems . this approach , defined as microbial resource management ( mrm ) , has been applied successfully in various environments and ecosystems , as wastewater treatments , prebiotics in humans , anaerobic digestion and so on . mrm foresees the proper management of the microbial resource present in a given ecosystem in order to solve practical problems through the use of microorganisms . in this review we present an interesting field for application for mrm concept , i.e. the microbial communities associated with arthropods and nematodes . several examples related to this field of applications are presented . insect microbiota can be manipulated : ( i ) to control insect pests for agriculture ; ( ii ) to control pathogens transmitted by insects to humans , animals and plants ; ( iii ) to protect beneficial insects from diseases and stresses . besides , we prospect further studies aimed to verify , improve and apply mrm by using the insect symbiont ecosystem as a model .

Introduction MRM of the insect microbiota Symbiont management in insect pests for agriculture Symbiont management in insect vectors to control the carried pathogens Symbiont management in the protection of beneficial insects Future perspectives

microbes and humans are strictly linked in every facet of the society ( evolution , economy , behaviour and lifestyle ) . on the contrary , there are several examples of beneficial interactions of microbes with plants , animals and humans , even in extreme conditions . , 2010 ) , or animal gut symbionts are positively involved in the stimulation of the host 's immune system and contribute to increase nutrient availability ( kinross et al . , although humans have unconsciously learnt to harness several microbial processes from the dawn of history , for example in the preparation of food ( leavening of dough ) , beverage ( fermentation of wine and beer ) and tissues ( soaking of linen ) , only from the second half of 1800 the development of microbiology slowly built up the awareness that it was possible to exploit the microbial metabolic capabilities for humans ' benefit ( rittmann et al . , willy verstraete theorized this concept and defined the microbial resource management ( mrm ) as the human ability to manage complex microbial systems and their associated metabolic capabilities in order to solve practical problems ( verstraete , 2007 ) . this led to the development of three parameters richness ( rr ) , dynamics ( dy ) and functional organization ( fo ) to describe the complex microbial community and to answer questions like who is there? this approach , originally designed for the ecological interpretation of raw fingerprinting patterns ( e.g. dgge , lhpcr , trflp ) , has been recently updated to be applied to the new molecular technologies ( i.e. pyrosequencing ) , thus allowing to provide a more accurate picture of the complexity and variability of the microbial communities ( read et al . besides proposing a series of parameters to assess the nature of a given microbial community , read and colleagues ( 2011 ) also proposed a practical mindset and a flow sheet based on the economical value of the approach , a clear determination of the endpoints , and an ecological survey to determine the proper microbial weapons , in order to logically identify the correct direction to proceed when implementing the big picture of mrm ( fig . this new approach inaugurated a more conscious phase of the microbial ecology , no longer dominated by the inductive method and based on empirical observations , but by the application of microbial ecology theories , capable to explain and predict the behaviour of a given microbial community . following this intuition , the concept of probiotic developed as the use of bacteria that could improve host health . these examples show that , even if mrm was initially conceived as a practical approach for the development of an elaborative system that would describe and drive the management of the resources associated to a given microbial community , the practical implementation for many environments is still complex ( read et al . this is mainly due to our limited understanding of those key factors that shape the composition and the activity of a microbial community in a complex environment . despite these limitations in fact , the recent literature in the entomological field ( a simplified environment as compared with the human gut ) can provide several examples in which the mrm concept has been used to practically solve real problems . the present work , after briefly discussing the biological role , sometime essential , of microbial simbionts in insects , aims to review these cases classifying them according to the purpose of the microbiota management : ( i ) for the control of insect pest for agriculture ; ( ii ) for the control of insecttransmitted pathogens ; ( iii ) for the protection of beneficial insects from diseases and stresses . moreover , this review will conclude analysing the possibility to develop future studies aimed to verify , improve and apply the mrm concept by using the insect one of the environmental hot topics in mrm is the gastrointestinal tract ( git ) , defined as an outside world inside the living animals ( verstraete , 2007 ) . , 2009 superorganism , whose components coevolved with the host , and play an essential role for the host 's health and the metabolic regulation . with regards to the invertebrate gut , the microbial communities are generally less complex if compared with those of mammals , with one or two orders of magnitude less in terms of richness . despite these differences microbes exert important and crucial functions for the survival and benefit of the host also in insects . for instance , in the case of aphids , we can find several examples of symbiontmediated protection . besides the obligate mutualistic symbiont buchnera aphidicola , the aphid acyrthosiphon pisum harbours one or more facultative symbionts , i.e. to exploit gut microbes in a mrm approach , firstly , the diversity of the gut microbiota is linked to the genotype , diet , developmental stage , sex and physiological conditions of the host ( dethlefsen et al . by the use of a metagenomic survey , it has been demonstrated that in the microbiota of healthy bees there is a predominance of alphaproteobacteria and firmicutes , which are not found when bee specimens affected by colony collapse disorder ( ccd ) are analysed . a phenomenon of dysbiosis occurs in this case and the restoration of a healthy microbiota could counteract the microbial disequilibrium . , 2004 ) and bioecological control ( bengmark , 2005 ) which make use of pre and probiotics ( or a combination of the two synbiotics ) in order to modulate the intestinal microbial community and improve the human health . in the next paragraph an elegant example of the manipulation of the insect microbiota is the management of the bacterial community associated to the mediterranean fruit fly , ceratitis capitata ( ben ami et al . , one of the strategies , commonly used to control this invasive pest , is the sterile insect technique ( sit ) that foresees , firstly , a mass rearing of overwhelming numbers of male individuals , followed by insect sterilization by gamma irradiation and finally their release in the target area . after releasing , the sterile males compete with the native males for the mating with wild females and , in a successful scenario , the reduction of the next fly generation is expected . this approach could be applied in order to extend the life span of sterile male insect and to enhance the success of sit programs . the reported examples show that the manipulation of the insect microbiota by the administration of members of the fly 's community can positively influences several aspects of the insect life . , 2011 ) , the authors were able to reach successful results by applying an mrm approach : they use molecular tools in order to : ( i ) evaluating the microbial community structure , satisfying the question who is there ; ( ii ) defining the key microorganisms , satisfying the question who is doing what ; and ( iii ) planning the strategy to restore the suitable climax community , satisfying the question who is with whom. another strategy proposed for the control of c. capitata foresees the use of cytoplasmic incompatibility ( ci)inducing wolbachia endosymbionts as a novel environmentalfriendly tool ( zabalou et al . results obtained by zabalou and colleagues ( 2004 ) evidenced that for the suppression of the insect pest a release of wolbachiainfected medflies could be successfully and efficiently used , as demonstrated by laboratory cage trials . for a successful implementation of iit it is mandatory to employ an efficient sexing strain of the insect pest , in order to release only the males . thus , a medfly line infected with ciinducing wolbachia and carrying the selectable marker temperature sensitive lethal ( tsl ) for the maleonly production has been developed by zabalou and colleagues ( 2009 ) . a microbial tool widely used in biocontrol programs of specific insect species is represented by the use of the entomopathogenic bacterium bacillus thuringiensis ( bt ) . bt ability has been used worldwide for the biocontrol of insect pests and for the development of transgenic crops ( van frankenhuyzen , 2009 ) . recently , the b. thuringiensis toxin specificity database has been designed to collect information on the biological specificity of the individual crystal proteins available in literature ( k. van frankenhuyzen and c. nystrom , http://www.glfc.forestry.ca/bacillus , january 2008 ; van frankenhuyzen , 2009 ) . the use of biopesticides as a component of integrated pest management ( ipm ) have been gaining acceptance over the world . however , in some cases , the lack of proper strategy and effective application methods are among the reasons why the usage of bt is not successful , as it has been recorded for bt ssp . the application of the mrm mindset in this field could enhance the exploitation of this microbial insecticide , which has proven to possess interesting features such as the safety for nontarget organisms , high specificity , easy productivity of the commercial formulates and realistic market positioning . in order to eliminate or block the diffusion of a pathogen , one of the recently proposed strategies is based on the exploitation of mutualistic symbiotic bacteria , which are associated to the host vector or to the pathogenic agent and which are essential for the host survival or pathogen reproduction . in this respect , they can be considered as the final target for chemotherapy treatments. generally , wolbachia is not a primary symbiont since it is not essential for the insect survival , though exceptions have been found , like in the case of the drosophila parasitoid , asobara tabida , where wolbachia is necessary for the wasp oogenesis ( dedeine et al . the therapeutic approach has been attested by multiple studies in which the antifilarial effects of antibiotics such as doxycycline or rifampicin on nematodes have been evaluated in laboratory conditions and by several clinical trials in humans ( bandi et al . this is a clear example of how the manipulation of the host microbiota , with the elimination of an essential primary endosymbiont , results in the impairing of a highly virulent and pathogenic parasite . essential for the transmission of a pathogen is that the pathogen spends a period of extrinsic incubation into the vector , in order to be transmitted . in order to get this achievement in mosquitotransmitted diseases , scientists firstly adapted wmelpop from drosophila in a mosquito cell culture for 3 years and then they microinjected the adapted wmelpop strain into naturally uninfected embryos of the major mosquito vector of dengue aedes aegypti . in this standpoint different scenario can be pictured : ( i ) wolbachia can be used as a gene driven agent , able to drive refractory genes into the vector population ( rasgon et al . , 2006 ) ; ( ii ) wolbachiainfected males can be released into the insect population and , through wolbachiainduced ci , it could be obtained a reduction of vector population ( see previous paragraph ) ; ( iii ) insect vectors with virulent or pathogenic strains of wolbachia can be released , as the case of the aforementioned wmelpop strain , able to shorter the host life span ( mcmeniman et al . moreover , it has been observed that wolbachia is able to exert an interference with transmitted pathogens , being able to inhibit plasmodium falciparum oocysts in mosquito midgut , or the development of the infectious stage of filarial nematodes ( kambris et al . also recombinant strains of m. anisopliae , expressing molecules whose targets were plasmodium sporozoites , in a variation of the so called paratransgenesis approach , resulted in a high inhibition of the malaria protozoan ( fang et al . specific formulations have been developed in order to prepare a more useful and persistent product under field conditions for the control of malariatransmitting anophelines ( bukhari et al . , it is not only important to evaluate the effective agent for the foreseen application , but also to consider the best carrier for the delivery of a product and the best delivery way ( where , when and how ) in order to scale up the procedure from the laboratory condition to the open field . paratransgenesis was firstly introduced with the study carried out on the triatomine rhodnius prolixus , the vector of the parasitic protozoan trypanosoma cruzi , the causative agent of the chagas disease ( beard et al . , a member of its microbial community , rodhococcus rhodnii , essential for the growth and development of the host , has been genetically modified ( gm ) to express trypanocidal genes and then it has been a formulation based on gm bacteria , named cruzigard , has been developed , at a laboratory scale , in order to introduce gm symbionts into its host , resulting in a successful application method . similarly , in the tsetse fly glossina morsitans , vector of trypanosoma brucei , the etiological agent of the sleeping sickness , its secondary symbiont sodalis has been proposed as a paratransgenic tool to block the transmission of the disease . asaia is tightly associated to different organs and tissues of the anopheles body , including salivary glands and midgut that represent key spots for the development and the transmission of the malarial pathogens . moreover , several features of asaia account for making it a powerful instrument in a applications of mrm applied to the insect microbiome : ( i ) the high prevalence and relative abundance in the mosquito individuals and populations ( favia et al . , 2010 ) ; ( ii ) the versatility to be transmitted by horizontal ( via cofeeding or venereal ) and vertical routes ( maternal or paternal ; damiani et al . 2011 ) ; ( iii ) the ability to efficiently spread through insects populations supported by the capacity of the bacterium to colonize and crosscolonize phylogenetically related or distant hosts ( crotti et al . similarly , very recently it has been proposed another symbiont of anopheles , the gammaproteobacterium pantoea agglomerans as a potential carrier of antagonistic factors against plasmodium ( riehle et al . wickerhamomyces anomalus has been identified in several anopheles and aedes species as a stably associated symbiont in the host midgut and reproductive systems . great attention is placed towards the use of a paratransgenesis approach based on genetically modified yeasts that , as eukaryotic organisms , could allow solving translation and folding biases of eukaryotic recombinant proteins . more precisely , research has been conducted on phytoplasmas , vectored by leafhoppers , liberibacter pathogens transmitted by psyllids , and the gammaproteobacterium xylella fastidiosa , spread by the glassywinged sharpshooter homalodisca vitripennis . disease control is commonly based on the control of the insects , i.e. , the final aim is to propose a biocontrol approach based on the management of the microbial symbionts associated to the vectors in order to counteract directly the pathogen or to reduce the vector competence . a culturable bacterial symbiont of the x. fastidiosa vector h. vitripennis has been isolated from the host foregut . denitrificans , was capable of colonizing the same niche , the foregut , occupied by x. fastidiosa indicating that it has the basic potential of counteracting the pathogen for instance by competitive exclusion during the colonization of the host foregut . by using a variant of the strain transformed with a plasmid for the expression of a fluorescent protein , it was possible to track the behaviour of the symbiont within the host body . a characteristic potentially very useful for the development of an approach of symbiotic control of the pierce 's disease is the versatility of the strain in colonizing different host type . however , these beneficial insects are coping with severe stresses , including both abiotic and biotic ones ( e.g. management of microbial symbionts could represent a mean to enhance the defences of beneficial insects from pathogens ' attacks . some microbial groups , as lab or acetic acid bacteria ( aab ) , have been reported as able to enhance innate immune system of bees or fruit flies ( evans and lopez , 2004 ; ryu et al . indeed , lab and aab are generating a lot of interest in apiculture , the former for the potential probiotic activity , the latter because it has been shown to be abundant and prevalent symbionts in healthy insects with sugarbased diets ( crotti et al . , we can say that this could open the possibility in mrm terms of acting on the microbial structure and functionality of a specific niche in order to reestablish a good balance of the microbiota with a benefit for the host . recently , by using artificial microcosms , it has been proved that microorganisms , once present in a suitable climax community , guarantee a high functionality of the system even during stressing events ( wittebolle et al . at the end of the treatment , the microbial community of all the larvae the study revealed that , compared with the microbiota of the larvae reared with the sterile artificial diet , those exposed to antibiotics , brussels sprouts and sinigrin were altered in their structure , resulting to be more susceptible to the invasion ( robinson et al . of particular utility are the ecological pareto value ( ep ) , which describes the optimal microbial community organization for a specific environment , and the community distortion factor ( cd ) that calculates the degree to which the community organization ( co ) is different from the ecological pareto value ( read et al . in both experiments , the cd factors resulted in a value different from the one of ep , indicating that the communities have a low resistance to the applied perturbations ( co values were 24.04 , 24.03 and 33.72 for the communities of the larvae fed with sinigrin , brussels sprouts and antibiotics respectively ) . in this review , we have evaluated the different possibilities in which the manipulation of the microbial community associated to the insects can be carried out in order to obtain multiple benefits . the influence of the microbial partners on the biology and evolution of a eukaryotic host is nowadays well recognized but the main drivers are frequently unknown . this theory considers the holobiont ( the host organism and its symbiotic microbiota ) with its hologenome ( the sum of the genetic information of the host and its microbiota ) acting in a consortium as a dynamic entity and a unit of selection in which some microorganisms multiply and other decrease in number as a function of local condition within the holobiont ( rosenberg and zilberrosenberg , 2011 ) . due to such a close relationship , the possibility of managing the microbial community opens several perspectives in terms of mrm in relation to the comprehensive characterization of the microbiota and the determination of its role in health and disease . on the one side , due to their relatively easy growth under controlled conditions , the possibility to manipulate both hosts and symbionts , the ability to determine precisely the kind of interactions between the partners and the possibility to measure the effects of these interactions , insects can be a more handy holobiont to study specific theories of microbial ecology and develop new aspects of mrm approach . this ecological aspect can be a promising field of application of mrm to control and manage the ecosystem symbiontinsect .

cancer represents a major public health problem and is a leading cause of death worldwide . as reported by the american cancer society , more than 1.6 million new cases were diagnosed in 2013 , and one in four deaths in the us is cancer related . the standard of care for many cancers typically includes surgery , chemotherapy , and radiation therapy . although these traditional clinical measures have proven their efficacy in cancer treatment , patients often experience debilitating side effects that significantly reduce their quality of life . in addition , cancer relapse with treatment resistance underscores the urgent need to identify novel molecular targets for the development of alternative therapies . about 13% of currently known drugs whose primary therapeutic targets are ion channels are being used for the treatment of a variety of human conditions , including cardiovascular and neurological disorders ( overington et al . , 2006 ) . although our knowledge of ion channel functions in the physiological and pathological conditions of excitable cells such as cardiomyocytes and neurons is vast , it is only recently that the manifold functions of ion channels in driving malignant cancer cell behaviors have been reported . potassium channels represent the most diverse group of ion channels , and have enormous potential as therapeutic targets for personalized cancer treatment , given their huge functional and structural diversity . in this review we summarize the dysregulated potassium channel expression in cancer , discuss the complex mechanisms potassium channels use for regulating cancer cell proliferation and migration , illustrate the therapeutic value for targeting potassium channel in preclinical models , and outline the major unanswered questions in the field . we direct the readers to excellent reviews on the functional roles of other ion channel classes in cancer ( sodium channels : roger et al . , 2006 ; djamgoz and onkal , 2013 ; fraser et al . , 2014 ; calcium channels : yang et al . , 2010 ; , 2011 ; santoni and farfariello , 2011 ; ouadid - ahidouch et al . , 2013 ; chloride channels : cuddapah and sontheimer , 2011 ) . the > 400 ion channel encoding genes represent 1.5% of the human genome , and ion channels display vast structural and functional diversity . these channels are central regulators of the distribution of potassium , sodium , calcium , and chloride ions for cellular ionic homeostasis and contribute to essentially all fundamental cellular processes . here we focus on potassium channels , which selectively conduct potassium ions across the membrane down their electrochemical gradient . because the sodium - potassium pump ( na - k - atpase ) transports two potassium molecules into the cell and three sodium molecules out of the cell at the cost of hydrolyzing one atp molecule , this results in a higher intracellular potassium concentration . once a potassium channel opens , it allows potassium ions to flow down its concentration gradient . this potassium conductance will drive the cell membrane potential toward the equilibrium potential for potassium . in nonexcitable cells or excitable cells at their basal state , the resting membrane potential is generally maintained at negative values ranging from 30 to 85 mv . the membrane potential can either become more negative ( a process called hyperpolarization ) or more positive ( a process called depolarization ) compared with the resting potential as various ion channels open . because potassium channels dominate the ion conductance at the resting state , the resting membrane potential is slightly more positive than the equilibrium potential for potassium . so , in general , potassium channel activation would cause hyperpolarization , whereas opening of sodium channels or chloride channels would cause depolarization . with 78 members , potassium channels can be divided into four main classes based on their domain structure and activation mechanisms ( fig . 1 ) . voltage - gated potassium channels ( kv ) , encoded by 40 genes in humans , are the largest subset of potassium channels gated by changes in the membrane potential . calcium - activated potassium channels ( kca ) are activated by intracellular calcium . according to their conductance , kca channels can be further divided into big conductance ( bk ) , intermediate conductance ( ik ) , and small conductance ( sk ) channels . kv and kca channels share a similar domain organization and are composed of four pore - lining subunits , each with six transmembrane domains ( tms ) and one pore - forming region , except for the bk channel , which has one additional transmembrane segment in the n terminus . the tm14 represents the voltage - sensing domains ( vsds ) for kv channels ( fig . 1 ) . inward rectifying potassium channels ( kir ) possess two transmembrane segments flanking one pore loop in each of the four subunits ( fig . 1 ) , and they pass potassium ions more easily in the inward direction ( into the cell ) than the outward direction ( out of the cell ) . two - pore domain potassium channels ( k2p ) have two pore domains per subunit , each pore domain with two transmembrane segments flanking a reentrant pore loop ( fig . 1 ) . two such subunits form a k2p channel that is usually constitutively open as a leak channel for maintaining a negative membrane potential . while the pore domain of all potassium channels possesses a highly conserved gyg / gfg signature motif , the molecular diversity of potassium channels is immense . potassium channels form homomeric or heteromeric complexes assembled with subunits from the same subfamily . the presence of auxiliary subunits can modify the functional properties and further diversify these channels . for example , a bk channel can be activated at resting membrane potential without an increase of intracellular calcium concentration by forming a complex with an auxiliary protein ( yan and aldrich , 2010 ) . potassium channel mrnas are subjected to rna editing ( bhalla et al . , 2004 ; garrett and rosenthal , 2012 ; ryan et al . , 2012 ) and alternative splicing ( schwarz et al . , 1988 ; xie and mccobb , 1998 ; sun et al . , 2009 ) , and the channel proteins can be further modified at the posttranslational level by phosphorylation ( park et al . , 2008 ) , palmitoylation ( shipston , 2011 ) , and glycosylation ( khanna et al . , 2001 ; gong et al . , 2002 ) . these rna and protein modifications together with the mix and match of channel subunits afford tremendous versatility of channel behaviors . ( top ) calcium - activated potassium channels ( kca ) and voltage - gated potassium ( kv ) channels are composed of four pore - lining subunits . whereas sk and ik channels resemble kv channels in having six transmembrane segments ( tm1tm6 ) and a reentrant pore loop between tm5 and tm6 per subunit , bk channels have one additional transmembrane segment in the n terminus of the subunit whereas its cytoplasmic c - terminal domain confers the channel s calcium sensitivity . the first four transmembrane segments ( tm1tm4 ) of kv channel subunit form the voltage sensor domain , whereas the remainder of the transmembrane domain corresponds to the pore domain conserved in all potassium channels ( isacoff et al . , 2013 ) . the inward rectifying potassium channel ( kir ) possesses two transmembrane domains with a pore - forming region ( p ) in between . the k2p channel is the so - called background potassium channel that is made of four transmembrane domains and two pore - forming regions . all potassium channels require a tetrameric arrangement of the pore - forming regions of the subunits to form the potassium - selective filter , therefore a complete conductive channel is a tetramer for the one - pore channels ( kv , kca , and kir ) or dimer for the k2p channels . the resting cell membrane potential is hyperpolarized due to the imbalance of potassium and sodium ion distribution caused by the electrogenic function of na - k - atpase . ( bottom ) phylogenetic dendrogram shows that the various potassium channels involved in oncogenic processes belong to all four classes . these potassium channels implicated in oncological processes belong to all four main classes ( fig . 1 ) . for example , overexpression of the voltage - gated potassium channel kv1.1 marks a subgroup of medulloblastoma ( taylor et al . , 2012 ) ; elevated kv1.3 expression is detected in a number of human malignancies including breast , colon , and prostate cancer ( comes et al . , 2013 ) ; high kv11.1 ( herg ) expression marks both solid and blood cancer ( pillozzi et al . , 2002 ; jehle et al . , 2011 ) ; and kv10.1 ( eag1 ) overexpression is found in > 70% human cancer types of various organs ( hemmerlein et al . , 2006 ) . overexpression of the g - protein activated inwardly rectifying potassium channel girk1 ( kcnj3 ) is correlated with the presence and degree of breast cancer lymph node metastases ( stringer et al . , 2001 ) . overexpression of a specific splice isoform of the bk channel correlates with the malignancy grade of glioma ( liu et al . , 2002 ) . the k2p channel kcnk9 is overexpressed in breast and lung cancer ( mu et al . , 2003 ) . potassium channel expression in cancer expression of various potassium channels that have been implicated in cancer . red boxes indicate that overexpression is reported ( in most cases ) and that the channel enhances tumorigenic processes , for example by driving proliferation , cell migration , or metastasis ( bielanska et al . , 2009 ; williams et al . , 2013 green boxes indicate that the channel expression is inversely correlated with tumor malignancy and clinical aggressiveness , such as kcna5 in glioma ( preussat et al . , 2003 ) and lymphoma ( bielanska et al . , 2009 ) , and kcnq1 in colorectal cancer ( than et al . , 2013 ) . the blue box indicates the presence of recurring somatic mutations in the kcnj5 gene detected in adrenal adenomas ( choi et al . , 2011 ) . does the dysregulated potassium channel expression ( mostly overexpression ) initiate tumorigenesis , confer advantages to malignant growth or metastatic spread once the tumor forms , or is the aberrant expression simply a consequence of neoplastic transformation without functional significance ? two potassium channels have been shown to possess tumorigenic capacity when ectopically expressed in heterologous systems . aggressive tumor growth is induced by transfecting eag1 into chinese hamster ovary ( cho ) cells ( pardo et al . , 1999 ) , or retroviral induction of kcnk9 expression in normal mouse mammary gland epithelial cells and mouse embryonic fibroblast cells ( mu et al . , 2003 so it is important to determine whether overexpression of a specific potassium channel in situ of an animal model is tumorigenic , which is a more stringent criterion for an oncogene . considering the fact that pharmacological inhibition or genetic suppression of potassium channels reduces growth in multiple cancer types ( pardo and sthmer , 2014 ; urrego et al . , 2014 ) , it is possible that most potassium channels take up a permissive role to mostly function as essential regulators for various cancer cell behaviors such as proliferation and migration , and that their up - regulation provides a repertoire for sufficient channel activity when necessary . strikingly , up - regulation of the plant orthologue of shaker - like voltage - gated potassium channel akt1 has been detected in arabidopsis thaliana tumors induced by bacterial infection , and the plant tumor growth is reduced in akt1 channel mutants ( deeken et al . , 2003 ) , highlighting the evolutionarily conserved mechanism for potassium channels to drive tumor growth . of note , although the expression of many potassium channels is up - regulated upon neoplastic transformation compared with adjacent normal tissue , low expression of the voltage - gated potassium channel kcnq1 is significantly associated with poor survival in patients with colorectal cancer ( than et al . , 2013 ) . consistently , loss of kcnq1 in a mouse model of intestinal tumor resulted in more aggressive cancer ( than et al . , this notion is further supported by the finding that misexpression of kcne1 , the regulatory accessory subunit that inhibits kcnq1 activity , leads to hyperproliferative and invasive phenotypes in xenopus laevis embryonic stem cells ( morokuma et al . , 2008 ) . recent work has demonstrated that oncogenic stress increases kcna1 expression and promotes its relocation from the cytoplasm to the plasma membrane , which is required for oncogene - induced senescence . ectopic expression of kcna1 inhibits ras - induced transformation , whereas a reduction of kcna1 expression correlates with an increase in breast cancer aggressiveness ( lallet - daher et al . , 2013 ) . these findings suggest that kcna1 may restrict tumor growth through a potassium channel dependent senescence pathway , and the functional significance of potassium channel during tumorigenesis could vary with the channel and/or cancer type . dysregulation of potassium channel expression has been identified at genomic , transcriptional , posttranslational , and epigenetic levels . for example , kcnk9 is amplified from 3- to 10-fold in 10% of breast tumors ( mu et al . , 2003 ) , and genomic amplification of eag1 was found in 15% of head and neck carcinoma ( menndez et al . , 2012 ) and 3.4% of human colorectal adenocarcinoma estrogen treatment can up - regulate kir channel transcription in human breast cancer cells to promote proliferation ( williams et al . , 2008 ) . an epigenome - wide study has shown that epigenetic silencing of herg expression by hypermethylation is a good prognostic marker in ovarian clear cell carcinoma ( cicek et al . , 2013 ) , interestingly , recent cross - cancer analysis of dna methylation patterns has revealed that voltage - gated potassium channels are frequently hypermethylated . for example , kcna3 hypermethylation is prominent in breast , lung , colorectal , kidney , ovarian , and prostate cancer ( brevet et al . the correlation between kcna3 hypermethylation to its expression and the clinical significance of this epigenetic alternation remains to be determined . as described above , kcnk9 amplification is detected in human carcinomas , and its ectopic expression confers tumorigenicity to otherwise nonneoplastic cell types ( mu et al . , 2003 ) . however , its expression correlates with prolonged survival after ovarian carcinoma resection ( innamaa et al . , 2013 ) . in addition , although herg channel blockers inhibit the growth of multiple cancer cell types , application of the herg channel activator also impairs mammary gland adenocarcinoma growth by inducing a senescence program ( lansu and gentile , 2013 ) . it is therefore important to note that the biological significance of potassium channel expression in cancer and whether its activation is tumorigenic or tumor suppressive is context dependent . historically , the involvement of potassium channels in cell proliferation was reported by the seminal work of decoursey et al . ( 1984 ) , who demonstrated that voltage - gated potassium channels are the predominant channels in proliferative human t lymphocytes . application of mitogenic factor promoted channel opening at a more negative membrane potential , and application of potassium channel blockers inhibited dna synthesis ( decoursey et al . , 1984 ) . indeed , early studies of unfertilized mouse oocytes have shown that a large - conductance potassium channel is active during the m and g1 phases and becomes inactivated during s and g2 phases . this temporal modulation of channel activity is not dependent on cell cycle specific protein synthesis . interestingly , the regulated potassium channel activity is correlated with a membrane potential oscillation , in which the cell is more depolarized at s and g2 ( day et al . , 1993 ) . since then the cell cycle phase - dependent potassium channel expression , localization , and activity have been found in multiple cell types ( takahashi et al . , 1993 ; arcangeli et al . , 1995 ; , 2004 ; huang et al . , 2012 ) , which suggests that it may be a general phenomenon that accompanies proliferation . how does a potassium channel regulate cell cycle progression ? emerging evidence has led to the proposal of four main mechanisms : setting up membrane potential oscillation , controlling the cell volume dynamics , regulating calcium signaling , and promoting malignant growth via noncanonical function independent of ion permeation ( fig . potassium channels may regulate cell proliferation by four mechanisms : setting up oscillating membrane potential , controlling cell volume dynamics , regulating calcium signaling , and promoting malignant growth via noncanonical function independent of ion permeation . depolarization can induce mitotic activity in several types of terminally differentiated cells . a transient depolarization at g2m transition has been observed . whether the potassium channel is required and the physiological significance of this depolarization at g2m transition remain unclear . ( 2012 ) : potassium channels can regulate cell proliferation via controlling cell volume dynamics throughout the cell cycle . this is exemplified by the voltage - gated potassium channel eag2 that exhibits cell cycle phase specific membrane localization . eag2 localizes intracellularly during interphase but enriches at the plasma membrane during late g2 and mitosis . this temporal eag2 membrane association promotes potassium efflux for premitotic condensation that is essential for mitotic entry , as well as regulating mitotic morphology for successful cell cycle progression . temporally and spatially regulated potassium channel functions therefore may be critical for passing the cell volume checkpoint for successful cell cycle progression . ( 3 ) potassium efflux through potassium channel can lead to membrane hyperpolarization , which increases the driving force for calcium entry through a calcium - permeable channel at the plasma membrane . this calcium entry can trigger calcium release from the internal store . increased intracellular calcium concentration ( [ ca]i ) ( 4 ) the potassium channel can regulate cell proliferation through a noncanonical function independent of its potassium ion permeability ( indicated by the channel with red asterisks ) . the potassium channel can interact with membrane protein ( x ) or intracellular protein ( y ) to initiate signaling cascade . alternatively spliced potassium channel lacking channel domains can enter the nucleus to modulate cell function ( sun et al . compared with the more hyperpolarized resting membrane potential in differentiated cell types such as neurons or cardiomyocytes ( 60 to 80 mv ) , rapidly proliferating embryonic cells , stem cells , or cancer cell are in general more depolarized , with a resting membrane potential at 20 to 40 mv ( yang and brackenbury , 2013 ) . therefore , one classical hypothesis is that membrane depolarization itself plays a functional role in tumor initiation or progression ( fig . 2 ) . in support of this hypothesis , sustained depolarization induces new dna synthesis , promotes mitotic activity in terminally differentiated central nervous system neurons cultured in vitro ( cone and cone , 1976 ) , and prevents mesenchymal stem cell differentiation ( sundelacruz et al . depolarized membrane potential also induces tumor - like growth and invasion in xenopus embryos ( morokuma et al . , 2008 ) . as potassium conductance is the predominant regulator for setting up the resting membrane potential , the potassium channel composition in cancer cells should accommodate the necessity of maintaining a relatively depolarized state . because the identities of potassium channels vary among different cancer types , the mechanism underlying the different potassium channels homeostatic control to achieve universal membrane depolarization remains to be elucidated . although the membrane potential of cancer cells tends to be more depolarized , cell cycle phase - specific changes in membrane potential have been reported . mcf-7 human mammary cells display a transient hyperpolarization at g1s transition , which was suggested to be a result of an increase in membrane permeability to potassium ( wonderlin et al . 2 ) . neuroblastoma cells exhibit hyperpolarization at g1s transition and depolarization at g2m transition , and the hyperpolarization phase correlates with increased potassium efflux ( boonstra et al . , 1981 ; fig . transition from quiescence to mitosis in mouse lymphocytes is accompanied by an initial phase of depolarization , followed by hyperpolarization during dna replication ( kiefer et al . , 1980 ; fig . 2 ) . because mouse oocytes display membrane depolarization at s and g2 phase ( day et al . , 1993 ) , the dynamic changes of membrane potential during cell cycle progression likely are cell type dependent . it is important to consider that , although cancer cells are in general more depolarized , a significant deviation from the range of their physiological membrane potential should also be detrimental . consistent with this notion , robust membrane depolarization mediated by optogenetic stimulation in cheta ( engineered channelrhodopsin-2 variant)-expressing glioma cells significantly reduced intracranial tumor growth and enhanced mouse survival ( yang et al . , 2013 ) . induction of the expression and activity of a constitutively open mutant sodium channel also leads to rapid and robust cell killing in multiple tumor cell types ( lemaire and halperin , 2009 ) , with the mechanisms potentially involving both membrane depolarization and osmotic shock . conversely , persistent hyperpolarization in glioma cells induced with kir4.1 channel expression results in significant growth arrest , a phenotype that can be inhibited by the application of the kir blocker barium ( higashimori and sontheimer , 2007 ) . besides the direct impact of membrane potential changes on tumorigenesis , potassium channel activity can hyperpolarize the membrane to increase the driving force for calcium entry through calcium - permeating channels ( fig . , pdgf up - regulates ik channel expression in vascular smooth muscle cells , and ik regulates pdgf - induced proliferation by promoting calcium entry and calcium - dependent signaling ( bi et al . , 2013 ) . interestingly , chronic pharmacological activation of ik channels paradoxically inhibits pdgf - induced proliferation by suppressing the rise of intracellular calcium concentration and its own expression ( bi et al . , 2013 ) , highlighting the importance of temporal and spatial regulation of potassium channel activity and calcium entry for successful cell proliferation . calcium is a major second messenger that can modulate a multitude of fundamental cellular functions such as proliferation , migration , survival , and apoptosis that have an impact on various aspects of tumor progression . recent excellent reviews have discussed the functional involvement of calcium signaling in cancer biology ( monteith et al . many potassium channels demonstrate cell cycle dependent expression level , trafficking , or activity . studying potassium channel function together with the advanced membrane potential monitoring and calcium imaging techniques throughout cell cycle progression in specific cell types comprehensive studies of volume dynamics have shown that the cells increase volume at interphase , rapidly condense cytoplasmic volume before mitotic entry ( premitotic cytoplasmic condensation ; pmc ) , and reach a minimal volume at metaphase to achieve mitotic rounding , followed by volume increase and cytokinesis to generate two daughter cells ( habela and sontheimer , 2007 ; boucrot and kirchhausen , 2008 ; fig . obligatory water flow across cell membrane , potassium channels are central regulators for cell volume . using a genome - wide microarray study , we have found that kv10.2 ( eag2 ) overexpression is a hallmark of a subset of medulloblastomas across different molecular and histological subgroups . the eag2 channel displays dynamic distribution throughout the cell cycle and enriches at the plasma membrane at g2 and m phase . this selective membrane association of eag2 is accompanied by elevated outward potassium current ( fig . consistent with the hypothesis that the outward potassium flow drives cell volume reduction for pmc and mitotic entry , genetic suppression of eag2 expression results in significantly increased cell volume at late g2 phase and induces g2 arrest . for the few medulloblastoma cells with deficient eag2 function that escape the g2 arrest and enter mitosis , they fail to achieve proper mitotic morphology and encounter mitotic catastrophe . conversely , overexpressing eag2 in heterologous systems ( hek293 and cos7 cells ) results in constitutive membrane localization of eag2 and sustained cell volume reduction , which also impairs cell cycle progression and leads to apoptosis ( huang et al . , 2012 ) . taken together , cell volume regulation involves concurrent flux of potassium and chloride ions to accompany water flow . while the identify of anion - conducting channels involved in medulloblastoma cell volume regulation remains to be determined , the clc3 chloride channel has been shown to conduct outward chloride current to drive regulatory volume reduction in glioma cells , and inhibiting clc3 channels reduces proliferation ( habela et al . , 2008 ) . because cell volume oscillation appears to be a universal phenomenon during cell cycle progression , identifying and targeting specific ion channels regulating this process offers great therapeutic potential . potassium channels form macromolecules at the plasma membrane . besides the membrane - spanning potassium ion permeation pathway it is therefore conceivable that some functional roles the potassium channels play during oncological process are mediated by these intracellular elements independent of their capacity to pass potassium ions ( fig . consistent with this notion , transfecting wild - type and nonconducting drosophila melanogaster eag channels into nih3t3 cells induced comparable levels of proliferation ( hegle et al . , 2006 ) . furthermore , expressing a nonconducting mutant eag1 ( downie et al . , 2008 ) or kv1.3 ( cidad et al . , 2012 ) is sufficient in promoting allografted tumor growth . however , the ability for kcnk9 ( pei et al . , 2003 ) to regulate cancer cell behaviors depends on its canonical role in potassium conductance , which suggests that there are multiple underlying mechanisms for the potassium channel to promote malignant phenotypes . fortunately , the conserved gyg / gfg signature motif in the pore domain provides a designated spot for mutational study in this regard . metastasis causes > 90% of human cancer deaths ( hanahan and weinberg , 2011 ) . the metastatic cascade is a multistep process that involves mobilization of primary tumor cells by migration , invasion into adjacent nontumoral regions , entry into the circulatory system , exit toward metastatic sites , and colonizing the secondary metastatic sites . as the first step , cell migration plays an integral role in metastasis and dictates the success of malignant dissemination . migratory cells are polarized with mobile lamellipodia at the leading edge and contracting cell body at the trailing edge . there are local cell volume changes during cell migration : increases at the leading edge with lamellipodia protrusion and decreases at cell rear as it retracts ( fig . 3 ) . the best - characterized functions of potassium channels in facilitating cell migration concern its capacity to cause volume changes by influencing potassium flow . an early study on transformed mdck - f renal epithelial cell migration showed that specific application of the ik channel blocker charybdotoxin to the trailing edge , but not the leading edge , inhibits migration and increases overall cell volume ( schwab et al . , 1999 ; schneider et al . , 2000 ) . however , ik calcium - activated potassium channels are enriched at the cell front ( schwab et al . , 2006 ) . one plausible scenario to account for these findings is that ik channels at the trailing edge may be preferentially activated , owing to a back - to - front calcium gradient in migrating cells ( schwab et al . , 2012 ) . it remains an open question whether potassium channels may be enriched at the trailing edge for local cell volume regulation ( fig . 3 ) . the dependence on kca channel activity in promoting cell migration has been repeatedly reported . sensitive potassium current . genetic suppression or pharmacological inhibition of bk channels impairs glioma cell migration ( weaver et al . , 2006 ) . treating the mice bearing ik channel expressing glioblastoma with tram-34 , a selective ik channel blocker , bk and sk channels can also promote breast cancer cell migration and are implicated in metastasis ( khaitan et al . the sk channel can form a complex with the orai1 calcium channel for localized calcium entry within lipid rafts , providing a mechanism for its specific activation to enhance cancer cell migration ( chantme et al . , 2013 ) . in addition , the bk channel can be functionally coupled with other ion channels , such as the clc3 chloride channel , within the lipid rafts of invadopodia in glioma cells in order to mediate both potassium and chloride ion efflux , and to facilitate invasion by regulatory volume decrease to enable cells to course through a confined space ( mcferrin and sontheimer , 2006 ) . interestingly , chronic application of a bk channel opener also results in defective glioma cell migration ( kraft et al . , 2003 ) , demonstrating the importance of temporal and spatial control of potassium channel activity for coordinated cell movement . recent studies have shown that ik channels modulate subventricular zone derived neuroblast migration along the rostral migratory stream to the olfactory bulb ( turner and sontheimer , 2013 ) , highlighting the function of potassium channels in regulating cell migration in both malignant and nonmalignant cell types . in addition to potassium channels , a variety of ion channels and transporters have been implicated in regulating local cell volume changes during cell migration ( fig . indeed , it has been recently shown that a net inflow of ions and water at the cell leading edge and outflow of ions and water at the cell trailing edge represents a major force for cell motility even in the absence of actin polymerization ( stroka et al . , 2014 ) . cell migration is characterized by the polarized cellular and molecular processes that promote the protrusion of leading edge ( local volume increase ) and retraction of the trailing edge ( local volume reduction ) . a net inflow of ions and water at the cell leading edge and a net outflow of ions and water at the cell trailing edge a number of ion channels and transporters display polarized subcellular distribution during cell migration . at the trailing edge , calcium entry through calcium - permeable channels can activate calcium - activated potassium channels to promote potassium efflux . the opening of chloride channels maintains electroneutrality and further enhances the outflow of ions , which is followed by obligatory water efflux through aquaporin . at the leading edge , an assortment of inward ion channels such as voltage - gated sodium channels ( nav ) or epithelial sodium channels ( enac ) , or ion transporters such as sodium - proton exchangers ( nhe1 ) or sodium - potassium - chloride cotransporters ( nkcc1 ) together with aquaporins , regulate ion and water influx . this ion and water influx drives local cell volume increase in the protruding lamellipodium for leading edge extension . the proton extrusion and acidification of extracellular microenvironment by nhe1 can further facilitate degradation of matrix proteins and tumor cell invasion ( brisson et al . , 2011 ) . kir4.2 localizes at the cell leading edge and enhances cell migration ( dehart et al . , 2008 ) . whether kir4.2 conducts an inward potassium flow , which will depend on the local electrochemical gradient for potassium at the leading edge , remains unclear . for comprehensive reviews on functional roles of the ion transportome during cell migration , the best - illustrated example is the herg channel , which can form a macromolecular complex with vegfr-1 and 1 integrin . the assembly of this protein complex confers a pro - migratory phenotype in leukemia , an effect that can be inhibited by application of a herg channel blocker . furthermore , herg expression in leukemia patients is associated with higher probability of relapse and shorter survival ( pillozzi et al . , 2007 ) . it has been shown that colocalization of 91 integrin with the kir4.2 channel in focal adhesions at the leading edge of migrating cells may promote local kir4.2 channel activity to induce the formation of a single dominant lamellipodial extension critical for persistent migration , although the underlying mechanism of how kir4.2 modulates lamellipodium formation remains unclear ( dehart et al . , 2008 ; fig . these findings highlight the potassium channel as an integral membrane protein that is functionally coupled with other signaling molecules to exert concerted regulation on cell migration . given the reported interactions between other potassium channels , focal adhesion molecules , and cytoskeleton proteins ( levite et al . , 2000 ; petrecca et al . , 2000 ; rezzonico et al . , 2003 ) , it is expected that future investigation will shed light on the novel noncanonical roles of potassium channels within the multiprotein complex during tumorigenic processes . being the most diverse class of ion channels that regulate a plethora of cellular behaviors in both excitable and nonexcitable cells , targeting the cell surface accessible potassium channels offers tremendous therapeutic potential for the treatment of human diseases that involve aberrant expression and function of specific potassium channels . for example , drugs targeting katp , kcnq , and herg channels have been successfully used in clinics for treating diabetes or hypertension , epilepsy , and cardiac arrhythmia , respectively . despite the increasing evidence of ion channel functions in oncology , therapeutic development of cancer treatment using ion channel targeting compounds the most comprehensive cancer therapeutic efficacy study on potassium channels has focused on the eag channel family , including eag1 , eag2 , and herg . eag1 overexpression confers tumorigenic potential , and its expression is correlated with poor patient survival in multiple cancer types ( pardo and sthmer , 2008 ) . small molecule eag1 blockers such as astemizole have shown efficacy in reducing eag1-expressing cancer cell growth in vitro and in vivo ( downie et al . , 2008 ) . pharmacological inhibition of herg channels reduces proliferation and impairs invasiveness in a variety of cancer cell types ( babcock and li , 2013 ) . importantly , targeting the herg channel with its specific blocker e-4031 impairs the function of the multiprotein complex consisting of herg , 1 integrin , and the chemokine receptor cxcr4 , which is essential for leukemic cell survival . e-4031 treatment inhibits leukemia progression in vivo and enhances mouse survival ( pillozzi et al . , 2011 ) . because the herg channel mediates the repolarization phase of cardiac action potential and eag channels share high structural similarity , one should be cautious about using these compounds for direct transition into treating human cancer patients , as herg channel blockade can induce potentially fatal cardiac arrhythmia ( long qt syndrome ) . nevertheless , it should be noted that a number of marketed drugs treating various human conditions are known herg inhibitors with well - characterized and acceptable toxicity profiles ( babcock et al . , 2013 ) . with careful treatment design and close monitoring of the potential adverse effects , these herg - blocking drugs may be repurposed for cancer treatment . generating monoclonal antibodies has proven to be an effective strategy to perturb the function of cell surface proteins . as a proof of principle , it has been demonstrated that a highly specific extracellular epitope - targeting eag1 monoclonal antibody exhibited antitumor activity in mouse ( gmez - varela et al . , 2007 ) . given the fact that eag1 is highly expressed in 70% of human cancers but minimally present in non - cns normal tissues , dye - tagged eag1 antibody also possesses the potential for selective visualization of tumor cells for diagnostic purposes ( pardo et al . , 2005 ) . eag1-specific antibodies can be further coupled with cytotoxic agents for selective killing of eag1-expressing cancer cells ( hartung et al . , 2011 ) . rational design of therapeutic targeting of potassium channels for cancer treatment will be based on our better understanding of their functions from mechanistic study . for example , cancer cells preferentially express an n - terminal truncated form of the herg channel ( crociani et al . , 2003 ) , providing an opportunity for generating an isoform - specific antagonist . strikingly , the disruption of lipid rafts by the alkyl - lipid ohmline leads to significantly reduced bone metastasis ( chantme et al . , 2013 ) , highlighting the effectiveness of indirectly targeting potassium channel protein complexes that influence key processes of cancer progression . herg channel protein maturation , stability , and current density can be enhanced by the stress - activated chaperone sigma 1 receptor , which physically associates with herg and represents a novel pharmacological target in a membrane signaling channel macrocomplex that is involved in cancer progression ( crotts et al . , 2011 ) . herg channel blockade reduces bone marrow mesenchymal stem cell induced chemotherapeutic resistance in leukemic cells ( pillozzi et al . , 2011 ) . ionizing radiation increases the open probability of bk channels without affecting their expression levels and contributes to the enhanced migration and invasiveness of glioma cells ( steinle et al . , 2011 ) . radiation treatment can also increase kv3.4 channel activity in leukemic cells to promote calcium entry and camkii - dependent cancer cell survival ( palme et al . , 2013 ) . low - dosage radiation induces voltage - dependent potassium channel activity that is otherwise not present in human lung adenocarcinoma cells ( kuo et al . , 1993 ) . these findings suggest that radiation - induced potassium channel activation may be a general response in different cancer types , and potassium channel inhibition may be a novel and promising component in combinatorial therapy for treating cancer patients . a growing body of evidence suggests that potassium channel dysregulation is associated with key aspects of cancer , and targeting potassium channel is a viable therapeutic approach . different cancers may use specific channel types for their malignant behaviors , depending on the cellular origin of the tumor and the driving oncogenic signal . since the identification of potassium channel involvement in t cell proliferation 30 years ago ( decoursey et al . , 1984 ) , much progress has been made , yet research into potassium channel function in cancer still represents a nascent field . first , there are many open questions regarding the possibility of heterogeneity in potassium channel expression or function in the cancer cell over space and time . for example , do cancer stem cells exploit different potassium channels for maintaining their unique properties such as enhanced tumorigenic potential and survival after irradiation and chemotherapy ? does a microenvironment such as hypoxia or cancer treatment induce differential potassium channel activity that confers drug resistance ? second , although the potassium channel expression level has been compared between tumor and nontumor tissues , the mutational landscape of potassium channels in various cancer types remains largely unexplored . the identification and quantification of recurrent mutations may offer another avenue to identify novel potassium channels involved in the oncogenic process , and stimulate the investigation of their functions . third , the majority of studies reported thus far are based on in vitro cell culture systems and xenograft models . future studies involving mutating potassium channels in the spontaneous cancer models established in various model organisms , such as fruit fly , zebrafish , and mouse , should reveal novel insights into potassium channel function during tumor initiation , progression , and evolution . fourth , studying the therapeutic value of potassium channel modulating drugs using genetic models of cancer should foster the rapid translation from preclinical discoveries to clinical trials . fifth , advanced high - throughput screening technology should be exploited for identifying novel compounds that modulate potassium channels implicated in cancer , while significant efforts can also be made toward elucidating the therapeutic value of repurposing potassium channel modulating drugs on the market for treating other human diseases . although no current fda - approved cancer drug is intended to function as a potassium channel blocker , the future holds great promise for targeting potassium channels in cancer .

potassium channels are pore - forming transmembrane proteins that regulate a multitude of biological processes by controlling potassium flow across cell membranes . aberrant potassium channel functions contribute to diseases such as epilepsy , cardiac arrhythmia , and neuromuscular symptoms collectively known as channelopathies . increasing evidence suggests that cancer constitutes another category of channelopathies associated with dysregulated channel expression . indeed , potassium channel modulating agents have demonstrated antitumor efficacy . potassium channels regulate cancer cell behaviors such as proliferation and migration through both canonical ion permeation dependent and noncanonical ion permeation independent functions . given their cell surface localization and well - known pharmacology , pharmacological strategies to target potassium channel could prove to be promising cancer therapeutics .

Introduction Potassium channels Dysregulated potassium channel expression in cancer Potassium channel in proliferation and tumor growth Potassium channels in cell migration and metastasis Therapeutic targeting of potassium channels in cancer Targeting potassium channels: Unanswered questions

although our knowledge of ion channel functions in the physiological and pathological conditions of excitable cells such as cardiomyocytes and neurons is vast , it is only recently that the manifold functions of ion channels in driving malignant cancer cell behaviors have been reported . potassium channels represent the most diverse group of ion channels , and have enormous potential as therapeutic targets for personalized cancer treatment , given their huge functional and structural diversity . in this review we summarize the dysregulated potassium channel expression in cancer , discuss the complex mechanisms potassium channels use for regulating cancer cell proliferation and migration , illustrate the therapeutic value for targeting potassium channel in preclinical models , and outline the major unanswered questions in the field . the > 400 ion channel encoding genes represent 1.5% of the human genome , and ion channels display vast structural and functional diversity . these channels are central regulators of the distribution of potassium , sodium , calcium , and chloride ions for cellular ionic homeostasis and contribute to essentially all fundamental cellular processes . so , in general , potassium channel activation would cause hyperpolarization , whereas opening of sodium channels or chloride channels would cause depolarization . with 78 members , potassium channels can be divided into four main classes based on their domain structure and activation mechanisms ( fig . voltage - gated potassium channels ( kv ) , encoded by 40 genes in humans , are the largest subset of potassium channels gated by changes in the membrane potential . kv and kca channels share a similar domain organization and are composed of four pore - lining subunits , each with six transmembrane domains ( tms ) and one pore - forming region , except for the bk channel , which has one additional transmembrane segment in the n terminus . 1 ) , and they pass potassium ions more easily in the inward direction ( into the cell ) than the outward direction ( out of the cell ) . two - pore domain potassium channels ( k2p ) have two pore domains per subunit , each pore domain with two transmembrane segments flanking a reentrant pore loop ( fig . while the pore domain of all potassium channels possesses a highly conserved gyg / gfg signature motif , the molecular diversity of potassium channels is immense . , 2009 ) , and the channel proteins can be further modified at the posttranslational level by phosphorylation ( park et al . ( top ) calcium - activated potassium channels ( kca ) and voltage - gated potassium ( kv ) channels are composed of four pore - lining subunits . the first four transmembrane segments ( tm1tm4 ) of kv channel subunit form the voltage sensor domain , whereas the remainder of the transmembrane domain corresponds to the pore domain conserved in all potassium channels ( isacoff et al . the inward rectifying potassium channel ( kir ) possesses two transmembrane domains with a pore - forming region ( p ) in between . the k2p channel is the so - called background potassium channel that is made of four transmembrane domains and two pore - forming regions . all potassium channels require a tetrameric arrangement of the pore - forming regions of the subunits to form the potassium - selective filter , therefore a complete conductive channel is a tetramer for the one - pore channels ( kv , kca , and kir ) or dimer for the k2p channels . these potassium channels implicated in oncological processes belong to all four main classes ( fig . overexpression of the g - protein activated inwardly rectifying potassium channel girk1 ( kcnj3 ) is correlated with the presence and degree of breast cancer lymph node metastases ( stringer et al . potassium channel expression in cancer expression of various potassium channels that have been implicated in cancer . , 2013 green boxes indicate that the channel expression is inversely correlated with tumor malignancy and clinical aggressiveness , such as kcna5 in glioma ( preussat et al . , 2009 ) , and kcnq1 in colorectal cancer ( than et al . does the dysregulated potassium channel expression ( mostly overexpression ) initiate tumorigenesis , confer advantages to malignant growth or metastatic spread once the tumor forms , or is the aberrant expression simply a consequence of neoplastic transformation without functional significance ? two potassium channels have been shown to possess tumorigenic capacity when ectopically expressed in heterologous systems . , 2003 so it is important to determine whether overexpression of a specific potassium channel in situ of an animal model is tumorigenic , which is a more stringent criterion for an oncogene . , 2014 ) , it is possible that most potassium channels take up a permissive role to mostly function as essential regulators for various cancer cell behaviors such as proliferation and migration , and that their up - regulation provides a repertoire for sufficient channel activity when necessary . strikingly , up - regulation of the plant orthologue of shaker - like voltage - gated potassium channel akt1 has been detected in arabidopsis thaliana tumors induced by bacterial infection , and the plant tumor growth is reduced in akt1 channel mutants ( deeken et al . , 2003 ) , highlighting the evolutionarily conserved mechanism for potassium channels to drive tumor growth . of note , although the expression of many potassium channels is up - regulated upon neoplastic transformation compared with adjacent normal tissue , low expression of the voltage - gated potassium channel kcnq1 is significantly associated with poor survival in patients with colorectal cancer ( than et al . these findings suggest that kcna1 may restrict tumor growth through a potassium channel dependent senescence pathway , and the functional significance of potassium channel during tumorigenesis could vary with the channel and/or cancer type . dysregulation of potassium channel expression has been identified at genomic , transcriptional , posttranslational , and epigenetic levels . , 2013 ) , interestingly , recent cross - cancer analysis of dna methylation patterns has revealed that voltage - gated potassium channels are frequently hypermethylated . as described above , kcnk9 amplification is detected in human carcinomas , and its ectopic expression confers tumorigenicity to otherwise nonneoplastic cell types ( mu et al . it is therefore important to note that the biological significance of potassium channel expression in cancer and whether its activation is tumorigenic or tumor suppressive is context dependent . ( 1984 ) , who demonstrated that voltage - gated potassium channels are the predominant channels in proliferative human t lymphocytes . application of mitogenic factor promoted channel opening at a more negative membrane potential , and application of potassium channel blockers inhibited dna synthesis ( decoursey et al . indeed , early studies of unfertilized mouse oocytes have shown that a large - conductance potassium channel is active during the m and g1 phases and becomes inactivated during s and g2 phases . since then the cell cycle phase - dependent potassium channel expression , localization , and activity have been found in multiple cell types ( takahashi et al . , 2012 ) , which suggests that it may be a general phenomenon that accompanies proliferation . emerging evidence has led to the proposal of four main mechanisms : setting up membrane potential oscillation , controlling the cell volume dynamics , regulating calcium signaling , and promoting malignant growth via noncanonical function independent of ion permeation ( fig . potassium channels may regulate cell proliferation by four mechanisms : setting up oscillating membrane potential , controlling cell volume dynamics , regulating calcium signaling , and promoting malignant growth via noncanonical function independent of ion permeation . whether the potassium channel is required and the physiological significance of this depolarization at g2m transition remain unclear . ( 2012 ) : potassium channels can regulate cell proliferation via controlling cell volume dynamics throughout the cell cycle . this is exemplified by the voltage - gated potassium channel eag2 that exhibits cell cycle phase specific membrane localization . temporally and spatially regulated potassium channel functions therefore may be critical for passing the cell volume checkpoint for successful cell cycle progression . increased intracellular calcium concentration ( [ ca]i ) ( 4 ) the potassium channel can regulate cell proliferation through a noncanonical function independent of its potassium ion permeability ( indicated by the channel with red asterisks ) . the potassium channel can interact with membrane protein ( x ) or intracellular protein ( y ) to initiate signaling cascade . alternatively spliced potassium channel lacking channel domains can enter the nucleus to modulate cell function ( sun et al . compared with the more hyperpolarized resting membrane potential in differentiated cell types such as neurons or cardiomyocytes ( 60 to 80 mv ) , rapidly proliferating embryonic cells , stem cells , or cancer cell are in general more depolarized , with a resting membrane potential at 20 to 40 mv ( yang and brackenbury , 2013 ) . as potassium conductance is the predominant regulator for setting up the resting membrane potential , the potassium channel composition in cancer cells should accommodate the necessity of maintaining a relatively depolarized state . because the identities of potassium channels vary among different cancer types , the mechanism underlying the different potassium channels homeostatic control to achieve universal membrane depolarization remains to be elucidated . mcf-7 human mammary cells display a transient hyperpolarization at g1s transition , which was suggested to be a result of an increase in membrane permeability to potassium ( wonderlin et al . neuroblastoma cells exhibit hyperpolarization at g1s transition and depolarization at g2m transition , and the hyperpolarization phase correlates with increased potassium efflux ( boonstra et al . conversely , persistent hyperpolarization in glioma cells induced with kir4.1 channel expression results in significant growth arrest , a phenotype that can be inhibited by the application of the kir blocker barium ( higashimori and sontheimer , 2007 ) . besides the direct impact of membrane potential changes on tumorigenesis , potassium channel activity can hyperpolarize the membrane to increase the driving force for calcium entry through calcium - permeating channels ( fig . , pdgf up - regulates ik channel expression in vascular smooth muscle cells , and ik regulates pdgf - induced proliferation by promoting calcium entry and calcium - dependent signaling ( bi et al . calcium is a major second messenger that can modulate a multitude of fundamental cellular functions such as proliferation , migration , survival , and apoptosis that have an impact on various aspects of tumor progression . studying potassium channel function together with the advanced membrane potential monitoring and calcium imaging techniques throughout cell cycle progression in specific cell types comprehensive studies of volume dynamics have shown that the cells increase volume at interphase , rapidly condense cytoplasmic volume before mitotic entry ( premitotic cytoplasmic condensation ; pmc ) , and reach a minimal volume at metaphase to achieve mitotic rounding , followed by volume increase and cytokinesis to generate two daughter cells ( habela and sontheimer , 2007 ; boucrot and kirchhausen , 2008 ; fig . obligatory water flow across cell membrane , potassium channels are central regulators for cell volume . consistent with the hypothesis that the outward potassium flow drives cell volume reduction for pmc and mitotic entry , genetic suppression of eag2 expression results in significantly increased cell volume at late g2 phase and induces g2 arrest . while the identify of anion - conducting channels involved in medulloblastoma cell volume regulation remains to be determined , the clc3 chloride channel has been shown to conduct outward chloride current to drive regulatory volume reduction in glioma cells , and inhibiting clc3 channels reduces proliferation ( habela et al . besides the membrane - spanning potassium ion permeation pathway it is therefore conceivable that some functional roles the potassium channels play during oncological process are mediated by these intracellular elements independent of their capacity to pass potassium ions ( fig . , 2003 ) to regulate cancer cell behaviors depends on its canonical role in potassium conductance , which suggests that there are multiple underlying mechanisms for the potassium channel to promote malignant phenotypes . the metastatic cascade is a multistep process that involves mobilization of primary tumor cells by migration , invasion into adjacent nontumoral regions , entry into the circulatory system , exit toward metastatic sites , and colonizing the secondary metastatic sites . the best - characterized functions of potassium channels in facilitating cell migration concern its capacity to cause volume changes by influencing potassium flow . however , ik calcium - activated potassium channels are enriched at the cell front ( schwab et al . it remains an open question whether potassium channels may be enriched at the trailing edge for local cell volume regulation ( fig . the sk channel can form a complex with the orai1 calcium channel for localized calcium entry within lipid rafts , providing a mechanism for its specific activation to enhance cancer cell migration ( chantme et al . in addition , the bk channel can be functionally coupled with other ion channels , such as the clc3 chloride channel , within the lipid rafts of invadopodia in glioma cells in order to mediate both potassium and chloride ion efflux , and to facilitate invasion by regulatory volume decrease to enable cells to course through a confined space ( mcferrin and sontheimer , 2006 ) . , 2003 ) , demonstrating the importance of temporal and spatial control of potassium channel activity for coordinated cell movement . recent studies have shown that ik channels modulate subventricular zone derived neuroblast migration along the rostral migratory stream to the olfactory bulb ( turner and sontheimer , 2013 ) , highlighting the function of potassium channels in regulating cell migration in both malignant and nonmalignant cell types . in addition to potassium channels , a variety of ion channels and transporters have been implicated in regulating local cell volume changes during cell migration ( fig . indeed , it has been recently shown that a net inflow of ions and water at the cell leading edge and outflow of ions and water at the cell trailing edge represents a major force for cell motility even in the absence of actin polymerization ( stroka et al . at the trailing edge , calcium entry through calcium - permeable channels can activate calcium - activated potassium channels to promote potassium efflux . at the leading edge , an assortment of inward ion channels such as voltage - gated sodium channels ( nav ) or epithelial sodium channels ( enac ) , or ion transporters such as sodium - proton exchangers ( nhe1 ) or sodium - potassium - chloride cotransporters ( nkcc1 ) together with aquaporins , regulate ion and water influx . whether kir4.2 conducts an inward potassium flow , which will depend on the local electrochemical gradient for potassium at the leading edge , remains unclear . furthermore , herg expression in leukemia patients is associated with higher probability of relapse and shorter survival ( pillozzi et al . given the reported interactions between other potassium channels , focal adhesion molecules , and cytoskeleton proteins ( levite et al . , 2003 ) , it is expected that future investigation will shed light on the novel noncanonical roles of potassium channels within the multiprotein complex during tumorigenic processes . being the most diverse class of ion channels that regulate a plethora of cellular behaviors in both excitable and nonexcitable cells , targeting the cell surface accessible potassium channels offers tremendous therapeutic potential for the treatment of human diseases that involve aberrant expression and function of specific potassium channels . for example , drugs targeting katp , kcnq , and herg channels have been successfully used in clinics for treating diabetes or hypertension , epilepsy , and cardiac arrhythmia , respectively . despite the increasing evidence of ion channel functions in oncology , therapeutic development of cancer treatment using ion channel targeting compounds the most comprehensive cancer therapeutic efficacy study on potassium channels has focused on the eag channel family , including eag1 , eag2 , and herg . eag1 overexpression confers tumorigenic potential , and its expression is correlated with poor patient survival in multiple cancer types ( pardo and sthmer , 2008 ) . small molecule eag1 blockers such as astemizole have shown efficacy in reducing eag1-expressing cancer cell growth in vitro and in vivo ( downie et al . pharmacological inhibition of herg channels reduces proliferation and impairs invasiveness in a variety of cancer cell types ( babcock and li , 2013 ) . importantly , targeting the herg channel with its specific blocker e-4031 impairs the function of the multiprotein complex consisting of herg , 1 integrin , and the chemokine receptor cxcr4 , which is essential for leukemic cell survival . because the herg channel mediates the repolarization phase of cardiac action potential and eag channels share high structural similarity , one should be cautious about using these compounds for direct transition into treating human cancer patients , as herg channel blockade can induce potentially fatal cardiac arrhythmia ( long qt syndrome ) . nevertheless , it should be noted that a number of marketed drugs treating various human conditions are known herg inhibitors with well - characterized and acceptable toxicity profiles ( babcock et al . generating monoclonal antibodies has proven to be an effective strategy to perturb the function of cell surface proteins . rational design of therapeutic targeting of potassium channels for cancer treatment will be based on our better understanding of their functions from mechanistic study . herg channel protein maturation , stability , and current density can be enhanced by the stress - activated chaperone sigma 1 receptor , which physically associates with herg and represents a novel pharmacological target in a membrane signaling channel macrocomplex that is involved in cancer progression ( crotts et al . radiation treatment can also increase kv3.4 channel activity in leukemic cells to promote calcium entry and camkii - dependent cancer cell survival ( palme et al . low - dosage radiation induces voltage - dependent potassium channel activity that is otherwise not present in human lung adenocarcinoma cells ( kuo et al . these findings suggest that radiation - induced potassium channel activation may be a general response in different cancer types , and potassium channel inhibition may be a novel and promising component in combinatorial therapy for treating cancer patients . a growing body of evidence suggests that potassium channel dysregulation is associated with key aspects of cancer , and targeting potassium channel is a viable therapeutic approach . first , there are many open questions regarding the possibility of heterogeneity in potassium channel expression or function in the cancer cell over space and time . for example , do cancer stem cells exploit different potassium channels for maintaining their unique properties such as enhanced tumorigenic potential and survival after irradiation and chemotherapy ? does a microenvironment such as hypoxia or cancer treatment induce differential potassium channel activity that confers drug resistance ? second , although the potassium channel expression level has been compared between tumor and nontumor tissues , the mutational landscape of potassium channels in various cancer types remains largely unexplored . the identification and quantification of recurrent mutations may offer another avenue to identify novel potassium channels involved in the oncogenic process , and stimulate the investigation of their functions . future studies involving mutating potassium channels in the spontaneous cancer models established in various model organisms , such as fruit fly , zebrafish , and mouse , should reveal novel insights into potassium channel function during tumor initiation , progression , and evolution . fourth , studying the therapeutic value of potassium channel modulating drugs using genetic models of cancer should foster the rapid translation from preclinical discoveries to clinical trials . fifth , advanced high - throughput screening technology should be exploited for identifying novel compounds that modulate potassium channels implicated in cancer , while significant efforts can also be made toward elucidating the therapeutic value of repurposing potassium channel modulating drugs on the market for treating other human diseases . although no current fda - approved cancer drug is intended to function as a potassium channel blocker , the future holds great promise for targeting potassium channels in cancer .

in 2011 renal cell carcinoma ( rcc ) still received a great deal of attention at the major urological and oncological congresses worldwide . though rcc is only the third most common urologic malignancy after prostatic and urothelial cancer , it accounts for more than 50,000 new cases in the usa and approximately 15,000 in germany each year . it takes a high death toll because of its often aggressive growth pattern , high rate of early metastasis , and the lack of curative therapy in advanced stages : in germany alone , about one third of all rcc patients ( n = 5,101 ) succumbed to their disease in 2008 . the incidence has increased significantly in the last 30 years.at the last congress of the american urological association ( aua ) , this significantly increased incidence was discussed by a st . it was evaluated in 63,843 rcc patients by a retrospective analysis of the seer ( surveillance epidemiology and end results ) database from 1975 to 2006 . the authors showed that the incidence increased in the usa from 7.4 to 17.6 per 100,000 population between 1975 and 2006 . thus , the mean annual increase in incidence was only 2.9% from 1991 to 2006 . analysis of the seer data also revealed that the increase in incidence mainly affected older patients up to 1990 and younger ones after 1991 . this also meant that , at the time of diagnosis , the majority of rcc patients were over 65 until 1991 , whereas 55.3% were under 65 in 2006 . the authors could not determine whether this shift towards younger age at diagnosis can be attributed to earlier tumor detection or earlier tumor development . most congress contributions dealing with localized rcc focused on the following questions.which rcc patients can and should be managed with an active surveillance strategy?when can and should a renal tumor biopsy be performed?what are the advantages and disadvantages of radical versus partial nephrectomy?what is the optimal access and best surgical procedure for partial nephrectomy?are there alternative local ablation techniques equivalent to surgery?what is the value of new predictive or prognostic markers for patients with localized rcc ? which rcc patients can and should be managed with an active surveillance strategy ? what are the advantages and disadvantages of radical versus partial nephrectomy ? what is the optimal access and best surgical procedure for partial nephrectomy ? what is the value of new predictive or prognostic markers for patients with localized rcc ? , renal function does not deteriorate , at least not primarily , since there is no loss of functioning nephrons by surgery / ablation . on the other hand , there are also thought - provoking arguments against an active surveillance strategy . living with a potentially malignant renal tumor then there are also the costs of regular follow - up imaging and the associated radiation exposure ( 3090 msv per ct scan ) . moreover , we still have no large prospective studies with the long - term results of a surveillance strategy in rcc , nor do we have markers that can adequately predict progression of a small localized kidney tumor under active surveillance . tsivian et al . from durham presented a retrospective monocenter analysis of data they collected from 2000 to 2008 in 243 patients who had received a partial nephrectomy for a small solid renal tumor . had a mean size of 2.9 cm in this patient population and were ultimately malignant in 73.7% of the cases . sixty percent of tumors < 2 cm and 77.6% of those > 2 cm were malignant in this cohort . the authors concluded that an active surveillance strategy can be justified particularly for patients with tumors < 2 cm since 40% of even the solid renal tumors were benign in this patient population . however , in a considerably larger study published in the journal of urology in 2003 , frank et al . reported that the percentage of malignant renal tumors increases significantly with the tumor size . small solid tumors < 1 cm were benign or malignant in approximately equal proportions , but only about 22% of renal tumors > 1 cm were benign . predictors of systemic metastasis of small solid renal tumors were investigated by the study group around smaldone from philadelphia . at the aua annual congress , they presented a meta - analysis of 6 studies comprising 880 patients with a follow - up of 15 to 48 months . the tumors had a mean initial size of 1.7 to 7.2 cm and an annual growth rate of 0.1 to 0.7 cm . this meta - analysis revealed a particularly high risk of systemic metastasis in patients with small solid tumors , who tended to be older , as well as in those whose tumors had a larger diameter or volume , or those with a higher annual growth rate ( 0.8 cm versus 0.3 cm ) . concluded that younger patients with small tumors as well as patients whose tumors have a low growth rate or none at all were optimal candidates for a surveillance strategy . however , nguyen and gill already demonstrated the danger of even small renal tumors in a publication based on seer database data from 2009 . < 1 cm and even 7.4% of all tumors with a diameter of 3 - 4 cm ( formally still stage t1a tumors ) had already metastasized at the time of diagnosis . the percentage increased with tumor size , ranging up to 22% for 6 - 7 cm tumors . approaching the problem from a completely different angle , chang et al . examined predictors of non - rcc - related death in patients with localized rcc in order to identify good candidates for an active surveillance strategy . their retrospective analysis of 1,101 patients with localized rcc demonstrated that , during a median follow - up of 62 months , non - tumor - related mortality was naturally higher among older patients ( particularly those > 70 ) as well as those in a reduced general condition with significant comorbidities ( asa score : 3/4 versus 1/2 , hazard ratio ( hr ) : 3.3 ; p = 0.003 ) . come to the conclusion that older patients with significant comorbidities have a higher risk of non - tumor - related death and are thus the optimal candidates for an active surveillance strategy . active treatment is still recommended for all other patients since about 85% of even the small solid tumors ultimately turn out to be malignant . moreover , a surveillance strategy involves a small but significant risk of progressive local tumor growth that can potentially render nephron - sparing surgery difficult or impossible . in addition , even in patients with small tumors , active surveillance is associated with a 1 - 2% annual risk of systemic metastasis , and potentially curable patients may thus become incurable [ 1012 ] . the aim of renal tumor biopsy is to avoid unnecessary surgery in patients with renal findings . it is always indicated when the radiological diagnosis of rcc appears questionable ( e.g. , differential diagnosis including lymphoma , metastasis , or abscess ) and should also precede thermal ablation . a number of studies published in 2011 dealt with the strengths and weaknesses of renal tumor biopsy [ 1315 ] . they all showed a close correlation between biopsy and resection tissue : 9193% for the histological subtype and 5478% for the degree of tumor differentiation . moreover , all studies revealed a very high positive predictive value ( ppv ) for renal tumor biopsy ( 95100% ) as well as relatively high specificity ( 69100% ) and an acceptable sensitivity ( 7289% ) . however , the negative predictive value is still poor ( npv , 11.7 to 31% ) , which means that still a negative biopsy can not reliably exclude a renal tumor and at least short - interval monitoring is required for patients with negative biopsies . diego published a retrospective analysis of the nis ( nation wide inpatient sample ) data base in the usa , which included 615,500 patients with renal tumor interventions from 1998 to 2008 . radical nephrectomy was performed in 81.3% of these patients , partial nephrectomy in 13.7% , and local tumor ablation in 5.1% ( cryotherapy or radiofrequency ablation ) . the proportion of nephron - sparing interventions increased over time but was still significantly lower than that of tumor nephrectomies , even among patients with chronic renal failure . at the 2011 annual meeting of the american society of clinical oncology ( asco ) , kates et al . from new york presented a seer database analysis covering 4,216 rcc patients who underwent surgery between 1998 and 2007 for tumors < 2 cm . partial nephrectomies were performed in the majority of cases , but the proportion of radical nephrectomies was still high at 45% , even in this patient population with tumors < 2 cm . however , the percentage of nephron - sparing interventions has increased significantly in recent years : from 27% in 1998 to 66% in 2007 . provided convincing evidence that radical nephrectomy per se significantly increases the mortality risk ( hr : 2.24 ; 95% ci : 1.752.84 ) . in the multivariate analysis , also the specific risk of cardiovascular mortality was significantly higher after radical than after partial nephrectomy ( hr : 2.53 ; 95% ci : 1.514.23 ) . on the other hand , dealt with tumor - specific survival after partial and radical nephrectomy in patients with pt1 tumors . within the framework of the saturn project involving 16 italian centers , they analyzed data of 3,320 pt1 patients surgically treated between 1995 and 2007 . their findings conclusively showed that tumor - specific survival was comparable after partial and radical nephrectomy . antonelli et al . thus clearly demonstrates longer overall survival after partial than after radical nephrectomy but comparable tumor - specific survival after both interventions . in order to completely exclude an oncological influence on the survival time after partial and radical nephrectomy , childs et al . performed a retrospective unicenter analysis of their patients who underwent surgery for an ultimately benign renal tumor between 1980 and 2008 . the average follow - up was 8.3 years in 442 patients with a unilateral benign lesion . were also able to show that even these patients had a 1.57 times higher risk of death after radical than after partial nephrectomy ( p = 0.029 ) . thus radical nephrectomy was confirmed as an independent predictor of early death in this study as well . to also elucidate the financial impact of the surgical procedure on the healthcare system , chang et al . used a cost analysis from the usa to demonstrate that performance of radical rather than partial nephrectomy in patients with pt1a rcc regardless of their age was associated with a significantly greater financial burden because of the higher incidence of chronic renal failure after surgery . thus , radical nephrectomy resulted in 24% , 28% , and 31% higher healthcare costs than partial nephrectomy in 55- , 65- , and 75-year - old patients . at the european association of urology ( eau ) congress in 2011 , sun et al . presented a study that retrospectively illuminated nis data from 1998 to 2007 with regard to complications associated with open versus laparoscopic renal tumor surgery . in short , the authors were able to demonstrate that complication rates were comparable for open and laparoscopic partial nephrectomy but that laparoscopic radical nephrectomy was associated with significantly lower morbidity than open radical nephrectomy . a small retrospective monocenter study was presented by hoff et al . from oslo on the occasion of the eau annual meeting . here 48 patients underwent either laparoscopic ( n = 30 ) or robot - assisted ( n = 18 ) partial nephrectomy between 2006 and 2010 . robot - assisted surgery was found to be superior with regard to the mean operating time ( 126 versus 163 min ) , the mean warm ischemia time ( 17 versus 24 min ) , and the mean blood loss ( 112 versus 305 ml ) . there is every indication that robot - assisted partial nephrectomy is technically easier and thus faster to perform than laparoscopic partial nephrectomy . in this small patient population , markedly fewer positive margins were seen after robot - assisted than after laparoscopic partial nephrectomy ( 11 versus 23% ) , though the overall rates seemed very high . to simplify the performance of partial nephrectomy , schnoeller et al . published a technique in which the capsular defect following open tumor enucleation of larger tumors is covered by modified porcine mucosa ( surgisis ) over hemostatic material applied to the parenchyma . the aim was to evenly distribute pressure across the wound surface for optimal hemostasis without further nephron loss due to a puckered suture line . the authors concluded that this technique can achieve blood - tight and , if necessary , urine - tight closure of even large defects and helps to facilitate the performance of partial nephrectomies . compared the effectiveness of local ablation techniques with the current standard of partial nephrectomy for patients with pt1a rcc . they identified 8,087 patients who underwent partial nephrectomy ( mean follow - up : 3.1 years ) and 836 who were submitted to a local ablation procedure ( mean follow - up , 1.9 years ) . multivariate analysis identified the following independent negative predictors of tumor - specific survival : older age ( hr : 1.9 ; 95% ci : 1.62.3 ) , unmarried status ( hr : 1.9 ; 95% ci : 1.32.9 ) , and tumors with > 1 cm growth rate ( hr 1.3 ; 95% ci : 1.01.5 ) . in addition , local ablation compared to partial nephrectomy was identified as an independent negative predictive factor for tumor - specific survival after the fourth postintervention year ( hr : 4.8 , 95% ci : 1.812.3 ) . whitson et al . concluded that partial nephrectomy is the more effective type of treatment for patients with a life expectancy of 4 years . published the first prospective follow - up study in patients with small renal tumors ( <3 cm ) who were treated with open partial nephrectomy , radiofrequency ablation ( rfa ) , or an active surveillance strategy . all patients were older ( > 70 years ) and had significant comorbidities ( asa 3/4 ) . this prospective study with a homogeneous patient population also had a long follow - up in all subgroups , that is , of 60.1 months in patients with open partial nephrectomy ( n = 27 ) , 62.1 months in those with rfa ( n = 24 ) , and 56.3 months in those with active surveillance ( n = 22 ) . in the course of this mean follow - up period of about five years , 92.6% of the patients who were treated by nephron - sparing surgery were found to be completely tumor - free , whereas only 8.3% of those submitted to rfa no longer showed any signs of residual tumor . however , another 70.1% were classified as having stable disease in this group . on the other hand , significant local tumor progression was documented for three patients ( 12.5% ) in the rfa and three ( 17% ) in the active surveillance group . a limitation of the study lies in the fact that no tumor biopsies were performed prior to its initiation in either the rfa or the active surveillance group . thus the inclusion of patients with benign renal tumors in these groups can not be reliably ruled out . the authors concluded that partial nephrectomy should still remain the standard treatment for small renal tumors . a local ablation procedure or an active surveillance strategy is only seen as a genuine alternative for patients with a high perioperative risk or a short overall life expectancy due to significant comorbidities . a meta - analysis published in european urology in 2011 by the study group of tobias klatte and mesut remzi from vienna compared the results of partial nephrectomy ( open or laparoscopic ) with those of laparoscopic cryotherapy . laparoscopic cryotherapy was used for comparison with the standard procedure ( partial nephrectomy ) because it is still considered to be the most effective local ablation technique . this meta - analysis by klatte et al . included 1,295 patients who received cryotherapy and 5,347 who underwent partial nephrectomy . cryotreated patients were significantly older ( 66.2 versus 60.6 years ) ; they also had smaller tumors ( 2.4 versus 3.0 cm ) and a markedly shorter follow - up ( 29.3 versus 57.3 months ) . multivariate analysis of this large patient population showed that , even with a considerably shorter follow - up time , the cryotherapy group had a 5.24 times higher local postinterventional progression rate than the partial nephrectomy group ( 8.5% versus 1.9% ) . on the other hand , the cryotherapy group had a significantly lower complication rate than the partial nephrectomy group ( 17 versus 23% ) . the fact that even local ablation techniques are not risk - free , however , was demonstrated at the aua annual congress by a group working with psutka from boston . here a retrospective analysis was performed to examine 274 patients with 311 renal tumors who underwent rfa between 1998 and 2008 ( mean follow - up : 4.1 years ) . the peri - interventional complication rate was 19.3% ; 14.2% of the complications were classified as minor ones and 5.1% as major ones according to the clavien system . they included 7 ureteral strictures , 5 hemorrhages requiring transfusion , and 3 perirenal abscesses . predictors of complications in this analysis were large tumor size and central tumor location . du et al . from china assessed the prognostic value of preoperative plasma fibrinogen levels in patients with rcc . they performed a retrospective monocenter analysis in 286 patients who underwent radical nephrectomy for rcc between 2000 and 2003 . moreover , multivariate analysis showed that a high fibrinogen level predicted metachronic metastases and poor tumor - specific survival . johnson et al . from atlanta assessed the prognostic value of another acute - phase protein , c - reactive protein ( crp ) . they showed that high crp values prior to radical nephrectomy could predict shorter survival for patients that had localized rcc at the time of surgery . however , it was not entirely understandable that the median survival times in this patient population with localized disease were only 43.4 , 41.8 , and 31.4 months for patients with a low , slightly increased and markedly increased crp value . in another publication , johnson et al . investigated whether not only serum crp but also intratumoral crp expression is of prognostic relevance for survival after radical nephrectomy . this analysis showed that intratumoral crp expression is also an independent prognostic marker for the survival of rcc patients . patients who had tumors that expressed little or no crp lived significantly longer ( mean of 40.5 or 44.2 months ) than those with high intratumoral crp expression ( 31.6 months ; hr : 11.9 ; 95% ci : 1.3111.2 ) . on the other hand , again , the short overall survival times were astonishing . for the italian saturn project , valotto et al . analyzed the prognostic relevance of necrotic areas in renal tumor specimens . the median follow - up was 40 months in 2,719 patients with clear cell rcc . multivariate analysis showed that the microscopically detectable tumor necrosis was significantly associated with poorer tumor - specific survival ( hr : 1.7 , p < 0.001 ) . accordingly , the 5-year tumor - specific survival was 89.4% without and 58.9% with tumor necrosis . investigated whether the number of aberrantly expressed mtor pathway components is of prognostic relevance for tumor - specific survival of patients with rcc . specimens obtained from 258 clear cell rcc patients between 1997 and 2008 were retrospectively examined . a prognostic score was developed based on the number of aberrant mtor pathway components ( mtor , raptor , p-4e - bp1 , p13 kinase , and pten ) . detection of less than three altered components was classified as a favorable biomarker , while three or more altered components indicated an unfavorable prognosis in univariate analysis . multivariate analysis also revealed that several alterations in the mtor pathway tended to be associated with reduced tumor - free survival ( hr : 2.9 ; p = 0.06 ) . the following are take - home messages regarding localized rcc.the risk of malignancy and metastatic spread as well as the aggressive potential of a localized solid renal tumor increases continuously with size.the active surveillance strategy as well as local ablation techniques can currently be recommended only in a selected patient population ( older patients with significant comorbidities ) . today , renal tumor biopsy is relatively harmless and has a high positive predictive value , though its negative predictive value is still low . if oncologically acceptable and technically feasible , nephron - sparing surgery should always be performed , regardless of tumor size . the access and surgical technique for partial nephrectomy should be geared mainly to the surgeon 's experience.possible prognostic parameters for localized rcc published in 2011 were among others : alterations in the mtor signal transduction pathway , microscopically visible intratumoral necrotic areas , and high preoperative crp and fibrinogen values . the risk of malignancy and metastatic spread as well as the aggressive potential of a localized solid renal tumor increases continuously with size . the active surveillance strategy as well as local ablation techniques can currently be recommended only in a selected patient population ( older patients with significant comorbidities ) . today , renal tumor biopsy is relatively harmless and has a high positive predictive value , though its negative predictive value is still low . if oncologically acceptable and technically feasible , nephron - sparing surgery should always be performed , regardless of tumor size . the access and surgical technique for partial nephrectomy should be geared mainly to the surgeon 's experience . possible prognostic parameters for localized rcc published in 2011 were among others : alterations in the mtor signal transduction pathway , microscopically visible intratumoral necrotic areas , and high preoperative crp and fibrinogen values . at the eau congress in vienna , hussain et al . from london presented a retrospective analysis comparing perioperative morbidity of 28 nonmetastatic with that of 22 metastatic rcc patients after preoperative sunitinib therapy . the authors concluded that surgery is technically more complex and difficult after upfront sunitinib therapy : the sunitinib - pretreated group had a significantly higher median blood loss ( 775 ml versus 320 ml ) , a longer operating time ( 195 min versus 128 min ) , and a higher number of intraoperative complications ( 5 versus 2 ) . on the other hand , wound - healing complications were not more common in the sunitinib - pretreated group . the oncological value of nephrectomy in patients with metastatic spread is currently being evaluated in two large prospective studies ( the carmena study and the surtime study ) . to make a definitive statement here , we will surely have to wait for an analysis of these studies . already in 2011 , however , several study groups examined the impact of cytoreductive nephrectomy on the overall survival of patients treated with targeted drugs . published a retrospective multicenter analysis in which 18 centers enrolled 351 patients who were treated with different antiangiogenic agents . here the patients who underwent nephrectomy in addition to systemic antiangiogenic therapy lived significantly longer than those who were not nephrectomized ( median : 38.1 months versus 16.4 months ; p < 0.001 ) . this survival advantage gained by nephrectomy was particularly marked in patients with an ecog performance status of 0 - 1 ( 43.3 versus 16.7 months , p = 0.03 ) . however , the difference in overall survival between patients with and without nephrectomy was no longer statistically significant for those with an ecog performance status of 2 - 3 ( 12.6 versus 8.0 months ; p = 0.8 ) . even using the mskcc risk group stratification , mainly mskcc good- or intermediate - risk patients derived benefit from nephrectomy in this large retrospective analysis ( 42.4 months with and 16.8 months without nephrectomy ; p = 0.02 ) . here too , overall survival did not differ among poor - risk patients , regardless of whether or not they were nephrectomized ( 5.2 months in either case ) . nozawa et al . performed a retrospective unicenter study comparing the response of in situ primary tumors and corresponding metastases to targeted systemic therapy . of the 17 patients included , 35% received first - line treatment with sunitinib , 35% with sorafenib , and 6% each with pazopanib and everolimus . the median maximum decrease in tumor size was 6% ( 48% to 13% ) for the primary rcc and 10% ( 100% to 76% ) for the metastases . in 29% of the patients , the authors concluded that ( a ) the response of the primary tumor and metastases very often differ markedly , ( b ) the metastases usually respond to systemic therapy somewhat better than the primary tumor itself , and thus ( c ) the response of the metastases can not be used to draw conclusions about the response of the primary tumor . at the last asco annual meeting , melichar et al . presented an open - label , single - arm , prospective multinational phase ii study ( bevlin study ) in which 147 nephrectomized mskcc good- and intermediate - risk patients with clear cell rcc received bevacizumab combined with low - dose ifn ( 3 miu ) . this patient population was compared with an avoren subgroup ( n = 272 ) in which patients were treated with a higher ifn dose ( 9 miu ) . median progression - free survival was longer for patients in the bevlin study than for those in the control arm or the avoren trial ( 14.8 versus 10.5 months ) . overall survival also tended to be longer for patients in the bevlin study ( hr : 0.74 ; 95% ci : 0.511.08 ) . these data were surprising in view of the fact that the response rate was markedly lower in the bevlin than in the avoren study ( 24.3% versus 35.9% ) . busch et al . performed a bicentric study to evaluate the response of patients to an mtor inhibitor after failure of treatment with one or more tyrosine kinase inhibitors . in this analysis , 32 patients were treated with everolimus and 29 with temsirolimus . in this retrospective nonrandomized analysis , both substances everolimus and temsirolimus showed comparable effectiveness with median progression - free periods of 5.9 and 5.1 months as well as median overall survival times of 18.7 and 18.0 months . disease stabilization was achieved in 53% of the patients with everolimus and in 52% with temsirolimus . the rate of grade 3 - 4 side effects did not differ significantly either ( 44 and 34% ) . presentation of the first results of the axis trial was one of the urooncology congress highlights of the asco annual meeting this year in chicago . this international prospective randomized phase iii study tested the tyrosine kinase inhibitor ( tki ) axitinib against sorafenib for effectiveness and toxicity in randomized patients who progressed after first - line therapy . previous phase ii studies had confirmed the effectiveness of axitinib another tki that targets mainly vegfr , pdgfr , and c - kit a total of 723 patients were randomized 1 : 1 to receive axitinib or sorafenib . they were treated with sorafenib 2 400 mg / day or axitinib 2 5 mg / day with the option of increasing the latter to 2 10 mg / day if well tolerated . the study included patients with metastatic clear cell rcc who were in good general condition ( ecog performance status 0 - 1 ) and had received previous therapy : sunitinib ( 54% ) , cytokines ( 35% ) , bevacizumab ( 8% ) , and temsirolimus ( 3% ) in both arms . the mskcc risk scores were also evenly distributed : 40% favorable , 54% intermediate , and 2% poor . for the total group , progression - free survival was 6.7 months in the axitinib arm and 4.7 months in the sorafenib arm ( hr : 0.67 , p < 0.001 ) . the partial remission rate was also significantly higher in the axitinib arm ( 19.4% versus 9.4% ) . there were no differences in the number of stable diseases ( 49.9% versus 54.4% ) or patients with primary progressive disease ( 21.6% versus 21.0% ) . however , it should be noted here that , in the course of the study , the dosage was increased at least temporarily in 36.8% of the axitinib patients . the following seemed to occur more frequently in the axitinib arm : grade 3/4 gastrointestinal side effects ( diarrhea 11 versus 7% , nausea and vomiting 7 versus 2% ) , arterial hypertension ( 16 versus 11% ) , and fatigue ( 11 versus 5% ) . on the other hand , sorafenib was more often associated with more severe hand - foot syndromes ( 16 versus 5% ) and erythema ( 4 versus , the first results of the axis trial clearly confirmed the effectiveness of axitinib ( and sorafenib ) in the ( second - line ) therapy of metastatic clear cell rcc . it seems to be a safe drug with toxicity comparable to that of other tkis . it achieved significantly better results than sorafenib in terms of objective remissions and progression - free survival . the latter improvement was most marked in the subgroup of cytokine - pretreated patients ( approx . 5 months ) , while a smaller difference was seen after failure of sunitinib ( approx . the axis trial yielded another very important result : it was the first prospective phase iii study to demonstrate that the tki - tki - sequence is a sensible and effective treatment option for patients with advanced renal cell carcinoma . several limitations should be noted : ( a ) the study was not blinded , ( b ) local and central radiology yielded in part dramatically conflicting results , and ( c ) only axitinib could be dose - escalated , although a number of publications have already confirmed that a dose escalation of other tkis can also lead to a higher response rate and longer progression - free survival . presented a small nonrandomized multicenter phase ii study in which 46 patients received pazopanib for second - line treatment of metastatic rcc after progression or intolerance under first - line treatment with sunitinib ( n = 34 ) or bevacizumab ( n = 12 ) . second - line pazopanib led to partial remission in 22% of all patients and to disease stabilization in another 48% . the median progression - free survival in the total patient population was 9 months ( 5.3 to 11.9 months ) . the most common side effects of pazopanib for second - line treatment corresponded to those known to occur during first - line treatment : grade 3/4 toxicity including fatigue ( 15% ) , arterial hypertension ( 11% ) , and proteinuria ( 11% ) . the authors concluded that second - line use of pazopanib after intolerance or progression of first - line antiangiogenetic treatment appears to be effective and well tolerated and that , in the future , pazopanib will also play a role in the sequence of vegf - targeted therapies . conducted a meta - analysis of 8 prospective studies to assess the hepatotoxicity of pazopanib monotherapy ( 800 mg / d ) . an alt increase was seen in 41.7% of the patients , and 8.2% even had a high - grade increase . interestingly , the incidence of pazopanib - related high - grade alt increases differed significantly between patients with rcc and those with other solid tumors ( 10.9 versus 5.7% ; p = 0.01 ) . ast increases were seen in 39.3% of the patients and high - grade increases in 6.4% . here no significant difference was found between patients with rcc and those with other malignant diseases ( 7.4% versus 4.8% , p = 0.22 ) . the authors concluded that pazopanib therapy is associated with a substantial risk of significant hepatotoxicity and thus requires close monitoring . it remains unclear why the incidence of pazopanib - related hepatotoxicity seems to be higher in patients with rcc than in those with other tumors . the following are take - home messages regarding advanced rcc.neoadjuvant or downsizing preoperative targeted therapy does not yet play a clearly defined role in the management of advanced / metastatic rcc.ifn used in combination with bevacizumab for first - line treatment of mskcc good- and intermediate - prognosis patients with clear cell rcc can ( and should ) be given at a lower dose.sequence therapy with tyrosine kinase inhibitors is an effective alternative to the tki / mtor inhibitor sequence.pazopanib also appears to be another effective tki also for second - line treatment . a positive phase iii study confirmed that axitinib has the potential for a standard second - line treatment , but it still lacks approval.no prognostic / predictive markers applicable in routine clinical practice are in the offing for patients with rcc or for a specific treatment . neoadjuvant or downsizing preoperative targeted therapy does not yet play a clearly defined role in the management of advanced / metastatic rcc . ifn used in combination with bevacizumab for first - line treatment of mskcc good- and intermediate - prognosis patients with clear cell rcc can ( and should ) be given at a lower dose . sequence therapy with tyrosine kinase inhibitors is an effective alternative to the tki / mtor inhibitor sequence . pazopanib also appears to be another effective tki also for second - line treatment . a positive phase iii study confirmed that axitinib has the potential for a standard second - line treatment , but it still lacks approval . no prognostic / predictive markers applicable in routine clinical practice are in the offing for patients with rcc or for a specific treatment .

renal cell carcinoma ( rcc ) is one of the most important urologic malignancies with a continuously growing incidence and health economic relevance . in 2011 , several hundred articles , abstracts , and lectures have been presented at the leading global urooncologic congresses . this review was composed to give an overview on the flood of novel findings dealing with diagnostics and therapy of both localized and advanced rcc . the most clinically relevant data are discussed in detail .

1. Incidence and Prevalence 2. Localized RCC 3. Advanced RCC

in 2011 renal cell carcinoma ( rcc ) still received a great deal of attention at the major urological and oncological congresses worldwide . though rcc is only the third most common urologic malignancy after prostatic and urothelial cancer , it accounts for more than 50,000 new cases in the usa and approximately 15,000 in germany each year . it takes a high death toll because of its often aggressive growth pattern , high rate of early metastasis , and the lack of curative therapy in advanced stages : in germany alone , about one third of all rcc patients ( n = 5,101 ) succumbed to their disease in 2008 . the incidence has increased significantly in the last 30 years.at the last congress of the american urological association ( aua ) , this significantly increased incidence was discussed by a st . it was evaluated in 63,843 rcc patients by a retrospective analysis of the seer ( surveillance epidemiology and end results ) database from 1975 to 2006 . analysis of the seer data also revealed that the increase in incidence mainly affected older patients up to 1990 and younger ones after 1991 . this also meant that , at the time of diagnosis , the majority of rcc patients were over 65 until 1991 , whereas 55.3% were under 65 in 2006 . most congress contributions dealing with localized rcc focused on the following questions.which rcc patients can and should be managed with an active surveillance strategy?when can and should a renal tumor biopsy be performed?what are the advantages and disadvantages of radical versus partial nephrectomy?what is the optimal access and best surgical procedure for partial nephrectomy?are there alternative local ablation techniques equivalent to surgery?what is the value of new predictive or prognostic markers for patients with localized rcc ? what is the optimal access and best surgical procedure for partial nephrectomy ? on the other hand , there are also thought - provoking arguments against an active surveillance strategy . living with a potentially malignant renal tumor then there are also the costs of regular follow - up imaging and the associated radiation exposure ( 3090 msv per ct scan ) . had a mean size of 2.9 cm in this patient population and were ultimately malignant in 73.7% of the cases . sixty percent of tumors < 2 cm and 77.6% of those > 2 cm were malignant in this cohort . reported that the percentage of malignant renal tumors increases significantly with the tumor size . at the aua annual congress , they presented a meta - analysis of 6 studies comprising 880 patients with a follow - up of 15 to 48 months . this meta - analysis revealed a particularly high risk of systemic metastasis in patients with small solid tumors , who tended to be older , as well as in those whose tumors had a larger diameter or volume , or those with a higher annual growth rate ( 0.8 cm versus 0.3 cm ) . < 1 cm and even 7.4% of all tumors with a diameter of 3 - 4 cm ( formally still stage t1a tumors ) had already metastasized at the time of diagnosis . approaching the problem from a completely different angle , chang et al . examined predictors of non - rcc - related death in patients with localized rcc in order to identify good candidates for an active surveillance strategy . moreover , a surveillance strategy involves a small but significant risk of progressive local tumor growth that can potentially render nephron - sparing surgery difficult or impossible . in addition , even in patients with small tumors , active surveillance is associated with a 1 - 2% annual risk of systemic metastasis , and potentially curable patients may thus become incurable [ 1012 ] . a number of studies published in 2011 dealt with the strengths and weaknesses of renal tumor biopsy [ 1315 ] . diego published a retrospective analysis of the nis ( nation wide inpatient sample ) data base in the usa , which included 615,500 patients with renal tumor interventions from 1998 to 2008 . radical nephrectomy was performed in 81.3% of these patients , partial nephrectomy in 13.7% , and local tumor ablation in 5.1% ( cryotherapy or radiofrequency ablation ) . at the 2011 annual meeting of the american society of clinical oncology ( asco ) , kates et al . in the multivariate analysis , also the specific risk of cardiovascular mortality was significantly higher after radical than after partial nephrectomy ( hr : 2.53 ; 95% ci : 1.514.23 ) . on the other hand , dealt with tumor - specific survival after partial and radical nephrectomy in patients with pt1 tumors . within the framework of the saturn project involving 16 italian centers , they analyzed data of 3,320 pt1 patients surgically treated between 1995 and 2007 . thus clearly demonstrates longer overall survival after partial than after radical nephrectomy but comparable tumor - specific survival after both interventions . in order to completely exclude an oncological influence on the survival time after partial and radical nephrectomy , childs et al . performed a retrospective unicenter analysis of their patients who underwent surgery for an ultimately benign renal tumor between 1980 and 2008 . the average follow - up was 8.3 years in 442 patients with a unilateral benign lesion . to also elucidate the financial impact of the surgical procedure on the healthcare system , chang et al . used a cost analysis from the usa to demonstrate that performance of radical rather than partial nephrectomy in patients with pt1a rcc regardless of their age was associated with a significantly greater financial burden because of the higher incidence of chronic renal failure after surgery . thus , radical nephrectomy resulted in 24% , 28% , and 31% higher healthcare costs than partial nephrectomy in 55- , 65- , and 75-year - old patients . at the european association of urology ( eau ) congress in 2011 , sun et al . from oslo on the occasion of the eau annual meeting . robot - assisted surgery was found to be superior with regard to the mean operating time ( 126 versus 163 min ) , the mean warm ischemia time ( 17 versus 24 min ) , and the mean blood loss ( 112 versus 305 ml ) . multivariate analysis identified the following independent negative predictors of tumor - specific survival : older age ( hr : 1.9 ; 95% ci : 1.62.3 ) , unmarried status ( hr : 1.9 ; 95% ci : 1.32.9 ) , and tumors with > 1 cm growth rate ( hr 1.3 ; 95% ci : 1.01.5 ) . whitson et al . concluded that partial nephrectomy is the more effective type of treatment for patients with a life expectancy of 4 years . published the first prospective follow - up study in patients with small renal tumors ( <3 cm ) who were treated with open partial nephrectomy , radiofrequency ablation ( rfa ) , or an active surveillance strategy . this prospective study with a homogeneous patient population also had a long follow - up in all subgroups , that is , of 60.1 months in patients with open partial nephrectomy ( n = 27 ) , 62.1 months in those with rfa ( n = 24 ) , and 56.3 months in those with active surveillance ( n = 22 ) . in the course of this mean follow - up period of about five years , 92.6% of the patients who were treated by nephron - sparing surgery were found to be completely tumor - free , whereas only 8.3% of those submitted to rfa no longer showed any signs of residual tumor . however , another 70.1% were classified as having stable disease in this group . on the other hand , significant local tumor progression was documented for three patients ( 12.5% ) in the rfa and three ( 17% ) in the active surveillance group . a limitation of the study lies in the fact that no tumor biopsies were performed prior to its initiation in either the rfa or the active surveillance group . the authors concluded that partial nephrectomy should still remain the standard treatment for small renal tumors . a local ablation procedure or an active surveillance strategy is only seen as a genuine alternative for patients with a high perioperative risk or a short overall life expectancy due to significant comorbidities . a meta - analysis published in european urology in 2011 by the study group of tobias klatte and mesut remzi from vienna compared the results of partial nephrectomy ( open or laparoscopic ) with those of laparoscopic cryotherapy . laparoscopic cryotherapy was used for comparison with the standard procedure ( partial nephrectomy ) because it is still considered to be the most effective local ablation technique . cryotreated patients were significantly older ( 66.2 versus 60.6 years ) ; they also had smaller tumors ( 2.4 versus 3.0 cm ) and a markedly shorter follow - up ( 29.3 versus 57.3 months ) . multivariate analysis of this large patient population showed that , even with a considerably shorter follow - up time , the cryotherapy group had a 5.24 times higher local postinterventional progression rate than the partial nephrectomy group ( 8.5% versus 1.9% ) . on the other hand , the cryotherapy group had a significantly lower complication rate than the partial nephrectomy group ( 17 versus 23% ) . the fact that even local ablation techniques are not risk - free , however , was demonstrated at the aua annual congress by a group working with psutka from boston . the peri - interventional complication rate was 19.3% ; 14.2% of the complications were classified as minor ones and 5.1% as major ones according to the clavien system . they included 7 ureteral strictures , 5 hemorrhages requiring transfusion , and 3 perirenal abscesses . predictors of complications in this analysis were large tumor size and central tumor location . from china assessed the prognostic value of preoperative plasma fibrinogen levels in patients with rcc . they performed a retrospective monocenter analysis in 286 patients who underwent radical nephrectomy for rcc between 2000 and 2003 . they showed that high crp values prior to radical nephrectomy could predict shorter survival for patients that had localized rcc at the time of surgery . however , it was not entirely understandable that the median survival times in this patient population with localized disease were only 43.4 , 41.8 , and 31.4 months for patients with a low , slightly increased and markedly increased crp value . on the other hand , again , the short overall survival times were astonishing . analyzed the prognostic relevance of necrotic areas in renal tumor specimens . specimens obtained from 258 clear cell rcc patients between 1997 and 2008 were retrospectively examined . a prognostic score was developed based on the number of aberrant mtor pathway components ( mtor , raptor , p-4e - bp1 , p13 kinase , and pten ) . the following are take - home messages regarding localized rcc.the risk of malignancy and metastatic spread as well as the aggressive potential of a localized solid renal tumor increases continuously with size.the active surveillance strategy as well as local ablation techniques can currently be recommended only in a selected patient population ( older patients with significant comorbidities ) . today , renal tumor biopsy is relatively harmless and has a high positive predictive value , though its negative predictive value is still low . if oncologically acceptable and technically feasible , nephron - sparing surgery should always be performed , regardless of tumor size . the access and surgical technique for partial nephrectomy should be geared mainly to the surgeon 's experience.possible prognostic parameters for localized rcc published in 2011 were among others : alterations in the mtor signal transduction pathway , microscopically visible intratumoral necrotic areas , and high preoperative crp and fibrinogen values . possible prognostic parameters for localized rcc published in 2011 were among others : alterations in the mtor signal transduction pathway , microscopically visible intratumoral necrotic areas , and high preoperative crp and fibrinogen values . at the eau congress in vienna , hussain et al . the authors concluded that surgery is technically more complex and difficult after upfront sunitinib therapy : the sunitinib - pretreated group had a significantly higher median blood loss ( 775 ml versus 320 ml ) , a longer operating time ( 195 min versus 128 min ) , and a higher number of intraoperative complications ( 5 versus 2 ) . on the other hand , wound - healing complications were not more common in the sunitinib - pretreated group . already in 2011 , however , several study groups examined the impact of cytoreductive nephrectomy on the overall survival of patients treated with targeted drugs . this survival advantage gained by nephrectomy was particularly marked in patients with an ecog performance status of 0 - 1 ( 43.3 versus 16.7 months , p = 0.03 ) . even using the mskcc risk group stratification , mainly mskcc good- or intermediate - risk patients derived benefit from nephrectomy in this large retrospective analysis ( 42.4 months with and 16.8 months without nephrectomy ; p = 0.02 ) . nozawa et al . performed a retrospective unicenter study comparing the response of in situ primary tumors and corresponding metastases to targeted systemic therapy . of the 17 patients included , 35% received first - line treatment with sunitinib , 35% with sorafenib , and 6% each with pazopanib and everolimus . in 29% of the patients , the authors concluded that ( a ) the response of the primary tumor and metastases very often differ markedly , ( b ) the metastases usually respond to systemic therapy somewhat better than the primary tumor itself , and thus ( c ) the response of the metastases can not be used to draw conclusions about the response of the primary tumor . at the last asco annual meeting , melichar et al . this patient population was compared with an avoren subgroup ( n = 272 ) in which patients were treated with a higher ifn dose ( 9 miu ) . these data were surprising in view of the fact that the response rate was markedly lower in the bevlin than in the avoren study ( 24.3% versus 35.9% ) . disease stabilization was achieved in 53% of the patients with everolimus and in 52% with temsirolimus . the rate of grade 3 - 4 side effects did not differ significantly either ( 44 and 34% ) . presentation of the first results of the axis trial was one of the urooncology congress highlights of the asco annual meeting this year in chicago . previous phase ii studies had confirmed the effectiveness of axitinib another tki that targets mainly vegfr , pdgfr , and c - kit a total of 723 patients were randomized 1 : 1 to receive axitinib or sorafenib . the study included patients with metastatic clear cell rcc who were in good general condition ( ecog performance status 0 - 1 ) and had received previous therapy : sunitinib ( 54% ) , cytokines ( 35% ) , bevacizumab ( 8% ) , and temsirolimus ( 3% ) in both arms . the mskcc risk scores were also evenly distributed : 40% favorable , 54% intermediate , and 2% poor . there were no differences in the number of stable diseases ( 49.9% versus 54.4% ) or patients with primary progressive disease ( 21.6% versus 21.0% ) . however , it should be noted here that , in the course of the study , the dosage was increased at least temporarily in 36.8% of the axitinib patients . the following seemed to occur more frequently in the axitinib arm : grade 3/4 gastrointestinal side effects ( diarrhea 11 versus 7% , nausea and vomiting 7 versus 2% ) , arterial hypertension ( 16 versus 11% ) , and fatigue ( 11 versus 5% ) . on the other hand , sorafenib was more often associated with more severe hand - foot syndromes ( 16 versus 5% ) and erythema ( 4 versus , the first results of the axis trial clearly confirmed the effectiveness of axitinib ( and sorafenib ) in the ( second - line ) therapy of metastatic clear cell rcc . it seems to be a safe drug with toxicity comparable to that of other tkis . the latter improvement was most marked in the subgroup of cytokine - pretreated patients ( approx . the axis trial yielded another very important result : it was the first prospective phase iii study to demonstrate that the tki - tki - sequence is a sensible and effective treatment option for patients with advanced renal cell carcinoma . several limitations should be noted : ( a ) the study was not blinded , ( b ) local and central radiology yielded in part dramatically conflicting results , and ( c ) only axitinib could be dose - escalated , although a number of publications have already confirmed that a dose escalation of other tkis can also lead to a higher response rate and longer progression - free survival . second - line pazopanib led to partial remission in 22% of all patients and to disease stabilization in another 48% . the median progression - free survival in the total patient population was 9 months ( 5.3 to 11.9 months ) . the most common side effects of pazopanib for second - line treatment corresponded to those known to occur during first - line treatment : grade 3/4 toxicity including fatigue ( 15% ) , arterial hypertension ( 11% ) , and proteinuria ( 11% ) . the authors concluded that second - line use of pazopanib after intolerance or progression of first - line antiangiogenetic treatment appears to be effective and well tolerated and that , in the future , pazopanib will also play a role in the sequence of vegf - targeted therapies . an alt increase was seen in 41.7% of the patients , and 8.2% even had a high - grade increase . ast increases were seen in 39.3% of the patients and high - grade increases in 6.4% . the authors concluded that pazopanib therapy is associated with a substantial risk of significant hepatotoxicity and thus requires close monitoring . the following are take - home messages regarding advanced rcc.neoadjuvant or downsizing preoperative targeted therapy does not yet play a clearly defined role in the management of advanced / metastatic rcc.ifn used in combination with bevacizumab for first - line treatment of mskcc good- and intermediate - prognosis patients with clear cell rcc can ( and should ) be given at a lower dose.sequence therapy with tyrosine kinase inhibitors is an effective alternative to the tki / mtor inhibitor sequence.pazopanib also appears to be another effective tki also for second - line treatment . neoadjuvant or downsizing preoperative targeted therapy does not yet play a clearly defined role in the management of advanced / metastatic rcc . no prognostic / predictive markers applicable in routine clinical practice are in the offing for patients with rcc or for a specific treatment .

in the united states , the number of hospitals providing percutaneous coronary intervention ( pci ) has increased in the past 2 decades.1 , 2 , 3 , 4 , 5 , 6 , 7 according to the american hospital association annual survey , 1176 us hospitals were capable of performing pci in 2001 ; this number increased to 1739 in 2008 , an increase of > 40%.4 , 5 to explain the rapid diffusion of pci adoption in us hospitals , vaughansarrazin et al8 suggested that pci is favored over coronary artery bypass graft ( cabg ) because pci is easy to learn and implement , has a high reimbursement rate , and requires only slight modifications of existing techniques , such as drugeluting stents . another study indicated that the newer and larger hospitals equipped with expensive medical technology were more likely to adopt pci.5 however , little is known about the differences between the characteristics of early and lateadopting hospitals . according to everett rogers theory of the diffusion of innovations , facilities can be classified into 5 categories based on their timing of adoption of new technology : innovators , early adopters , early majority , late majority , and laggards.9 the first objective of this study was to examine whether the characteristics of the lateadopting hospitals providing pci differed from those of the earlyadopting hospitals . as indicated by rogers , diffusion of innovations is a means to an ultimate end : consequences that result from adopting an innovation . consequences are the changes that occur to an individual or a social system from adapting or rejecting an innovation.9 in terms of diffusion of pci , the end goals are to improve pci accessibility , provide highquality pci in all areas , and in the long run , decrease the overall mortality rate in patients with coronary artery disease . however , as argued by concannon et al,5 in an era of flat use of elective pci procedure , systematic duplication ( ie , implementing new pci programs targeting neighborhoods already served by an existing program ) reduces average pci volumes and results in lower procedure quality and worse outcomes for patients with coronary artery disease . the second objective of this study was to examine whether late adopting hospitals had different effects on pci volume and access compared with their earlyadopting counterparts . the main data source for this study was obtained from the taiwan national health insurance research database ( nhird ) , which is maintained by the national health research institutes , taiwan . the nhi program was introduced in 1995 and covers more than 99% of the taiwanese population . the nhird includes outpatient and inpatient claims data ( including data on diagnosis and procedure codes , patient characteristics , physicians , clinical settings , and various service fees ) for 19972012.10 because all patients receiving pci in taiwan were required to be hospitalized for observation , we only used the inpatient claims data from the nhrid . the second data source we used was the medical facility open information query system maintained by the ministry of health and welfare ; it was used to obtain information on hospital characteristics unavailable in the nhird.11 this study was approved by the institutional review board of cheng hsin general hospital ( chghirb no . ( 424)1031 ) . as this study used secondary administrative data the requirement that subjects should give informed consent was waived . we used the international classification of disease , the ninth revision , clinical modification ( icd9cm ) codes 36.01 , 36.02 , 36.05 , 36.06 , and 36.09 ( taiwan nhi does not use the icd9cm codes 00.66 , 36.00 , and 36.07 for pci , which are used in us hospitals ) to determine the number of pcis performed per hospital per year . according to innovation diffusion theory , we classified the pci hospitals into 4 categories on the basis of the hospitals firstyear claims data on pci reimbursements : ( 1 ) the early adopters , which started providing pci before 1998 ; ( 2 ) the early majority , which started providing pci between 1998 and 2002 ; ( 3 ) the late majority , which started providing pci between 2003 and 2007 ; and ( 4 ) the laggards , which started providing pci between 2008 and 2012 . information on the number of hospitals providing pci before 1997 were derived from a historical review and personal communication.12 the characteristics of hospitals included level of hospitals ( tertiary referral hospital , secondary referral hospital , and community district hospital ) , ownership of hospital ( public versus private ) , number of hospital beds , number of pci performed , having cabg surgery facility ( icd9cm code 36.1x and 36.2 ) , percentage of pci performed with principal discharge diagnosis as acute myocardial infarction ( ami ) . we did not distinguish stelevation myocardial infarction versus nonstelevation myocardial infarction because of unreliable coding . the information on the number of hospital beds was obtained from the query system of ministry of health and welfare.11 the information on other characteristics was derived from the inpatient claims data . we used icd9cm codes 36.1x and 36.2 to identify hospital having cabg surgery facility , and icd9cm code 410.xx for ami diagnosis . we first calculated the pci volumes per hospital per year to examine the differences in pci volumes between the early and late adopting hospitals.13 , 14 we then used a geographic information system ( gis ) to geocode these hospitals to determine whether public accessibility to pci improved between 1997 and 2012 . if the gisestimated linear distance ( crow 's fly distance ) between the district centroid and the nearest pci hospital was < 40 km ( 25 miles ) , the entire population of that district was considered to have appropriate access to pci , and if this distance > 40 km , the entire population of the district was considered to be inappropriate access to pci . we then computed the percentages of the taiwanese population with appropriate access to pci in 1997 , 2002 , 2007 , and 2012 . study in the us used 40 miles as threshold to assess the accessibility.6 as taiwan is a small island with highdensity population , we used 25 miles as the threshold to assess the accessibility of pci services . moreover , we calculated the gisestimated linear distance between new pci hospitals and the nearest old pci hospitals to assess the problem of duplication of pci hospitals . taiwan ( 35 980 km ) is similar in size to the us state of maryland ( 32 133 km ) . however , the population of taiwan ( 23 million ) is nearly 4 times that of maryland ( 6 million ) . except in certain mountainous regions , the national health insurance administration , thus , classified taiwan into 6 medical regions , namely taipei , northern , central , southern , kauping , and eastern ; this classification is similar to the 306 hospital referral regions in the united states . to assess regional disparities in the abundance of pci hospitals , we calculated the number of pci hospitals per 1 million persons in each medical region in 1997 , 2002 , 2007 , and 2012 . we used the chisquare test to examine if there were significant differences in the characteristics ( level of hospital , ownership of hospital , bed capacity of hospital , percentage of hospitals with cabg support in 2012 , and the percentage of pci performed in 2012 with ami as principal diagnosis ) of the 4 pci hospital categories , in which the innovators and early adopters were combined into a single category . we used the kruskalwallis test to assess whether the median pci volumes and median distance between new hospitals with existing hospitals in 2012 differed among the 4 pci hospitals categories . the main data source for this study was obtained from the taiwan national health insurance research database ( nhird ) , which is maintained by the national health research institutes , taiwan . the nhi program was introduced in 1995 and covers more than 99% of the taiwanese population . the nhird includes outpatient and inpatient claims data ( including data on diagnosis and procedure codes , patient characteristics , physicians , clinical settings , and various service fees ) for 19972012.10 because all patients receiving pci in taiwan were required to be hospitalized for observation , we only used the inpatient claims data from the nhrid . the second data source we used was the medical facility open information query system maintained by the ministry of health and welfare ; it was used to obtain information on hospital characteristics unavailable in the nhird.11 this study was approved by the institutional review board of cheng hsin general hospital ( chghirb no . ( 424)1031 ) . as this study used secondary administrative data the requirement that subjects should give informed consent was waived . we used the international classification of disease , the ninth revision , clinical modification ( icd9cm ) codes 36.01 , 36.02 , 36.05 , 36.06 , and 36.09 ( taiwan nhi does not use the icd9cm codes 00.66 , 36.00 , and 36.07 for pci , which are used in us hospitals ) to determine the number of pcis performed per hospital per year . according to innovation diffusion theory , we classified the pci hospitals into 4 categories on the basis of the hospitals firstyear claims data on pci reimbursements : ( 1 ) the early adopters , which started providing pci before 1998 ; ( 2 ) the early majority , which started providing pci between 1998 and 2002 ; ( 3 ) the late majority , which started providing pci between 2003 and 2007 ; and ( 4 ) the laggards , which started providing pci between 2008 and 2012 . information on the number of hospitals providing pci before 1997 were derived from a historical review and personal communication.12 the characteristics of hospitals included level of hospitals ( tertiary referral hospital , secondary referral hospital , and community district hospital ) , ownership of hospital ( public versus private ) , number of hospital beds , number of pci performed , having cabg surgery facility ( icd9cm code 36.1x and 36.2 ) , percentage of pci performed with principal discharge diagnosis as acute myocardial infarction ( ami ) . we did not distinguish stelevation myocardial infarction versus nonstelevation myocardial infarction because of unreliable coding . the information on the number of hospital beds was obtained from the query system of ministry of health and welfare.11 the information on other characteristics was derived from the inpatient claims data . we used icd9cm codes 36.1x and 36.2 to identify hospital having cabg surgery facility , and icd9cm code 410.xx for ami diagnosis . we first calculated the pci volumes per hospital per year to examine the differences in pci volumes between the early and late adopting hospitals.13 , 14 we then used a geographic information system ( gis ) to geocode these hospitals to determine whether public accessibility to pci improved between 1997 and 2012 . if the gisestimated linear distance ( crow 's fly distance ) between the district centroid and the nearest pci hospital was < 40 km ( 25 miles ) , the entire population of that district was considered to have appropriate access to pci , and if this distance > 40 km , the entire population of the district was considered to be inappropriate access to pci . we then computed the percentages of the taiwanese population with appropriate access to pci in 1997 , 2002 , 2007 , and 2012 . study in the us used 40 miles as threshold to assess the accessibility.6 as taiwan is a small island with highdensity population , we used 25 miles as the threshold to assess the accessibility of pci services . moreover , we calculated the gisestimated linear distance between new pci hospitals and the nearest old pci hospitals to assess the problem of duplication of pci hospitals . taiwan ( 35 980 km ) is similar in size to the us state of maryland ( 32 133 km ) . however , the population of taiwan ( 23 million ) is nearly 4 times that of maryland ( 6 million ) . except in certain mountainous regions , the national health insurance administration , thus , classified taiwan into 6 medical regions , namely taipei , northern , central , southern , kauping , and eastern ; this classification is similar to the 306 hospital referral regions in the united states . to assess regional disparities in the abundance of pci hospitals , we calculated the number of pci hospitals per 1 million persons in each medical region in 1997 , 2002 , 2007 , and 2012 . we used the chisquare test to examine if there were significant differences in the characteristics ( level of hospital , ownership of hospital , bed capacity of hospital , percentage of hospitals with cabg support in 2012 , and the percentage of pci performed in 2012 with ami as principal diagnosis ) of the 4 pci hospital categories , in which the innovators and early adopters were combined into a single category . we used the kruskalwallis test to assess whether the median pci volumes and median distance between new hospitals with existing hospitals in 2012 differed among the 4 pci hospitals categories . the number of hospitals adopting pci by year is illustrated in figure 1 ; 2 surges were observed , the first was in 19961997 and the second in 20102011 . according to the claims data , 32 hospitals provided pci in 1997 , and depending on the year that hospitals started providing pci , 32 were classified as early adopters ( before 1998 ) , 23 as early majority ( 19982002 ) , 24 as late majority ( 20032007 ) , and 16 as laggards ( 20082012 ) . the characteristics of the 4 pci hospital groups are presented in table 1 . a prominent increase in district community hospitals ( n=7 ) providing pci services was noted since 2008 . the later the hospitals adopted pci , the smaller their sizes and the closer they were to existing pci hospitals , the lower the percentage with cabg surgery support , the lower the volume of pci performed , and the higher the percentage of pci performed with ami as principal discharge diagnosis . characteristics of hospitals providing percutaneous coronary intervention ( pci ) by year providing pci services in taiwan ami indicates acute myocardial infarction ; cabg , coronary artery bypass graft . the descriptive statistics of the pci volumes in 2012 for the 4 pci hospital categories are illustrated in figure 2 . the minimum , median , and maximum pci volumes in 2012 were 197 , 706 , and 1605 for the early adopters ; 100 , 338 , and 836 for the early majority ; 34 , 138 , and 625 for the late majority ; and 15 , 81 , and 365 for the laggards , respectively . the annual pci volumes of 15 hospitals was < 100 , and 13 of these hospitals were laggards . one laggard pci hospital adopted pci in 2009 but stopped offering the procedure in 2012 . the percentage of pci volume100% , 75% , 50% , 25% , and 0%observed in early adopters , early majority , late majority , and laggard pci hospitals , respectively , in 2012 in taiwan . to assess whether the new pci hospitals were located in an area where neighboring hospitals already offered similar procedures , we tracked each hospital providing pci on a map ( figure 3 ) . as shown in figure 3b , between 1998 and 2002 , only 2 early majority hospitals had a distance between the new pci hospital and its nearest existing pci hospital higher than 20 km ( 12.4 miles ) : one each in the eastern ( 81.3 km , 50.5 miles ) and kaupin regions ( 20.8 km , 12.9 miles ) . between 2003 and 2007 , no late majority hospitals had a distance exceeding 20 km from the nearest existing pci hospital , and 6 new pci hospitals had distances between 10 and 20 km ( figure 3c ) . between 2008 and 2012 , only one laggard hospital in the central region had a distance exceeding 20 km ( 29.7 km , 17.3 miles ) , and 3 new pci hospitals had distances between 10 and 20 km ( figure 3d ) . locations of hospitals ( based on the pci starting year ) offering pci in taiwan ( a ) early adopters ( before 1998 ) ; ( b ) early majority ( 19982002 ) ; ( c ) late majority ( 20032007 ) ; ( d ) laggards ( 20082012 ) . the number in parentheses in each medical region is the number of pci hospitals per 1 million persons in that region for the years 1997 ( a ) , 2002 ( b ) , 2007 ( c ) and 2012 ( d ) . the red dot indicates the location of new pci hospitals and the gray dot indicates the location of existing pci hospitals in the given study period . with regard to public accessibility to pci , in 1997 , 95.7% of the population lived within a distance of 40 km ( 25 miles ) from a pci hospital ; this percentage was 98.0% in 2002 , 98.0% in 2007 , and 98.2% in 2012 . table 2 illustrates regional variations in pci hospitals per 1 million population by year . the variation in pci center density among 6 regions was small in 1997 ( 1.11.9 ) and a little bit larger in 2012 ( 3.15.3 ) . number of hospitals providing percutaneous coronary intervention ( pci ) per 1 million population in 6 medical regions in taiwan the findings of this nationwide descriptive study in taiwan indicate that the characteristics of the earlyadopting pci hospitals differed from those of the lateadopting hospitals . the lateadopting hospitals were smaller in size and had lower pci volumes and lower percentage of having cabg surgery support ; yet had higher percentage of pci performed with ami as principal discharge diagnosis . despite the mild increase in population access to pci access during the past decade , some of laggard pciadopting hospitals are in remote areas and provide needed and timely services for ami patients . in contrast to the united states , where 1739 hospitals provided pci in 2008,5 only 88 hospitals provided pci in taiwan in 2012 . following rogers classification , 3 innovators ( ie , taipei veterans general hospital , chang gang memorial hospital , and national taiwan university hospital ) started offering pci in 1980 . by the early 1990s , the adoption of pci accelerated after baremetal stents were introduced to taiwan in 1995 , many large secondary referral hospitals started to provide pci services . a slowdown of increase was noted in 19981999 because of the change of pci insurance reimbursement from feeforservices ( ffs ) to diagnosisrelated groups ( drg ) in 1999.15 as the total payments under drg regime were better than ffs for pci services , a return to the growth curve in 20102011 was noted . the second surge in the number of hospitals offering pci in 20102011 ( figure 1 ) , which was initiated by the government . the doortoballoon alliance , which included 15 leading pci hospitals , initiated by the taiwan joint commission on hospital accreditation was launched in 2008.16 the hospitals ability to provide primary pci facilities for patients with stemi was mandated by the commission as a criteria for obtaining a high emergency accreditation level . furthermore , the ministry of health and welfare encouraged district hospitals in remote areas to provide pci services . regarding the minimum pci volume standard , the 2013 american college of cardiology / american heart association / society for cardiovascular angiography and interventions clinical competence statement on pci recommended that pci be performed only by operators with an annual volume of > 50 procedures at hospitals with an annual volume exceeding 200 procedures.17 in 2012 , > onethird ( 32 of 88 ) of all pci hospitals in taiwan had annual volumes < 200 . furthermore , 9 of the 16 laggard hospitals had an annual pci volume of less than 100 . hence , regular monitoring of the pci quality at the laggard pci hospitals seems warranted . however , many of these low pci volume laggard hospitals are in remote areas with small populations and with relatively low pci demand . according to table 1 , the laggard hospitals had a statistically significant higher percentage of pci performed with ami as principal discharge diagnosis than the earlyadopting hospitals , the difference in percentage was not huge . these lowpci volume laggard hospitals play an important role in providing needed and timely services for people with ami in remote areas . actually , the inhospital mortality rate of ami patients has been reduced significantly in those areas that have utilized this approach.18 regarding pci accessibility , 88% of the us population lived within a distance of 64 km ( 40 mi ) from a pci hospital in 1997 , and the percentage increased to 93% in 2008.5 a study in michigan reported that of the 12 hospitals that were upgraded to provide primary pci procedures , only 3 were located at least 32 km ( 20 miles ) from another primary pci hospital , accounting for a 4.3% improvement in public accessibility , whereas the remaining 9 hospitals increased the accessibility by only 0.5%.19 geographically , taiwan is a small country with a highdensity population and a convenient public transportation system . the percentage of the population living within a range of 40 km ( 25 miles ) from a pci hospital was already high ( 95.7% ) in 1997 , and only 32 earlyadopter hospitals provided pci . the percentage increased to 98.0% in 2002 , after 23 hospitals started providing pci between 1998 and 2002 . between 2002 and 2012 , 40 new hospitals started providing pci ; however , no substantial improvement in public accessibility to pci was observed . another measure of pci accessibility was pci center density : the number of pci hospitals per 1 million population in a given region . a us study reported a wide statetostate variation in pci center density from 3.2 in vermont and 3.6 in minnesota to 12.1 in west virginia and 11.6 in louisiana , with a median of 6.95.7 contrarily , no such substantial regional variation in pci center density , which ranged from 3.12 in the northern region to 5.33 in the eastern region in 2012 , was noted in taiwan ( table 2 and figure 3 ) . first , the arbitrary use of the year that each hospital started providing pci into 4 time periods ( 1997 , 19982002 , 20032007 , and 20082012 ) for classifying the pci hospitals was the first limitation . however , we could not find any superior classification method ; rogers also arbitrarily used statistical standard deviation to classify the underlying population of adopters into 5 categories.9 second , we used the crowfly distance instead of road distance or drive time between the population and the nearest pci hospitals as a proxy measure of pci accessibility , which might generate results that are different from approaches that use actual transportation time as the basis of measurement , which is important for treatment of patients with ami . however , the transport time to a pci facility can be affected by many other factors , such as traffic , time of the day , and other factors , which vary considerably.20 third , as the geographic size of the 368 districts in taiwan varied greatly from 9 persons per km in soulin township , hualien county to 21 950 persons per km in central district in taichung city , which had different implications with regard to the distance of 40 km used for assessing access . furthermore , caution should be exercised in interpreting pci center density as an indicator of population accessibility to pci . the population in the eastern region was 0.56 million in 2012 , which is relatively small compared with the other regions in taiwan : 7.5 million in the taipei region , 3.5 million in the northern region , 4.5 million in the central region , 3.4 million in the southern region , and 3.7 million in the kaupin region . in contrast to the united states , where 1739 hospitals provided pci in 2008,5 only 88 hospitals provided pci in taiwan in 2012 . following rogers classification , 3 innovators ( ie , taipei veterans general hospital , chang gang memorial hospital , and national taiwan university hospital ) started offering pci in 1980 . by the early 1990s , almost all tertiary referral hospitals with > 1000 hospital beds have provided pci services . the adoption of pci accelerated after baremetal stents were introduced to taiwan in 1995 , many large secondary referral hospitals started to provide pci services . a slowdown of increase was noted in 19981999 because of the change of pci insurance reimbursement from feeforservices ( ffs ) to diagnosisrelated groups ( drg ) in 1999.15 as the total payments under drg regime were better than ffs for pci services , a return to the growth curve in 20102011 was noted . the second surge in the number of hospitals offering pci in 20102011 ( figure 1 ) , which was initiated by the government . the doortoballoon alliance , which included 15 leading pci hospitals , initiated by the taiwan joint commission on hospital accreditation was launched in 2008.16 the hospitals ability to provide primary pci facilities for patients with stemi was mandated by the commission as a criteria for obtaining a high emergency accreditation level . furthermore , the ministry of health and welfare encouraged district hospitals in remote areas to provide pci services . regarding the minimum pci volume standard , the 2013 american college of cardiology / american heart association / society for cardiovascular angiography and interventions clinical competence statement on pci recommended that pci be performed only by operators with an annual volume of > 50 procedures at hospitals with an annual volume exceeding 200 procedures.17 in 2012 , > onethird ( 32 of 88 ) of all pci hospitals in taiwan had annual volumes < 200 . furthermore , 9 of the 16 laggard hospitals had an annual pci volume of less than 100 . hence , regular monitoring of the pci quality at the laggard pci hospitals seems warranted . however , many of these low pci volume laggard hospitals are in remote areas with small populations and with relatively low pci demand . according to table 1 , the laggard hospitals had a statistically significant higher percentage of pci performed with ami as principal discharge diagnosis than the earlyadopting hospitals , the difference in percentage was not huge . these lowpci volume laggard hospitals play an important role in providing needed and timely services for people with ami in remote areas . actually , the inhospital mortality rate of ami patients has been reduced significantly in those areas that have utilized this approach.18 regarding pci accessibility , 88% of the us population lived within a distance of 64 km ( 40 mi ) from a pci hospital in 1997 , and the percentage increased to 93% in 2008.5 a study in michigan reported that of the 12 hospitals that were upgraded to provide primary pci procedures , only 3 were located at least 32 km ( 20 miles ) from another primary pci hospital , accounting for a 4.3% improvement in public accessibility , whereas the remaining 9 hospitals increased the accessibility by only 0.5%.19 geographically , taiwan is a small country with a highdensity population and a convenient public transportation system . the percentage of the population living within a range of 40 km ( 25 miles ) from a pci hospital was already high ( 95.7% ) in 1997 , and only 32 earlyadopter hospitals provided pci . the percentage increased to 98.0% in 2002 , after 23 hospitals started providing pci between 1998 and 2002 . between 2002 and 2012 , 40 new hospitals started providing pci ; however , no substantial improvement in public accessibility to pci was observed . another measure of pci accessibility was pci center density : the number of pci hospitals per 1 million population in a given region . a us study reported a wide statetostate variation in pci center density from 3.2 in vermont and 3.6 in minnesota to 12.1 in west virginia and 11.6 in louisiana , with a median of 6.95.7 contrarily , no such substantial regional variation in pci center density , which ranged from 3.12 in the northern region to 5.33 in the eastern region in 2012 , was noted in taiwan ( table 2 and figure 3 ) . first , the arbitrary use of the year that each hospital started providing pci into 4 time periods ( 1997 , 19982002 , 20032007 , and 20082012 ) for classifying the pci hospitals was the first limitation . however , we could not find any superior classification method ; rogers also arbitrarily used statistical standard deviation to classify the underlying population of adopters into 5 categories.9 second , we used the crowfly distance instead of road distance or drive time between the population and the nearest pci hospitals as a proxy measure of pci accessibility , which might generate results that are different from approaches that use actual transportation time as the basis of measurement , which is important for treatment of patients with ami . however , the transport time to a pci facility can be affected by many other factors , such as traffic , time of the day , and other factors , which vary considerably.20 third , as the geographic size of the 368 districts in taiwan varied greatly from 9 persons per km in soulin township , hualien county to 21 950 persons per km in central district in taichung city , which had different implications with regard to the distance of 40 km used for assessing access . furthermore , caution should be exercised in interpreting pci center density as an indicator of population accessibility to pci . the population in the eastern region was 0.56 million in 2012 , which is relatively small compared with the other regions in taiwan : 7.5 million in the taipei region , 3.5 million in the northern region , 4.5 million in the central region , 3.4 million in the southern region , and 3.7 million in the kaupin region . many evidencebased innovations in health care , as indicated by berwick,21 implemented successfully in one location often disseminate slowly . by contrast , the diffusion of pci in the united states and taiwan occurred quite rapidly . as the theory suggests that diffusion depends upon many things beyond the innovation itself ( pci in this case ) , but also upon the adopters ( explored in this paper ) , communication channels , time , and the social system ( initiation by government in taiwan ) . however , as suggested by rogers , concern about the consequences of the diffusion of innovations should not be neglected . our data reveal that the lateadopting hospitals were smaller and had lower pci volumes performed . however , these low pci volume laggard hospitals played an important role in providing needed and timely services for people with ami in remove areas . the ministry of health and social welfare and national health insurance administration should provide needed support for these lowpci volume hospitals in remote areas to ensure the quality of pci performed .

backgroundstudies in the united states suggested that the characteristics of hospitals providing percutaneous coronary intervention ( pci ) differed from those not providing pci . however , little is known on the differences between the characteristics of earlyadopting hospitals and those of lateadopting hospitals , and on their potential impacts on pci volume and access.methods and resultswe used inpatient claims data from 1997 to 2012 from the taiwan national health insurance program to identify the hospitals offering pci . geographic information systems ( gis ) were used to determine the population access to pci hospital . as of 2012 , 88 hospitals were capable of providing pci . on the basis of the year that the hospitals started providing pci , 32 hospitals were designated as early adopters ( before 1998 ) , 23 as early majority ( 19982002 ) , 24 as late majority ( 20032007 ) , and 16 as laggards ( 20082012 ) . hospitals that adopted pci later were smaller in size and closer to an existing pci hospital and had lower pci volumes performed and less bypass surgery support . the median pci volumes in 2012 were n=706 , 330 , 138 , and 81 in early adopters , early majority , late majority , and laggards , respectively . despite the low volume of pci performed in laggard hospitals , the percentage with stelevation myocardial infarction and acute myocardial infarction as principal discharge diagnosis was higher than their earlyadopting hospital counterparts . the percentage of the taiwanese population living within 40 km of pci hospitals ( appropriate access defined in this study ) was 95.7% in 1997 and 98.0% in 2002 , and this has remained unchanged since 2002.conclusionsthe characteristics of earlyadopting hospitals differed from those of lateadopting hospitals . despite lower pci volume performed in lateadopting hospitals , many of them are in remote areas and provide needed and timely services for patients with acute myocardial infarction .

Introduction Methods Data Sources PCI Hospitals PCI Volume and Access Statistical Analyses Results Discussion Diffusion of PCI Adopting PCI Volume PCI Access Limitations Conclusions Disclosures

in the united states , the number of hospitals providing percutaneous coronary intervention ( pci ) has increased in the past 2 decades.1 , 2 , 3 , 4 , 5 , 6 , 7 according to the american hospital association annual survey , 1176 us hospitals were capable of performing pci in 2001 ; this number increased to 1739 in 2008 , an increase of > 40%.4 , 5 to explain the rapid diffusion of pci adoption in us hospitals , vaughansarrazin et al8 suggested that pci is favored over coronary artery bypass graft ( cabg ) because pci is easy to learn and implement , has a high reimbursement rate , and requires only slight modifications of existing techniques , such as drugeluting stents . another study indicated that the newer and larger hospitals equipped with expensive medical technology were more likely to adopt pci.5 however , little is known about the differences between the characteristics of early and lateadopting hospitals . according to everett rogers theory of the diffusion of innovations , facilities can be classified into 5 categories based on their timing of adoption of new technology : innovators , early adopters , early majority , late majority , and laggards.9 the first objective of this study was to examine whether the characteristics of the lateadopting hospitals providing pci differed from those of the earlyadopting hospitals . consequences are the changes that occur to an individual or a social system from adapting or rejecting an innovation.9 in terms of diffusion of pci , the end goals are to improve pci accessibility , provide highquality pci in all areas , and in the long run , decrease the overall mortality rate in patients with coronary artery disease . the second objective of this study was to examine whether late adopting hospitals had different effects on pci volume and access compared with their earlyadopting counterparts . the main data source for this study was obtained from the taiwan national health insurance research database ( nhird ) , which is maintained by the national health research institutes , taiwan . we used the international classification of disease , the ninth revision , clinical modification ( icd9cm ) codes 36.01 , 36.02 , 36.05 , 36.06 , and 36.09 ( taiwan nhi does not use the icd9cm codes 00.66 , 36.00 , and 36.07 for pci , which are used in us hospitals ) to determine the number of pcis performed per hospital per year . according to innovation diffusion theory , we classified the pci hospitals into 4 categories on the basis of the hospitals firstyear claims data on pci reimbursements : ( 1 ) the early adopters , which started providing pci before 1998 ; ( 2 ) the early majority , which started providing pci between 1998 and 2002 ; ( 3 ) the late majority , which started providing pci between 2003 and 2007 ; and ( 4 ) the laggards , which started providing pci between 2008 and 2012 . information on the number of hospitals providing pci before 1997 were derived from a historical review and personal communication.12 the characteristics of hospitals included level of hospitals ( tertiary referral hospital , secondary referral hospital , and community district hospital ) , ownership of hospital ( public versus private ) , number of hospital beds , number of pci performed , having cabg surgery facility ( icd9cm code 36.1x and 36.2 ) , percentage of pci performed with principal discharge diagnosis as acute myocardial infarction ( ami ) . we first calculated the pci volumes per hospital per year to examine the differences in pci volumes between the early and late adopting hospitals.13 , 14 we then used a geographic information system ( gis ) to geocode these hospitals to determine whether public accessibility to pci improved between 1997 and 2012 . if the gisestimated linear distance ( crow 's fly distance ) between the district centroid and the nearest pci hospital was < 40 km ( 25 miles ) , the entire population of that district was considered to have appropriate access to pci , and if this distance > 40 km , the entire population of the district was considered to be inappropriate access to pci . we then computed the percentages of the taiwanese population with appropriate access to pci in 1997 , 2002 , 2007 , and 2012 . except in certain mountainous regions , the national health insurance administration , thus , classified taiwan into 6 medical regions , namely taipei , northern , central , southern , kauping , and eastern ; this classification is similar to the 306 hospital referral regions in the united states . to assess regional disparities in the abundance of pci hospitals , we calculated the number of pci hospitals per 1 million persons in each medical region in 1997 , 2002 , 2007 , and 2012 . we used the chisquare test to examine if there were significant differences in the characteristics ( level of hospital , ownership of hospital , bed capacity of hospital , percentage of hospitals with cabg support in 2012 , and the percentage of pci performed in 2012 with ami as principal diagnosis ) of the 4 pci hospital categories , in which the innovators and early adopters were combined into a single category . the main data source for this study was obtained from the taiwan national health insurance research database ( nhird ) , which is maintained by the national health research institutes , taiwan . the nhird includes outpatient and inpatient claims data ( including data on diagnosis and procedure codes , patient characteristics , physicians , clinical settings , and various service fees ) for 19972012.10 because all patients receiving pci in taiwan were required to be hospitalized for observation , we only used the inpatient claims data from the nhrid . we used the international classification of disease , the ninth revision , clinical modification ( icd9cm ) codes 36.01 , 36.02 , 36.05 , 36.06 , and 36.09 ( taiwan nhi does not use the icd9cm codes 00.66 , 36.00 , and 36.07 for pci , which are used in us hospitals ) to determine the number of pcis performed per hospital per year . according to innovation diffusion theory , we classified the pci hospitals into 4 categories on the basis of the hospitals firstyear claims data on pci reimbursements : ( 1 ) the early adopters , which started providing pci before 1998 ; ( 2 ) the early majority , which started providing pci between 1998 and 2002 ; ( 3 ) the late majority , which started providing pci between 2003 and 2007 ; and ( 4 ) the laggards , which started providing pci between 2008 and 2012 . information on the number of hospitals providing pci before 1997 were derived from a historical review and personal communication.12 the characteristics of hospitals included level of hospitals ( tertiary referral hospital , secondary referral hospital , and community district hospital ) , ownership of hospital ( public versus private ) , number of hospital beds , number of pci performed , having cabg surgery facility ( icd9cm code 36.1x and 36.2 ) , percentage of pci performed with principal discharge diagnosis as acute myocardial infarction ( ami ) . we first calculated the pci volumes per hospital per year to examine the differences in pci volumes between the early and late adopting hospitals.13 , 14 we then used a geographic information system ( gis ) to geocode these hospitals to determine whether public accessibility to pci improved between 1997 and 2012 . if the gisestimated linear distance ( crow 's fly distance ) between the district centroid and the nearest pci hospital was < 40 km ( 25 miles ) , the entire population of that district was considered to have appropriate access to pci , and if this distance > 40 km , the entire population of the district was considered to be inappropriate access to pci . we then computed the percentages of the taiwanese population with appropriate access to pci in 1997 , 2002 , 2007 , and 2012 . except in certain mountainous regions , the national health insurance administration , thus , classified taiwan into 6 medical regions , namely taipei , northern , central , southern , kauping , and eastern ; this classification is similar to the 306 hospital referral regions in the united states . to assess regional disparities in the abundance of pci hospitals , we calculated the number of pci hospitals per 1 million persons in each medical region in 1997 , 2002 , 2007 , and 2012 . we used the chisquare test to examine if there were significant differences in the characteristics ( level of hospital , ownership of hospital , bed capacity of hospital , percentage of hospitals with cabg support in 2012 , and the percentage of pci performed in 2012 with ami as principal diagnosis ) of the 4 pci hospital categories , in which the innovators and early adopters were combined into a single category . according to the claims data , 32 hospitals provided pci in 1997 , and depending on the year that hospitals started providing pci , 32 were classified as early adopters ( before 1998 ) , 23 as early majority ( 19982002 ) , 24 as late majority ( 20032007 ) , and 16 as laggards ( 20082012 ) . the later the hospitals adopted pci , the smaller their sizes and the closer they were to existing pci hospitals , the lower the percentage with cabg surgery support , the lower the volume of pci performed , and the higher the percentage of pci performed with ami as principal discharge diagnosis . characteristics of hospitals providing percutaneous coronary intervention ( pci ) by year providing pci services in taiwan ami indicates acute myocardial infarction ; cabg , coronary artery bypass graft . the descriptive statistics of the pci volumes in 2012 for the 4 pci hospital categories are illustrated in figure 2 . the minimum , median , and maximum pci volumes in 2012 were 197 , 706 , and 1605 for the early adopters ; 100 , 338 , and 836 for the early majority ; 34 , 138 , and 625 for the late majority ; and 15 , 81 , and 365 for the laggards , respectively . the percentage of pci volume100% , 75% , 50% , 25% , and 0%observed in early adopters , early majority , late majority , and laggard pci hospitals , respectively , in 2012 in taiwan . as shown in figure 3b , between 1998 and 2002 , only 2 early majority hospitals had a distance between the new pci hospital and its nearest existing pci hospital higher than 20 km ( 12.4 miles ) : one each in the eastern ( 81.3 km , 50.5 miles ) and kaupin regions ( 20.8 km , 12.9 miles ) . between 2003 and 2007 , no late majority hospitals had a distance exceeding 20 km from the nearest existing pci hospital , and 6 new pci hospitals had distances between 10 and 20 km ( figure 3c ) . between 2008 and 2012 , only one laggard hospital in the central region had a distance exceeding 20 km ( 29.7 km , 17.3 miles ) , and 3 new pci hospitals had distances between 10 and 20 km ( figure 3d ) . locations of hospitals ( based on the pci starting year ) offering pci in taiwan ( a ) early adopters ( before 1998 ) ; ( b ) early majority ( 19982002 ) ; ( c ) late majority ( 20032007 ) ; ( d ) laggards ( 20082012 ) . with regard to public accessibility to pci , in 1997 , 95.7% of the population lived within a distance of 40 km ( 25 miles ) from a pci hospital ; this percentage was 98.0% in 2002 , 98.0% in 2007 , and 98.2% in 2012 . number of hospitals providing percutaneous coronary intervention ( pci ) per 1 million population in 6 medical regions in taiwan the findings of this nationwide descriptive study in taiwan indicate that the characteristics of the earlyadopting pci hospitals differed from those of the lateadopting hospitals . the lateadopting hospitals were smaller in size and had lower pci volumes and lower percentage of having cabg surgery support ; yet had higher percentage of pci performed with ami as principal discharge diagnosis . despite the mild increase in population access to pci access during the past decade , some of laggard pciadopting hospitals are in remote areas and provide needed and timely services for ami patients . the doortoballoon alliance , which included 15 leading pci hospitals , initiated by the taiwan joint commission on hospital accreditation was launched in 2008.16 the hospitals ability to provide primary pci facilities for patients with stemi was mandated by the commission as a criteria for obtaining a high emergency accreditation level . regarding the minimum pci volume standard , the 2013 american college of cardiology / american heart association / society for cardiovascular angiography and interventions clinical competence statement on pci recommended that pci be performed only by operators with an annual volume of > 50 procedures at hospitals with an annual volume exceeding 200 procedures.17 in 2012 , > onethird ( 32 of 88 ) of all pci hospitals in taiwan had annual volumes < 200 . however , many of these low pci volume laggard hospitals are in remote areas with small populations and with relatively low pci demand . according to table 1 , the laggard hospitals had a statistically significant higher percentage of pci performed with ami as principal discharge diagnosis than the earlyadopting hospitals , the difference in percentage was not huge . these lowpci volume laggard hospitals play an important role in providing needed and timely services for people with ami in remote areas . actually , the inhospital mortality rate of ami patients has been reduced significantly in those areas that have utilized this approach.18 regarding pci accessibility , 88% of the us population lived within a distance of 64 km ( 40 mi ) from a pci hospital in 1997 , and the percentage increased to 93% in 2008.5 a study in michigan reported that of the 12 hospitals that were upgraded to provide primary pci procedures , only 3 were located at least 32 km ( 20 miles ) from another primary pci hospital , accounting for a 4.3% improvement in public accessibility , whereas the remaining 9 hospitals increased the accessibility by only 0.5%.19 geographically , taiwan is a small country with a highdensity population and a convenient public transportation system . the percentage of the population living within a range of 40 km ( 25 miles ) from a pci hospital was already high ( 95.7% ) in 1997 , and only 32 earlyadopter hospitals provided pci . the percentage increased to 98.0% in 2002 , after 23 hospitals started providing pci between 1998 and 2002 . between 2002 and 2012 , 40 new hospitals started providing pci ; however , no substantial improvement in public accessibility to pci was observed . first , the arbitrary use of the year that each hospital started providing pci into 4 time periods ( 1997 , 19982002 , 20032007 , and 20082012 ) for classifying the pci hospitals was the first limitation . however , we could not find any superior classification method ; rogers also arbitrarily used statistical standard deviation to classify the underlying population of adopters into 5 categories.9 second , we used the crowfly distance instead of road distance or drive time between the population and the nearest pci hospitals as a proxy measure of pci accessibility , which might generate results that are different from approaches that use actual transportation time as the basis of measurement , which is important for treatment of patients with ami . however , the transport time to a pci facility can be affected by many other factors , such as traffic , time of the day , and other factors , which vary considerably.20 third , as the geographic size of the 368 districts in taiwan varied greatly from 9 persons per km in soulin township , hualien county to 21 950 persons per km in central district in taichung city , which had different implications with regard to the distance of 40 km used for assessing access . the population in the eastern region was 0.56 million in 2012 , which is relatively small compared with the other regions in taiwan : 7.5 million in the taipei region , 3.5 million in the northern region , 4.5 million in the central region , 3.4 million in the southern region , and 3.7 million in the kaupin region . the second surge in the number of hospitals offering pci in 20102011 ( figure 1 ) , which was initiated by the government . the doortoballoon alliance , which included 15 leading pci hospitals , initiated by the taiwan joint commission on hospital accreditation was launched in 2008.16 the hospitals ability to provide primary pci facilities for patients with stemi was mandated by the commission as a criteria for obtaining a high emergency accreditation level . regarding the minimum pci volume standard , the 2013 american college of cardiology / american heart association / society for cardiovascular angiography and interventions clinical competence statement on pci recommended that pci be performed only by operators with an annual volume of > 50 procedures at hospitals with an annual volume exceeding 200 procedures.17 in 2012 , > onethird ( 32 of 88 ) of all pci hospitals in taiwan had annual volumes < 200 . however , many of these low pci volume laggard hospitals are in remote areas with small populations and with relatively low pci demand . according to table 1 , the laggard hospitals had a statistically significant higher percentage of pci performed with ami as principal discharge diagnosis than the earlyadopting hospitals , the difference in percentage was not huge . these lowpci volume laggard hospitals play an important role in providing needed and timely services for people with ami in remote areas . actually , the inhospital mortality rate of ami patients has been reduced significantly in those areas that have utilized this approach.18 regarding pci accessibility , 88% of the us population lived within a distance of 64 km ( 40 mi ) from a pci hospital in 1997 , and the percentage increased to 93% in 2008.5 a study in michigan reported that of the 12 hospitals that were upgraded to provide primary pci procedures , only 3 were located at least 32 km ( 20 miles ) from another primary pci hospital , accounting for a 4.3% improvement in public accessibility , whereas the remaining 9 hospitals increased the accessibility by only 0.5%.19 geographically , taiwan is a small country with a highdensity population and a convenient public transportation system . the percentage of the population living within a range of 40 km ( 25 miles ) from a pci hospital was already high ( 95.7% ) in 1997 , and only 32 earlyadopter hospitals provided pci . the percentage increased to 98.0% in 2002 , after 23 hospitals started providing pci between 1998 and 2002 . between 2002 and 2012 , 40 new hospitals started providing pci ; however , no substantial improvement in public accessibility to pci was observed . first , the arbitrary use of the year that each hospital started providing pci into 4 time periods ( 1997 , 19982002 , 20032007 , and 20082012 ) for classifying the pci hospitals was the first limitation . however , we could not find any superior classification method ; rogers also arbitrarily used statistical standard deviation to classify the underlying population of adopters into 5 categories.9 second , we used the crowfly distance instead of road distance or drive time between the population and the nearest pci hospitals as a proxy measure of pci accessibility , which might generate results that are different from approaches that use actual transportation time as the basis of measurement , which is important for treatment of patients with ami . as the theory suggests that diffusion depends upon many things beyond the innovation itself ( pci in this case ) , but also upon the adopters ( explored in this paper ) , communication channels , time , and the social system ( initiation by government in taiwan ) . our data reveal that the lateadopting hospitals were smaller and had lower pci volumes performed . however , these low pci volume laggard hospitals played an important role in providing needed and timely services for people with ami in remove areas . the ministry of health and social welfare and national health insurance administration should provide needed support for these lowpci volume hospitals in remote areas to ensure the quality of pci performed .

the microenvironment is a dynamic system that is largely composed of inflammatory cells and cytokines , including tumor cells and stroma ( the latter of which consists of immune cells and the surrounding extracellular matrix ) [ 1 , 2 ] . under specific stimulation conditions , inflammation can result in the production of antiangiogenic cytokines , which suppress tumor growth . despite being antagonistic to the action of tumor suppression , in most tumors , inflammatory cells can promote angiogenesis , growth , tumor cell migration , tissue degradation , and remodeling steps that are characteristic of the chronic inflammation associated with intense tissue disorders typical of acute inflammation [ 1 , 37 ] . the transcription factor nuclear kappa b ( nf-b ) plays a major role in innate immunity and inflammatory responses and it is considered the major link between inflammation and tumorigenesis . this factor activates antiapoptotic genes [ 911 ] and could promote the transcription of genes associated with growth , invasion , and metastasis , being the key factor that allows precancerous and malignant cells to escape apoptosis . interleukins ( ils ) could stimulate many signaling pathways and thus regulate the transcription of target genes involved in cellular proliferation and survival . besides that , they act as immune mediators and have been linked to cancer , playing a key role in the recruitment and positioning of leukocytes in carcinogenesis . the recruitment and activation of immune cells and the antitumor effects of il-4 , an anti - inflammatory cytokine , are involved in the inhibition of angiogenesis , inflammation , thrombosis , growth , and invasion in some cancers [ 16 , 17 ] . in addition to these effects on stromal cells , some studies have reported that il-4 has antitumor functions by inducing apoptosis in several types of tumor cells including breast cancer , renal cell carcinoma , and hepatocellular carcinoma . il-10 is a cytokine produced by th1 and th2 cells as well as by tumor cells and has potent immunosuppressive properties . originally known as an inhibitor factor of the synthesis of cytokines , il-10 suppresses the production of il-1 , il-1 , tumor necrosis factor- ( tnf- ) , il-6 , il-8 , il-12 , il-18 , granulocytes , inflammatory macrophages , and nf-b [ 7 , 21 , 22 ] . this cytokine acts concurrently with il-3 and il-4 to stimulate the proliferation of mast cells , peripheral blood lymphocytes , and macrophages , and their anti - inflammatory action has been suggested in tumor microenvironment . on the other hand , il-6 has strong procarcinogenic activity due to its role in tumor cell proliferation , survival , angiogenesis , inflammation , and metastasis [ 7 , 23 ] . in addition , il-6 is one of the activation signals for nf-b , promoting cell differentiation and subsequent metastasis , thus reinforcing the close link between inflammatory mediators and neoplastic progression . therefore , tnf- induces proinflammatory cytokines such as il-1 and il-6 [ 26 , 27 ] , during the inflammatory response [ 2628 ] . the interaction of this cytokine with its receptor ( tnfr ) induces apoptotic and antiapoptotic effects in different cell systems , and it can promote cell proliferation and inhibits apoptosis in the tumor microenvironment , at least indirectly , through the induction of nf-b . a better understanding of the complexity of these biological factors ' network , together with the characterization of new genes , aids in uncovering the molecular mechanisms of inflammatory diseases and cancer . in this way , the objective of this work was to ascertain the role of anti - inflammatory cytokines , il-4 and il-10 , and proinflammatory cytokines , il-6 and tnf- , in mammary tumors of female dogs and to correlate these results with the gene and protein expression of nf-b . in addition , we sought to verify the correlation of these cytokines with clinicopathological parameters , cancer progression , and overall survival , to determine their prognostic value in mammary tumors . peripheral blood and tumor samples were obtained from 30 female dogs with malignant mammary neoplasia ( test group ) attended to in so jos do rio preto , sp , brazil , veterinary clinics in the years 2011 and 2012 . animals with mammary tumors were included regardless of breed , weight , or age . these animals were followed up for 540 days after tumor excision and blood collection . for control group , peripheral blood samples from 30 female dogs and five samples of mammary tissue adjacent to mammary cancer were obtained from animals that underwent elective hysterectomy as a preventive measure , after completion of the consent form by their owners . the criteria for the control group were rigorously followed and included animals outside the estrus period , animals with no tumor history , and animals with no detectable disease / inflammation in the period or surgery in the next study period . in addition , animals that died of causes other than tumor recurrence or metastasis were excluded from the study . peripheral blood ( 3 ml ) was collected in a corvac serum separator tube ( labor import , so paulo , sp ) , stored at 4c , processed by centrifugation ( 1000 g , 25 min ) , and cryopreserved at 80c for posterior enzyme linked immunosorbent assay ( elisa ) and xmap protein analysis . the first one was fixed in 10% formaldehyde buffer for 24 hours and paraffin embedded for histopathology classification after hematoxylin - and - eosin ( he ) stain . tumor grading was ascertained according to misdorp et al . by the armed forces institute of pathology ( afip ) . the second piece was collected in a falcon tube containing the rna - stabilizing solution , rnalater ( life technologies ) , stored at room temperature for 24 hours , and posteriorly used for rna extraction and qpcr analysis . female dogs from the test group were evaluated with respect to physical ( age ) , pathological ( time course ( interval between tumor diagnosis and surgical removal ) , tumor nodules location , lymph node involvement , tumor mass size , clinical staging , ulceration , and vascularization ) , and clinical ( metastasis , local recurrence , and death ) characteristics ( table 1 ) . the parameters employed for the classification of clinical tumor grading were in accordance with the tnm ( size , lymph node involvement , and metastasis ) system established by the world health organization ( who ) for canine mammary gland tumors . the clinical grade was assigned as i , ii , iii , or iv according to the extent of the tumor and the prognosis . the age of the animals varied from 7 to 14 years ( mean = 10 years ) and there was predominance of poodle ( 19% ) and dachshund ( 24% ) races . in terms of the clinicopathological characteristics , most animals had a tumor for more than six months ( 54% ) and were of clinical stage iii or iv ( 54% ) . the proportions of animals with lymph node involvement , animals with metastasis / local recurrence , or animals that died during the study were 16% , 27% , and 27% , respectively ( table 1 ) . for total rna extraction , the tumor samples were manually processed into smaller pieces ( 100 mg / piece ) , and the pieces were then immersed in trizol reagent ( invitrogen life technologies ) and macerated with a politron ( dremel , mexico ) . previously , tumor samples were treated with dnase and 10x buffer ( 10 mm tris - hcl , ph 7.5 ; 50 mm cacl2 ; and 10 mm mgcl2 ) ( rneasy mini kit , qiagen ) followed by the addition of ethylenediaminetetraacetic acid ( edta ) to deactivate or eliminate proteins binding to template dna . the qpcr was performed in triplicate using the stepone plus real time pcr system ( applied biosystems ) and taqman universal master mix ( applied biosystems ) . the following specific primers were used : nf-b , cf02622551_m1 ( applied biosystems ) ; il-4 , cf02623112_m1 ( applied biosystems ) ; il-6 , cf02624153_m1 ( applied biosystems ) ; il-10 , cf02624265_m1 ( applied biosystems ) ; and tnf- , cf02628236_m1 ( applied biosystems ) . the following qpcr conditions were used : an initial step of 2 minutes at 50c ; denaturation for 10 minutes at 95c ; 40 cycles at 95c for 15 seconds and 60c for one minute to anneal primers and extend the chains , respectively ; and 35 seconds at 65c to collect the signal . the dissociation curve was generated after amplification and included a step of 15 seconds at 95c followed by 1 min at 60c . each transcript level was normalized to the expression of rps19 ( sense ( 5-gcc ttc ctc aaa aag tct ggg-3 ) , antisense ( 5-gct tgc tcc cta cga tga gaa c-3 ) , and probe ( 5-ccc tga atg ggt gga c-3 ) ( applied biosystems ) ) and rpl8 , cf02663820_m1 ( applied biosystems ) , which were used as endogenous control genes . the relative quantification ( rq ) value of the expression of interest genes was determined with dataassist v3.0 , by the quantification method related to the average of the normalizing genes used as endogenous controls ( ct ) and showed by log10 . the gene expression of each sample was separately analyzed and classified as underexpressed ( samples giving < 0 log10 measurement ) and overexpressed ( samples with quantification > 0 log10 ) . nf-b ( dog nuclear factor ( nf ) kappa b p105 subunit ( nf-b1 ) , cusabio biotech co. , hubei , china ) and il-4 ( uscn life science inc . , houston , tx , usa ) protein expression analysis was performed according to the manufacturers recommendations . first of all , the plate was precoated with a specific antibody for nf-b and il-4 . then , the standards and samples were added in each well and incubated for 2 hours at 37c . a biotin - conjugated antibody and a horseradish peroxidase- ( hrp- ) avidin conjugate were added to the plate to amplify the signal and to increase the detectability of the target molecule . a chromogenic substrate ( 3 - 3 - 5 - 5-tetramethylbenzidine , tmb ) finally , sulfuric acid ( stop solution ) was added to stabilize the color development to enable accurate measurement of the intensity at 450 nm . the analysis of il-6 , il-10 , and tnf- proteins was performed using the milliplex map kit ( ccytomag-90k ; millipore corporation , usa ) , and the protein levels were analyzed using the luminex xmap magpix ( millipore corporation , usa ) . the standard , control , and samples were added to the appropriate wells as were magnetic beads coated with specific antibodies for il-6 , il-10 , and tnf-. the plate was sealed and incubated overnight at 4c with agitation on a plate shaker . measurements were performed at 635 nm to excite the magnetic beads and 525 nm to detect phycoerythrin . the calculation of od was determined through the adjustment curve four - parameter logistic ( 4-pl ) using the skanit software for the multiskan fc 2.5.1 . clinicopathological characteristics , gene expression , and protein quantification were analyzed in the test and control groups , and the differences between these groups were determined by student 's t - test or anova followed by the bonferroni test . the cutoff points for the protein analyses were established by the receiver operating characteristic ( roc ) curve . for the roc curve , the protein expression levels from female dogs that died were compared with those that survived until the end of the followup period . survival curves were plotted by the kaplan - meier method and the differences between the curves were evaluated by a log - rank test and hazard function . spearman 's rank correlation analysis was performed to describe the correlation between proinflammatory and anti - inflammatory cytokines protein and gene expression with nf-b . in addition , the mutual relationship between proinflammatory and anti - inflammatory cytokines was analyzed . a multivariate logistic regression analysis was employed to evaluate the simultaneous influence of the prognostic factors on animal death . the nf-b gene was significantly overexpressed in animals with tumor time course greater than six months ( p < 0.0001 ) ; multiple tumor locations ( p = 0.005 ) ; abundant tumor vascularization ( p = 0.009 ) ; tumor mass size greater than three centimeters ( cm ) ( p = 0.005 ) ; lymph node involvement ( p = 0.008 ) ; metastasis ( p = 0.02 ) ; recurrence ( p = 0.01 ) ; and clinical stage iii or iv ( p < 0.0001 ) ( figure 1(a ) ) . in the same way , the nf-b protein expression was significantly higher in animals older than 10 years ( p = 0.0001 ) , as well as in animals with a tumor time course of less than six months ( p = 0.001 ) , multiple tumor locations ( p = 0.01 ) , abundant tumor vascularization ( p = 0.03 ) , and metastasis ( p = 0.0005 ) ( figure 1(b ) ) . the best cutoff point for nf-b established by the roc curve to discriminate the high risk of death was 219.5 pg / ml ( sensitivity ( 95% ci ) = 71% , specificity ( 95% ci ) = 41% ) . no correlation was observed between nf-b serum levels and survival time ( or 2.003 ; 95% ci = 0.5553 to 8.983 ; p = 0.29 ) ( data not shown ) . il-4 was significantly overexpressed in animals aged 10 years ( p = 0.02 ) and animals with tumor time course of less than six months ( p = 0.01 ) ; single tumor location ( p = 0.0001 ) ; moderate tumor vascularization ( p = 0.0002 ) ; tumor mass size less than three centimeters ( p = 0.0006 ) ; no lymph node involvement ( p = 0.001 ) ; metastasis ( p < 0.0001 ) ; recurrence ( p = 0.0003 ) ; and clinical stage i or ii ( p < 0.0001 ) and animals that were still alive at the end of the followup period ( p < 0.0001 ) ( figure 2(a ) ) . therefore , il-4 protein expression was significantly higher in animals aged 10 years ( p < 0.0001 ) and animals with tumor time course of less than six months ( p = 0.04 ) ; moderate tumor vascularization ( p = 0.03 ) ; tumor mass size of less than three centimeters ( p = 0.001 ) ; no metastasis ( p = 0.01 ) ; no recurrence ( p = 0.02 ) ; and clinical stage i or ii ( p = 0.008 ) and animals that were still alive at the end of the followup period ( p = 0.005 ) ( figure 2(b ) ) . using the roc curve , the best cutoff point for il-4 to discriminate the high risk of death was 368.4 pg / ml ( sensitivity ( 95% ci ) = 88% , specificity ( 95% ci ) = 95% ) . the kaplan - meier test demonstrated a positive correlation between il-4 level and survival time ( or 0.068184 ; 95% ci = 0.783029 to 1 ; p = 0.007 ) ( figure 3 ) . for il-10 , significant gene overexpression was also correlated with tumor time course of less than six months ( p = 0.003 ) ; moderate tumor vascularization ( p = 0.02 ) ; no lymph node involvement ( p = 0.0005 ) ; no metastasis ( p = 0.01 ) ; no recurrence ( p = 0.01 ) ; clinical stage i or ii ( p = 0.02 ) ; and animals that were still alive at the end of the followup period ( p = 0.009 ) ( figure 4(a ) ) . likewise , il-10 protein expression was significantly higher in animals with tumor time course of less than six months ( p = 0.003 ) ; single tumor location ( p = 0.0009 ) ; moderate vascularization ( p = 0.04 ) ; no recurrence ( p = 0.04 ) , and clinical stage i or ii ( p = 0.03 ) and animals that were still alive during the followup ( p = 0.04 ) ( figure 4(b ) ) . for the roc curve for il-10 , the best cutoff point to discriminate the high risk of death was 243 pg / ml ( sensitivity ( 95% ci ) = 100% , specificity ( 95% ci ) = 53% ) . no correlation was observed between il-10 protein expression and survival time ( or 0.3267 ; 95% ci = 0.06811 to 1.564 ; p = 0.16 ) ( data not shown ) . in contrast , il-6 was significantly overexpressed in animals aged > 10 years ( p = 0.03 ) and animals with multiple tumor locations ( p = 0.0005 ) ; abundant tumor vascularization ( p = 0.003 ) ; tumor mass size greater than three centimeters ( p = 0.01 ) ; lymph node involvement ( p = 0.04 ) ; metastasis ( p = 0.003 ) ; recurrence ( p = 0.03 ) ; and clinical stage iii or iv ( p = 0.02 ) and animals that died during followup ( p = 0.008 ) ( figure 5(a ) ) . similarly , il-6 protein expression was significantly higher in animals with tumor time course greater than six months ( p = 0.001 ) ; multiple tumor locations ( p = 0.01 ) ; abundant vascularization ( p = 0.02 ) ; tumor mass size greater than three centimeters ( p = 0.001 ) ; lymph node involvement ( p < 0.0001 ) ; metastasis ( p < 0.0001 ) ; recurrence ( p = 0.007 ) ; and clinical stage iii or iv ( p = 0.001 ) and animals that died during followup ( p = 0.04 ) ( figure 5(b ) ) . the best cutoff point for il-6 established by the roc curve to discriminate the high risk of death was 76.95 pg / ml ( sensitivity ( 95% ci ) = 75% , specificity ( 95% ci ) = 86% ) . no correlation was observed between il-6 protein expression and survival time ( or 1.719 ; 95% ci = 0.4045 to 9.580 ; p = 0.43 ) ( data not shown ) . tnf- was also overexpressed in animals with tumor time course greater than six months ( p = 0.01 ) ; multiple tumor locations ( p = 0.005 ) ; abundant tumor vascularization ( p = 0.007 ) ; tumor mass size greater than three centimeters ( p = 0.02 ) ; metastasis ( p = 0.001 ) ; recurrence ( p = 0.003 ) ; and clinical stage iii or iv ( p = 0.0002 ) and animals that died during followup ( p = 0.005 ) ( figure 6(a ) ) . the protein expression of tnf- was significantly higher in animals aged > 10 years ( p = 0.04 ) and animals with tumor time course greater than six months ( p = 0.004 ) ; abundant vascularization ( p = 0.01 ) ; lymph node involvement ( p = 0.0007 ) ; metastasis ( p = 0.02 ) ; and recurrence ( p = 0.01 ) and animals that died during followup ( p = 0.04 ) ( figure 6(b ) ) . the best cutoff point to discriminate the high risk of death established by the roc curve was 16.3 pg / ml ( sensitivity ( 95% ci ) = 87% , specificity ( 95% ci ) = 62% ) . this analysis demonstrated a negative correlation between tnf- protein expression and survival time ( or 5.906 ; 95% ci = 1.387 to 25.6 ; p = 0.01 ) ( figure 7 ) . spearman 's rank correlation demonstrated that proinflammatory cytokines il-6 and tnf- are mediated by nf-b . there was a positive correlation between the proinflammatory tnf- protein and gene expression when compared to nf-b protein and gene expression ( r = 0.5233 , p = 0.003 ; r = 0.6645 , p < 0.0001 ; figures 8(a ) and 8(b ) , resp . ) . in the same way , there was a positive correlation between il-6 and nf-b gene expression ( r = 0.4012 ; p = 0.02 ) ( figure 8(b ) ) . furthermore , the mutual balance between proinflammatory cytokines and anti - inflammatory ones was analyzed by spearman 's rank correlation and showed no correlation ( p > 0.05 ; data not shown ) . the multivariate analysis of high levels of the proinflammatory cytokines , low levels of the anti - inflammatory cytokines , and clinicopathological features of poor prognosis with regard to an increased risk of death showed no correlation ( p > 0.05 ) ( table 2 ) . nf-b is a crucial factor between chronic inflammation and cancer development and it regulates both anti- and proinflammatory cytokines at various stages of tumorigenesis and inflammation [ 35 , 36 ] . we observed high gene and protein expression levels of nf-b in animals with tumor progression for greater than six months , multiple tumor locations , abundant vascularization , and metastasis , indicating a role of this factor in angiogenesis support and inhibition of apoptosis in tumor cells . in accordance with our prediction , kiliccioglu et al . observed that nf-b inhibition , through proteasome inhibition , induces apoptosis in human prostate cancer cell line ( pc3 ) , resulting in significantly increased protein levels of caspase-3 , which plays a central role in the execution phase of cell apoptosis . similarly , chien et al . reported that pancreatic cancer cell growth is suppressed by inhibiting the nf-b pathway . in the same way , high protein levels of nf-b were observed in female dogs above 10 years old which could indicate the correlation between this nuclear factor and cell oxidative stress , leading to tissue damage and chronic inflammation . corroborating with this , a review of oxidative stress and breast cancer , recently published by nourazarian et al . , suggests that the increase of free radical levels in tumor cells occurs under the influence of the increased expression of cellular enzymes and enhanced activity of some dependent tumor cells , such as cancer - associated fibroblasts ( cafs ) and tumor - associated macrophages ( tams ) [ 39 , 40 ] . once nf-b has been activated through one of many pathways , crucial properties of the malignant phenotype are activated , promoting tumor cell adaptation to the tumor environment ; then , we also reported nf-b overexpressed in female dogs with tumor masses greater than three centimeters , lymph node involvement , recurrence , and clinical stage iii or iv , leading to a poor survival rate of the animals . it is known that nf-b is required for the expression of many cytokine genes that encode proteins associated with inflammatory mediators . then , through spearman 's rank correlation , we showed that high expression of nf-b modulates positively the il-6 and tnf- expression levels in mammary tumors . the proinflammatory cytokines il-1 , il-6 , and tnf- could be modulated by lipopolysaccharides ( lps ) administration as it occurs in inflammatory diseases . according to cheon et al . , some drugs , as saxifragin , inhibited the lps - induced nuclear translocation of p65 and the activation of caspase-1 in raw 264 cells , suggesting that the nf-b - regulated gene transcription is responsible for anti - inflammatory response of macrophages . in addition , osnes et al . affirmed that the inhibitory synthesis of tnf- by drugs as acetylsalicylic acid and sodium salicylate in human monocytes is due to interfering of nuclear translocation of nf-b / c - rel proteins . besides that , corroborating with our study , al - halabi et al . showed that the inhibition of nf-b significantly lowers microvessel density ( cd31 ) and mrna expression levels of il-6 , tnf- , and il-1 , as well as protein levels of proliferation marker ( ki-67 ) and vascular endothelial growth factor- ( vegf- ) a in human colon cancer , confirming the primordial role of nf-b in angiogenesis and , therefore , tumor progression . regarding anti - inflammatory interleukins , our study sustains a protector action of the anti - inflammatory cytokines il-4 and il-10 in female dogs ' mammary tumors . il-4 was highly expressed in gene and protein levels in female dogs with a better prognostic . this cytokine acts on endothelial cells , inhibiting tumor - induced vascularization and starving tumor cells , and also induces secretion of il-10 , the th2 cytokine with a strong suppressive effect on tumors [ 46 , 47 ] , which could explain high levels of this cytokine in animals that are still alive after the followup period and in overall survival . furthermore , according to okada and kuwashima , the sustained expression of il-4 may provide effective means for therapy of a variety of diseases , including cancer . in addition , high gene and protein expression of il-10 were correlated with characteristics of better prognosis . il-10 downregulates proinflammatory cytokine expression [ 48 , 49 ] and inhibits the expression of cd31 . lin and karin reported that il-10 could modulate apoptosis and suppress angiogenesis during tumor regression , downregulating vegf , tnf- , and il-6 production by tams . jindal and borges showed that il-10 inhibits tumor growth by preventing chemokine expression and angiogenesis when administered at higher doses in cancer animal models . correlated high il-10 expression in female dogs with no metastasis or recurrence and high overall survival . contrarily , gene overexpression of il-10 in animals that had metastasis could suggest posttranscriptional regulation of this cytokine , once the protein levels do not show this correlation . il-6 mediates a plethora of physiological functions , including the developmental differentiation of lymphocytes , cell proliferation , and cell survival during tumorigenesis [ 48 , 53 ] . this cytokine had high gene and protein expression correlated with poor prognosis and a negative correlation was found in animals that died during the followup . in the same way , a study by madeddu et al . demonstrated high expression of il-6 through activation of akt / pi3k / mtor cascade due to elevated oxidative stress , promoting oncogenesis and tumor progression . owing to its association with poor prognosis , researchers have proposed il-6 as a therapeutic target in cancer . several phase i / ii clinical trials are currently evaluating antibodies against il-6 or il-6r as therapeutic alternatives for prostate cancer [ 53 , 55 ] and renal cancer [ 56 , 57 ] . these results suggest that further studies are needed to determine the appropriate use of anti - interleukin monoclonal antibodies as a therapeutic treatment , and studies similar to ours , evaluating interleukin expression , may be useful as a foundation for further clinical trials of therapy using cytokines . increasing lines of evidence suggest that tnf- regulates many of the critical processes of tumor promotion and progression [ 48 , 58 ] . in our study , high protein levels of tnf- were found in animals that died during the followup and correlated with worse prognostic features . its cytokine has been implicated yet as a tumor promoter in different tumor types like ovarian cancer , gallbladder cancer , and oral squamous cell carcinoma . tnf- binds to two receptors , namely , the ubiquitously expressed tnf receptor 1 ( tnfr1 ) and hematopoietically restricted tnfr2 , and modulates a signaling cascade that induces mediators of transcriptional regulation that are key to cell survival , invasion , angiogenesis , and impairment of immune surveillance in tumor biology [ 49 , 58 , 59 , 62 ] . altogether , our data showed the effect of cytokines on tumor development and progression , as well as positive modulation of nf-b and proinflammatory interleukins in mammary tumors . analysis of the active genes and proteins in cancer can be used to target treatment more specifically , and our results suggest the use of these biological factors as prognostic biomarkers in cancer . our results are of utmost importance for a better understanding of the dynamic network of cytokines and the influence of nf-b on tumorigenesis . these cytokines could be employed as noninvasive prognostic markers in female dogs with mammary tumors and could be useful for predicting disease progression and tumor recurrence . there is lack of studies devoted to diagnostic , therapeutic , and prognostic markers in canine mammary tumors ; so , this study defines potential prognostic and predictive markers for routine use in the clinic , allowing the detection of recurrence and metastasis , which could allow the early adoption of more precise conduct to improve the overall survival of animals .

inflammation results in the production of cytokines , such as interleukin- ( il- ) 4 and il-10 with immunosuppressive properties or il-6 and tnf- with procarcinogenic activity . furthermore , nf-b is the major link between inflammation and tumorigenesis . this study verified the interaction between active inflammatory cytokines in the tumor microenvironment and serum of female dogs with mammary tumors and their correlation with the clinicopathological characteristics and overall survival . measurement of gene expression was performed by qpcr and protein levels by elisa / luminex . high gene and protein expression levels of nf-b , il-6 , and tnf- were found in association with characteristics that reflect worse prognosis and a negative correlation between tnf- protein expression and survival time was observed ( p < 0.05 ) . in contrast , high gene and protein expression levels of il-4 and il-10 were associated with characteristics of better prognosis and an increased level of il-4 and a longer survival time of animals were obtained ( p < 0.05 ) . in addition , there was a positive correlation between tnf- and il-6 expression in association with nf-b . the results show a significant correlation of these cytokines with tumor development , associated with nf-b expression and cytokines promodulation , showing that these biological factors could be used as predictive and prognostic markers in breast cancer .

1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusion

the microenvironment is a dynamic system that is largely composed of inflammatory cells and cytokines , including tumor cells and stroma ( the latter of which consists of immune cells and the surrounding extracellular matrix ) [ 1 , 2 ] . under specific stimulation conditions , inflammation can result in the production of antiangiogenic cytokines , which suppress tumor growth . the transcription factor nuclear kappa b ( nf-b ) plays a major role in innate immunity and inflammatory responses and it is considered the major link between inflammation and tumorigenesis . the recruitment and activation of immune cells and the antitumor effects of il-4 , an anti - inflammatory cytokine , are involved in the inhibition of angiogenesis , inflammation , thrombosis , growth , and invasion in some cancers [ 16 , 17 ] . in addition to these effects on stromal cells , some studies have reported that il-4 has antitumor functions by inducing apoptosis in several types of tumor cells including breast cancer , renal cell carcinoma , and hepatocellular carcinoma . originally known as an inhibitor factor of the synthesis of cytokines , il-10 suppresses the production of il-1 , il-1 , tumor necrosis factor- ( tnf- ) , il-6 , il-8 , il-12 , il-18 , granulocytes , inflammatory macrophages , and nf-b [ 7 , 21 , 22 ] . this cytokine acts concurrently with il-3 and il-4 to stimulate the proliferation of mast cells , peripheral blood lymphocytes , and macrophages , and their anti - inflammatory action has been suggested in tumor microenvironment . on the other hand , il-6 has strong procarcinogenic activity due to its role in tumor cell proliferation , survival , angiogenesis , inflammation , and metastasis [ 7 , 23 ] . in addition , il-6 is one of the activation signals for nf-b , promoting cell differentiation and subsequent metastasis , thus reinforcing the close link between inflammatory mediators and neoplastic progression . the interaction of this cytokine with its receptor ( tnfr ) induces apoptotic and antiapoptotic effects in different cell systems , and it can promote cell proliferation and inhibits apoptosis in the tumor microenvironment , at least indirectly , through the induction of nf-b . a better understanding of the complexity of these biological factors ' network , together with the characterization of new genes , aids in uncovering the molecular mechanisms of inflammatory diseases and cancer . in this way , the objective of this work was to ascertain the role of anti - inflammatory cytokines , il-4 and il-10 , and proinflammatory cytokines , il-6 and tnf- , in mammary tumors of female dogs and to correlate these results with the gene and protein expression of nf-b . in addition , we sought to verify the correlation of these cytokines with clinicopathological parameters , cancer progression , and overall survival , to determine their prognostic value in mammary tumors . peripheral blood and tumor samples were obtained from 30 female dogs with malignant mammary neoplasia ( test group ) attended to in so jos do rio preto , sp , brazil , veterinary clinics in the years 2011 and 2012 . animals with mammary tumors were included regardless of breed , weight , or age . for control group , peripheral blood samples from 30 female dogs and five samples of mammary tissue adjacent to mammary cancer were obtained from animals that underwent elective hysterectomy as a preventive measure , after completion of the consent form by their owners . the criteria for the control group were rigorously followed and included animals outside the estrus period , animals with no tumor history , and animals with no detectable disease / inflammation in the period or surgery in the next study period . in addition , animals that died of causes other than tumor recurrence or metastasis were excluded from the study . female dogs from the test group were evaluated with respect to physical ( age ) , pathological ( time course ( interval between tumor diagnosis and surgical removal ) , tumor nodules location , lymph node involvement , tumor mass size , clinical staging , ulceration , and vascularization ) , and clinical ( metastasis , local recurrence , and death ) characteristics ( table 1 ) . in terms of the clinicopathological characteristics , most animals had a tumor for more than six months ( 54% ) and were of clinical stage iii or iv ( 54% ) . the proportions of animals with lymph node involvement , animals with metastasis / local recurrence , or animals that died during the study were 16% , 27% , and 27% , respectively ( table 1 ) . the following specific primers were used : nf-b , cf02622551_m1 ( applied biosystems ) ; il-4 , cf02623112_m1 ( applied biosystems ) ; il-6 , cf02624153_m1 ( applied biosystems ) ; il-10 , cf02624265_m1 ( applied biosystems ) ; and tnf- , cf02628236_m1 ( applied biosystems ) . each transcript level was normalized to the expression of rps19 ( sense ( 5-gcc ttc ctc aaa aag tct ggg-3 ) , antisense ( 5-gct tgc tcc cta cga tga gaa c-3 ) , and probe ( 5-ccc tga atg ggt gga c-3 ) ( applied biosystems ) ) and rpl8 , cf02663820_m1 ( applied biosystems ) , which were used as endogenous control genes . the analysis of il-6 , il-10 , and tnf- proteins was performed using the milliplex map kit ( ccytomag-90k ; millipore corporation , usa ) , and the protein levels were analyzed using the luminex xmap magpix ( millipore corporation , usa ) . the standard , control , and samples were added to the appropriate wells as were magnetic beads coated with specific antibodies for il-6 , il-10 , and tnf-. clinicopathological characteristics , gene expression , and protein quantification were analyzed in the test and control groups , and the differences between these groups were determined by student 's t - test or anova followed by the bonferroni test . for the roc curve , the protein expression levels from female dogs that died were compared with those that survived until the end of the followup period . spearman 's rank correlation analysis was performed to describe the correlation between proinflammatory and anti - inflammatory cytokines protein and gene expression with nf-b . in addition , the mutual relationship between proinflammatory and anti - inflammatory cytokines was analyzed . the nf-b gene was significantly overexpressed in animals with tumor time course greater than six months ( p < 0.0001 ) ; multiple tumor locations ( p = 0.005 ) ; abundant tumor vascularization ( p = 0.009 ) ; tumor mass size greater than three centimeters ( cm ) ( p = 0.005 ) ; lymph node involvement ( p = 0.008 ) ; metastasis ( p = 0.02 ) ; recurrence ( p = 0.01 ) ; and clinical stage iii or iv ( p < 0.0001 ) ( figure 1(a ) ) . in the same way , the nf-b protein expression was significantly higher in animals older than 10 years ( p = 0.0001 ) , as well as in animals with a tumor time course of less than six months ( p = 0.001 ) , multiple tumor locations ( p = 0.01 ) , abundant tumor vascularization ( p = 0.03 ) , and metastasis ( p = 0.0005 ) ( figure 1(b ) ) . no correlation was observed between nf-b serum levels and survival time ( or 2.003 ; 95% ci = 0.5553 to 8.983 ; p = 0.29 ) ( data not shown ) . il-4 was significantly overexpressed in animals aged 10 years ( p = 0.02 ) and animals with tumor time course of less than six months ( p = 0.01 ) ; single tumor location ( p = 0.0001 ) ; moderate tumor vascularization ( p = 0.0002 ) ; tumor mass size less than three centimeters ( p = 0.0006 ) ; no lymph node involvement ( p = 0.001 ) ; metastasis ( p < 0.0001 ) ; recurrence ( p = 0.0003 ) ; and clinical stage i or ii ( p < 0.0001 ) and animals that were still alive at the end of the followup period ( p < 0.0001 ) ( figure 2(a ) ) . therefore , il-4 protein expression was significantly higher in animals aged 10 years ( p < 0.0001 ) and animals with tumor time course of less than six months ( p = 0.04 ) ; moderate tumor vascularization ( p = 0.03 ) ; tumor mass size of less than three centimeters ( p = 0.001 ) ; no metastasis ( p = 0.01 ) ; no recurrence ( p = 0.02 ) ; and clinical stage i or ii ( p = 0.008 ) and animals that were still alive at the end of the followup period ( p = 0.005 ) ( figure 2(b ) ) . the kaplan - meier test demonstrated a positive correlation between il-4 level and survival time ( or 0.068184 ; 95% ci = 0.783029 to 1 ; p = 0.007 ) ( figure 3 ) . for il-10 , significant gene overexpression was also correlated with tumor time course of less than six months ( p = 0.003 ) ; moderate tumor vascularization ( p = 0.02 ) ; no lymph node involvement ( p = 0.0005 ) ; no metastasis ( p = 0.01 ) ; no recurrence ( p = 0.01 ) ; clinical stage i or ii ( p = 0.02 ) ; and animals that were still alive at the end of the followup period ( p = 0.009 ) ( figure 4(a ) ) . likewise , il-10 protein expression was significantly higher in animals with tumor time course of less than six months ( p = 0.003 ) ; single tumor location ( p = 0.0009 ) ; moderate vascularization ( p = 0.04 ) ; no recurrence ( p = 0.04 ) , and clinical stage i or ii ( p = 0.03 ) and animals that were still alive during the followup ( p = 0.04 ) ( figure 4(b ) ) . no correlation was observed between il-10 protein expression and survival time ( or 0.3267 ; 95% ci = 0.06811 to 1.564 ; p = 0.16 ) ( data not shown ) . in contrast , il-6 was significantly overexpressed in animals aged > 10 years ( p = 0.03 ) and animals with multiple tumor locations ( p = 0.0005 ) ; abundant tumor vascularization ( p = 0.003 ) ; tumor mass size greater than three centimeters ( p = 0.01 ) ; lymph node involvement ( p = 0.04 ) ; metastasis ( p = 0.003 ) ; recurrence ( p = 0.03 ) ; and clinical stage iii or iv ( p = 0.02 ) and animals that died during followup ( p = 0.008 ) ( figure 5(a ) ) . similarly , il-6 protein expression was significantly higher in animals with tumor time course greater than six months ( p = 0.001 ) ; multiple tumor locations ( p = 0.01 ) ; abundant vascularization ( p = 0.02 ) ; tumor mass size greater than three centimeters ( p = 0.001 ) ; lymph node involvement ( p < 0.0001 ) ; metastasis ( p < 0.0001 ) ; recurrence ( p = 0.007 ) ; and clinical stage iii or iv ( p = 0.001 ) and animals that died during followup ( p = 0.04 ) ( figure 5(b ) ) . no correlation was observed between il-6 protein expression and survival time ( or 1.719 ; 95% ci = 0.4045 to 9.580 ; p = 0.43 ) ( data not shown ) . tnf- was also overexpressed in animals with tumor time course greater than six months ( p = 0.01 ) ; multiple tumor locations ( p = 0.005 ) ; abundant tumor vascularization ( p = 0.007 ) ; tumor mass size greater than three centimeters ( p = 0.02 ) ; metastasis ( p = 0.001 ) ; recurrence ( p = 0.003 ) ; and clinical stage iii or iv ( p = 0.0002 ) and animals that died during followup ( p = 0.005 ) ( figure 6(a ) ) . the protein expression of tnf- was significantly higher in animals aged > 10 years ( p = 0.04 ) and animals with tumor time course greater than six months ( p = 0.004 ) ; abundant vascularization ( p = 0.01 ) ; lymph node involvement ( p = 0.0007 ) ; metastasis ( p = 0.02 ) ; and recurrence ( p = 0.01 ) and animals that died during followup ( p = 0.04 ) ( figure 6(b ) ) . this analysis demonstrated a negative correlation between tnf- protein expression and survival time ( or 5.906 ; 95% ci = 1.387 to 25.6 ; p = 0.01 ) ( figure 7 ) . spearman 's rank correlation demonstrated that proinflammatory cytokines il-6 and tnf- are mediated by nf-b . there was a positive correlation between the proinflammatory tnf- protein and gene expression when compared to nf-b protein and gene expression ( r = 0.5233 , p = 0.003 ; r = 0.6645 , p < 0.0001 ; figures 8(a ) and 8(b ) , resp . ) in the same way , there was a positive correlation between il-6 and nf-b gene expression ( r = 0.4012 ; p = 0.02 ) ( figure 8(b ) ) . furthermore , the mutual balance between proinflammatory cytokines and anti - inflammatory ones was analyzed by spearman 's rank correlation and showed no correlation ( p > 0.05 ; data not shown ) . the multivariate analysis of high levels of the proinflammatory cytokines , low levels of the anti - inflammatory cytokines , and clinicopathological features of poor prognosis with regard to an increased risk of death showed no correlation ( p > 0.05 ) ( table 2 ) . we observed high gene and protein expression levels of nf-b in animals with tumor progression for greater than six months , multiple tumor locations , abundant vascularization , and metastasis , indicating a role of this factor in angiogenesis support and inhibition of apoptosis in tumor cells . observed that nf-b inhibition , through proteasome inhibition , induces apoptosis in human prostate cancer cell line ( pc3 ) , resulting in significantly increased protein levels of caspase-3 , which plays a central role in the execution phase of cell apoptosis . in the same way , high protein levels of nf-b were observed in female dogs above 10 years old which could indicate the correlation between this nuclear factor and cell oxidative stress , leading to tissue damage and chronic inflammation . , suggests that the increase of free radical levels in tumor cells occurs under the influence of the increased expression of cellular enzymes and enhanced activity of some dependent tumor cells , such as cancer - associated fibroblasts ( cafs ) and tumor - associated macrophages ( tams ) [ 39 , 40 ] . once nf-b has been activated through one of many pathways , crucial properties of the malignant phenotype are activated , promoting tumor cell adaptation to the tumor environment ; then , we also reported nf-b overexpressed in female dogs with tumor masses greater than three centimeters , lymph node involvement , recurrence , and clinical stage iii or iv , leading to a poor survival rate of the animals . it is known that nf-b is required for the expression of many cytokine genes that encode proteins associated with inflammatory mediators . then , through spearman 's rank correlation , we showed that high expression of nf-b modulates positively the il-6 and tnf- expression levels in mammary tumors . the proinflammatory cytokines il-1 , il-6 , and tnf- could be modulated by lipopolysaccharides ( lps ) administration as it occurs in inflammatory diseases . showed that the inhibition of nf-b significantly lowers microvessel density ( cd31 ) and mrna expression levels of il-6 , tnf- , and il-1 , as well as protein levels of proliferation marker ( ki-67 ) and vascular endothelial growth factor- ( vegf- ) a in human colon cancer , confirming the primordial role of nf-b in angiogenesis and , therefore , tumor progression . regarding anti - inflammatory interleukins , our study sustains a protector action of the anti - inflammatory cytokines il-4 and il-10 in female dogs ' mammary tumors . il-4 was highly expressed in gene and protein levels in female dogs with a better prognostic . this cytokine acts on endothelial cells , inhibiting tumor - induced vascularization and starving tumor cells , and also induces secretion of il-10 , the th2 cytokine with a strong suppressive effect on tumors [ 46 , 47 ] , which could explain high levels of this cytokine in animals that are still alive after the followup period and in overall survival . furthermore , according to okada and kuwashima , the sustained expression of il-4 may provide effective means for therapy of a variety of diseases , including cancer . in addition , high gene and protein expression of il-10 were correlated with characteristics of better prognosis . lin and karin reported that il-10 could modulate apoptosis and suppress angiogenesis during tumor regression , downregulating vegf , tnf- , and il-6 production by tams . correlated high il-10 expression in female dogs with no metastasis or recurrence and high overall survival . this cytokine had high gene and protein expression correlated with poor prognosis and a negative correlation was found in animals that died during the followup . in our study , high protein levels of tnf- were found in animals that died during the followup and correlated with worse prognostic features . altogether , our data showed the effect of cytokines on tumor development and progression , as well as positive modulation of nf-b and proinflammatory interleukins in mammary tumors . analysis of the active genes and proteins in cancer can be used to target treatment more specifically , and our results suggest the use of these biological factors as prognostic biomarkers in cancer . our results are of utmost importance for a better understanding of the dynamic network of cytokines and the influence of nf-b on tumorigenesis . these cytokines could be employed as noninvasive prognostic markers in female dogs with mammary tumors and could be useful for predicting disease progression and tumor recurrence . there is lack of studies devoted to diagnostic , therapeutic , and prognostic markers in canine mammary tumors ; so , this study defines potential prognostic and predictive markers for routine use in the clinic , allowing the detection of recurrence and metastasis , which could allow the early adoption of more precise conduct to improve the overall survival of animals .

we performed a pubmed search and selected risk scores for their relative novelty and their applicability to the swiss population . the score from the swiss diabetes association , available on the internet ( 8) , also was assessed . this score actually is an adaptation of the finnish diabetes risk score ( findrisc ) score ( 7 ) . overall , seven risk scores , including clinical ( c ) or clinical and biological variables ( cb ) were studied : 10-year risk scores from kahn et al . ( 3 ) ( c and cb ) ; 8-year risk score from wilson et al . ( 4 ) ( cb ) ; 9-year risk score from balkau et al . ( 6 ) ( c ) ; the prevalent undiagnosed diabetes risk score from griffin et al . ( 5 ) ( c ) ; the risk score from the swiss diabetes association ( 8) ; and the findrisc ( c ) , which is a 5- to 10-year risk score ( 7 ) . the characteristics of the studies , where the scores were developed , and the variables included in each score are summarized in supplementary tables 1 and 2 . from this point on , the scores will be referenced by the name of the first author , with a further differentiation by c or cb in the case of the kahn and balkau scores . we used the thresholds recommended by the authors to define participants at high risk of developing type 2 diabetes ( supplementary table 1 ) . these thresholds were defined differently according to the study : kahn ( 3 ) , wilson ( 4 ) , and balkau ( 6 ) used a probability , whereas the swiss diabetes association and findrisc used a score above a given number of points . the initial publication from griffin et al . ( 5 ) provided no threshold ; hence , we used the 37% probability , which was used in another study ( 12 ) . the scores from the swiss diabetes association and findrisc included regular consumption of selected foods ( fruits , vegetables , berries , and brown bread ) and familial history of diabetes for second - degree parents ( grandparents , cousins , and uncles ) . because these data were not available in our study , the scoring system was adapted by reducing by one point the cutoff value for high - risk participants . the colaus study is a cross - sectional study in the caucasian population of lausanne , switzerland , a town of 117,161 inhabitants , of which 79,420 are of a swiss nationality . the study was designed to assess the prevalence and to identify the molecular determinants of cardiovascular risk factors . the population of lausanne can be considered as representative of the whole country because a considerable proportion is non - swiss or comes from other cantons ( political regions of switzerland ) . in 2006 , of 128,231 lausanne inhabitants , 38% were non - swiss , 30% came from other cantons ( including italian and german - speaking cantons ) , and only 32% were actually from the vaud canton ( 13 ) . the sampling procedure of the colaus study has been described previously ( 14 ) . a complete list of lausanne inhabitants , aged 3575 years ( n = 56,694 ) the following inclusion criteria were applied : 1 ) provided written informed consent ; 2 ) was aged 3575 years ; 3 ) was willing to take part in the examination and donate blood samples ; and 4 ) was of caucasian origin , defined as having both parents and grandparents born in a restricted list of countries ( available from the authors ) . participation rate was 41% , and 6,188 caucasian participants ( 3,251 women and 2,937 men ) took part in the study . all participants attended the outpatient clinic of the university hospital of lausanne in the morning after an overnight fast ( minimum fasting time 8 h ) . the participants first received a questionnaire to record information about their lifestyle factors , namely tobacco use , alcohol use , and physical activity . according to their smoking histories , participants were classified as never , current , or former smokers . current smokers were defined as giving a positive answer to the statement i currently smoke , i do nt smoke anymore , and never smokers were defined as giving a positive answer to the statement i have never smoked . alcohol consumption included past and current drinking status as well as the number of alcoholic beverage units ( wine , beer , and spirits ) consumed over the week preceding the interview . a participant was considered to be physically active if he / she reported practicing at least 2 h of leisure - time physical activity per week . during a second face - to - face meeting , the participants were asked if they or their first - degree family ( i.e. , parents , children ) had presented with diabetes . the participants also were asked if they had been diagnosed with hypertension or if they currently were being treated for hypertension . personal medicines , including prescription and self - prescribed drugs , were collected , together with their main indications . only corticosteroids , being of systemic or topical use , were considered for testing the scores . despite the fact that other medications , such as hydrochlorothiazide or ace inhibitors , have been shown to influence diabetes status ( 15 ) , body weight and height were measured with participants standing without shoes in light indoor clothes . body weight was measured in kilograms to the nearest 100 g , using a seca scale , which was calibrated regularly . waist was measured with a nonstretchable tape over the unclothed abdomen at the narrowest point between the lowest rib and the iliac crest . two measures were made , and the mean ( expressed in centimeters ) was used for analyses . blood pressure and resting pulse were measured three times using an omron hem-907 automated oscillometric sphygmomanometer on the left arm , with an appropriately sized cuff , after at least 10 min rest in the seated position . the average of the last two measurements was used for analyses . fasting plasma glucose , hdl cholesterol , ldl cholesterol , triglycerides , and uric acid levels were measured by the centre hospitalier universitaire vaudois clinical laboratory using fresh blood samples within 2 h of blood collection . all measurements were conducted in a modular p apparatus ( roche diagnostics , basel , switzerland ) . the following analytical procedures ( with maximum interbatch and intrabatch coefficients of variation ) were used : cholesterol by cholesterol oxidase - peroxide + 4-aminophenazone + phenol ( pap ) ( 1.61.7% ) ; hdl cholesterol by cholesterol oxidase - pap plus polyethylene - glycol plus cyclodextrin ( 3.60.9% ) ; glucose by glucose dehydrogenase ( 2.11.0% ) ; triglycerides by glucose oxidase - pap ( 2.91.5% ) ; and uric acid by uricase - pap ( 1.00.5% ) . diabetes was defined as fasting plasma glucose 7.0 mmol / l and/or the presence of oral hypoglycemic or insulin treatment . type 2 diabetes was defined in cases of diabetes without self - reported type 1 diabetes . mmol / l without antidiabetes treatment . of the initial 6,188 participants , 21 ( 0.3% ) had missing data for the variables of interest , 407 ( 6.6% ) had diabetes , and 655 had impaired fasting glucose ( 10.6% ) . diabetic participants were excluded , and the remaining 5,760 ( 93.1% ) participants were used in the analyses . the prevalence of participants at risk for developing type 2 diabetes according to each score was determined and expressed in percentages and 95% cis . the number of participants at risk in switzerland was then estimated for each score by applying the sex - specific and 10-year age - group specific prevalence obtained to the corresponding diabetes - free population numbers , obtained by averaging the population estimates between 2003 and 2006 , provided by the swiss federal statistical office ( www.statistique.admin.ch ) . to assess the number of subjects without diabetes in the swiss population , we assumed that the proportion of nondiabetic patients in our study was representative of the whole country . all statistical analyses were made using stata version 11.1 ( stata , college station , tx ) . we performed a pubmed search and selected risk scores for their relative novelty and their applicability to the swiss population . the score from the swiss diabetes association , available on the internet ( 8) , also was assessed . this score actually is an adaptation of the finnish diabetes risk score ( findrisc ) score ( 7 ) . overall , seven risk scores , including clinical ( c ) or clinical and biological variables ( cb ) were studied : 10-year risk scores from kahn et al . ( 3 ) ( c and cb ) ; 8-year risk score from wilson et al . ( 4 ) ( cb ) ; 9-year risk score from balkau et al . ( 6 ) ( c ) ; the prevalent undiagnosed diabetes risk score from griffin et al . ( 5 ) ( c ) ; the risk score from the swiss diabetes association ( 8) ; and the findrisc ( c ) , which is a 5- to 10-year risk score ( 7 ) . the characteristics of the studies , where the scores were developed , and the variables included in each score are summarized in supplementary tables 1 and 2 . from this point on , the scores will be referenced by the name of the first author , with a further differentiation by c or cb in the case of the kahn and balkau scores . we used the thresholds recommended by the authors to define participants at high risk of developing type 2 diabetes ( supplementary table 1 ) . these thresholds were defined differently according to the study : kahn ( 3 ) , wilson ( 4 ) , and balkau ( 6 ) used a probability , whereas the swiss diabetes association and findrisc used a score above a given number of points . the initial publication from griffin et al . ( 5 ) provided no threshold ; hence , we used the 37% probability , which was used in another study ( 12 ) . the scores from the swiss diabetes association and findrisc included regular consumption of selected foods ( fruits , vegetables , berries , and brown bread ) and familial history of diabetes for second - degree parents ( grandparents , cousins , and uncles ) . because these data were not available in our study , the scoring system was adapted by reducing by one point the cutoff value for high - risk participants . the colaus study is a cross - sectional study in the caucasian population of lausanne , switzerland , a town of 117,161 inhabitants , of which 79,420 are of a swiss nationality . the study was designed to assess the prevalence and to identify the molecular determinants of cardiovascular risk factors . the population of lausanne can be considered as representative of the whole country because a considerable proportion is non - swiss or comes from other cantons ( political regions of switzerland ) . in 2006 , of 128,231 lausanne inhabitants , 38% were non - swiss , 30% came from other cantons ( including italian and german - speaking cantons ) , and only 32% were actually from the vaud canton ( 13 ) . the sampling procedure of the colaus study has been described previously ( 14 ) . a complete list of lausanne inhabitants , aged 3575 years ( n = 56,694 ) , was provided by the population registry of the city . the following inclusion criteria were applied : 1 ) provided written informed consent ; 2 ) was aged 3575 years ; 3 ) was willing to take part in the examination and donate blood samples ; and 4 ) was of caucasian origin , defined as having both parents and grandparents born in a restricted list of countries ( available from the authors ) . participation rate was 41% , and 6,188 caucasian participants ( 3,251 women and 2,937 men ) took part in the study . all participants attended the outpatient clinic of the university hospital of lausanne in the morning after an overnight fast ( minimum fasting time 8 h ) . the participants first received a questionnaire to record information about their lifestyle factors , namely tobacco use , alcohol use , and physical activity . according to their smoking histories , participants were classified as never , current , or former smokers . current smokers were defined as giving a positive answer to the statement i currently smoke , i do nt smoke anymore , and never smokers were defined as giving a positive answer to the statement i have never smoked . alcohol consumption included past and current drinking status as well as the number of alcoholic beverage units ( wine , beer , and spirits ) consumed over the week preceding the interview . a participant was considered to be physically active if he / she reported practicing at least 2 h of leisure - time physical activity per week . during a second face - to - face meeting , the participants were asked if they or their first - degree family ( i.e. , parents , children ) had presented with diabetes . the participants also were asked if they had been diagnosed with hypertension or if they currently were being treated for hypertension . personal medicines , including prescription and self - prescribed drugs , were collected , together with their main indications . only corticosteroids , being of systemic or topical use , were considered for testing the scores . despite the fact that other medications , such as hydrochlorothiazide or ace inhibitors , have been shown to influence diabetes status ( 15 ) , body weight and height were measured with participants standing without shoes in light indoor clothes . body weight was measured in kilograms to the nearest 100 g , using a seca scale , which was calibrated regularly . waist was measured with a nonstretchable tape over the unclothed abdomen at the narrowest point between the lowest rib and the iliac crest . two measures were made , and the mean ( expressed in centimeters ) was used for analyses . blood pressure and resting pulse were measured three times using an omron hem-907 automated oscillometric sphygmomanometer on the left arm , with an appropriately sized cuff , after at least 10 min rest in the seated position . fasting plasma glucose , hdl cholesterol , ldl cholesterol , triglycerides , and uric acid levels were measured by the centre hospitalier universitaire vaudois clinical laboratory using fresh blood samples within 2 h of blood collection . all measurements were conducted in a modular p apparatus ( roche diagnostics , basel , switzerland ) . the following analytical procedures ( with maximum interbatch and intrabatch coefficients of variation ) were used : cholesterol by cholesterol oxidase - peroxide + 4-aminophenazone + phenol ( pap ) ( 1.61.7% ) ; hdl cholesterol by cholesterol oxidase - pap plus polyethylene - glycol plus cyclodextrin ( 3.60.9% ) ; glucose by glucose dehydrogenase ( 2.11.0% ) ; triglycerides by glucose oxidase - pap ( 2.91.5% ) ; and uric acid by uricase - pap ( 1.00.5% ) . diabetes was defined as fasting plasma glucose 7.0 mmol / l and/or the presence of oral hypoglycemic or insulin treatment . type 2 diabetes was defined in cases of diabetes without self - reported type 1 diabetes . impaired fasting glucose was defined as fasting plasma glucose between 6.1 and 6.9 mmol / l without antidiabetes treatment . of the initial 6,188 participants , 21 ( 0.3% ) had missing data for the variables of interest , 407 ( 6.6% ) had diabetes , and 655 had impaired fasting glucose ( 10.6% ) . diabetic participants were excluded , and the remaining 5,760 ( 93.1% ) participants were used in the analyses . the prevalence of participants at risk for developing type 2 diabetes according to each score was determined and expressed in percentages and 95% cis . the number of participants at risk in switzerland was then estimated for each score by applying the sex - specific and 10-year age - group specific prevalence obtained to the corresponding diabetes - free population numbers , obtained by averaging the population estimates between 2003 and 2006 , provided by the swiss federal statistical office ( www.statistique.admin.ch ) . to assess the number of subjects without diabetes in the swiss population , we assumed that the proportion of nondiabetic patients in our study was representative of the whole country . all statistical analyses were made using stata version 11.1 ( stata , college station , tx ) . the prevalence of participants at risk for developing type 2 diabetes is shown in fig . 1 . in men , the prevalence of participants at high risk of developing type 2 diabetes was the following : 1.6% ( 1.22.2 ) ( wilson ) ; 4.2% ( 3.45.0 ) ( balkau ) ; 12.8% ( 11.514.1 ) ( kahn [ c ] ) ; 13.5% ( 12.214.9 ) ( swiss diabetes association ) ; 13.7% ( 12.415.0 ) ( kahn [ cb ] ) ; 22.9% ( 21.324.5 ) ( findrisc ) ; and 24.9% ( 23.426.6 ) ( griffin ) . in women , the corresponding values were 1.1% ( 0.81.6 ) ( wilson ) ; 2.9% ( 2.43.6 ) ( balkau ) ; 6.0% ( 5.26.9 ) ( kahn [ c ] ) ; 11.1% ( 10.012.3 ) ( swiss diabetes association ) ; 6.1% ( 5.37.0 ) ( kahn [ cb ] ) ; 15.7% ( 14.517.1 ) ( findrisc ) ; and 10.7% ( 9.611.8 ) ( griffin ) . overall , men tended to present a higher risk of type 2 diabetes than women . extrapolated to the swiss population of the same age , the number of subjects at risk ranged from 46,708 to 636,841 , more than a 13-fold variation ( table 1 ) . restricting the analysis to participants aged < 65 years showed either slight increases ( wilson ) or decreases ( findrisc ; griffin ) in the prevalence of subjects at risk for developing type 2 diabetes ( supplementary table 4 ) . likewise , excluding from the analysis the 11 women with a possible pregnancy at examination did not change the results ( data not shown ) . proportion of participants at high risk of developing type 2 diabetes according to each score , by sex . c and cb are only specified in the case of various equations provided by the authors . number of participants in the swiss population at high risk of developing type 2 diabetes according to each score , by sex and age - group c and cb are only specified in the case of various equations provided by the authors . we also checked whether the same participants were considered to be at risk according to the different scores . for this , we compared the participants at risk for type 2 diabetes according to the scores that led to the lowest prevalence ( wilson and balkau ) and also according to the scores that included the same clinical variables ( balkau and kahn [ c ] ) . 2 . the scores classified a total of 612 participants as being at risk : n = 78 , wilson ; n = 201 , balkau ; and n = 558 , kahn ( c ) . of 78 participants at risk according to wilson et al . ( 4 ) , only 21 ( 26.9% ) also were considered at risk according to balkau . likewise , of 201 participants at risk according to balkau , only 145 ( 72% ) also were considered at risk according to kahn ( c ) . number of patients at high risk of developing type 2 diabetes according to three scores . the prevalence of participants at risk for developing type 2 diabetes is shown in fig . 1 . in men , the prevalence of participants at high risk of developing type 2 diabetes was the following : 1.6% ( 1.22.2 ) ( wilson ) ; 4.2% ( 3.45.0 ) ( balkau ) ; 12.8% ( 11.514.1 ) ( kahn [ c ] ) ; 13.5% ( 12.214.9 ) ( swiss diabetes association ) ; 13.7% ( 12.415.0 ) ( kahn [ cb ] ) ; 22.9% ( 21.324.5 ) ( findrisc ) ; and 24.9% ( 23.426.6 ) ( griffin ) . in women , the corresponding values were 1.1% ( 0.81.6 ) ( wilson ) ; 2.9% ( 2.43.6 ) ( balkau ) ; 6.0% ( 5.26.9 ) ( kahn [ c ] ) ; 11.1% ( 10.012.3 ) ( swiss diabetes association ) ; 6.1% ( 5.37.0 ) ( kahn [ cb ] ) ; 15.7% ( 14.517.1 ) ( findrisc ) ; and 10.7% ( 9.611.8 ) ( griffin ) . overall , men tended to present a higher risk of type 2 diabetes than women . extrapolated to the swiss population of the same age , the number of subjects at risk ranged from 46,708 to 636,841 , more than a 13-fold variation ( table 1 ) . restricting the analysis to participants aged < 65 years showed either slight increases ( wilson ) or decreases ( findrisc ; griffin ) in the prevalence of subjects at risk for developing type 2 diabetes ( supplementary table 4 ) . likewise , excluding from the analysis the 11 women with a possible pregnancy at examination did not change the results ( data not shown ) . proportion of participants at high risk of developing type 2 diabetes according to each score , by sex . c and cb are only specified in the case of various equations provided by the authors . number of participants in the swiss population at high risk of developing type 2 diabetes according to each score , by sex and age - group c and cb are only specified in the case of various equations provided by the authors . we also checked whether the same participants were considered to be at risk according to the different scores . for this , we compared the participants at risk for type 2 diabetes according to the scores that led to the lowest prevalence ( wilson and balkau ) and also according to the scores that included the same clinical variables ( balkau and kahn [ c ] ) . 2 . the scores classified a total of 612 participants as being at risk : n = 78 , wilson ; n = 201 , balkau ; and n = 558 , kahn ( c ) . ( 4 ) , only 21 ( 26.9% ) also were considered at risk according to balkau . likewise , of 201 participants at risk according to balkau , only 145 ( 72% ) also were considered at risk according to kahn ( c ) . number of patients at high risk of developing type 2 diabetes according to three scores . to our knowledge , this is one of the few studies that assessed the effect of differing type 2 diabetes risk scoring systems in a given population . in agreement with previous studies ( 10,16 ) , our results indicate that the prevalence of subjects at risk for developing type 2 diabetes varies considerably according to the scoring system used . this has a considerable impact in the number of subjects susceptible of benefiting from measures regarding the primary prevention of type 2 diabetes . the risk - scoring systems compared in this study shared several types of variables ( supplementary table 1 ) . for instance , all of them included a genetic background ( personal or family history ) , which can be explained by the association between certain genes and diabetes ( 17 ) , and most of them also included age , which has been shown to be related to the risk of diabetes . most scores also included obesity markers , such as bmi or waist circumference , as well as cardiovascular risk factors , such as hypertension and dyslipidemia , all of which are involved in the metabolic syndrome definition ( 18 ) . finally , some scores included lifestyle habits , whether protective , such as alcohol consumption and physical activity , or deleterious , such as smoking , also in agreement with previous findings ( 19 ) . it should be noticed that in some studies nondrinkers and former drinkers were included in the same group ( 3 ) and that the nonlinear , u - shaped association between alcohol consumption and the risk of developing type 2 diabetes ( 20 ) was not considered . overall , these findings suggest that any generic scoring system that included clinical variables known to be related to type 2 diabetes ( age , obesity , cardiovascular risk factors , and lifestyle ) could be used to derive diabetes risk scores but that the relative weight of each variable might be different according to the population considered . for instance , age , obesity , and the other factors mentioned vary by country , and this may result in a differential importance to predict diabetes . finally , the inclusion of other variables , such as biological and genetic markers , also should be considered but is beyond the scope of our study ( 21 ) . the two scoring systems that did not include age ( i.e. , wilson and balkau ) gave the lowest prevalence rates of subjects at risk for type 2 diabetes , whereas the griffin score , which includes a linear relationship with age ( 5 ) , provided the greatest prevalence . it is interesting to note that the scoring systems using age - groups instead of age ( 3,8,9 ) provided intermediate prevalence rates . considering how easy it is to collect age and the increasing prevalence of type 2 diabetes with age ( 18 ) , we can postulate that this variable should be included in any risk - scoring system . another possible explanation for the low prevalence rates of participants at risk using the wilson score is the fact that it includes a low hdl cholesterol level , whose prevalence was 36.9% in the original study . however , in the colaus study ( 14 ) , the prevalence of low hdl cholesterol was only 2.8% , which might lead to spurious results . overall , these findings further stress the importance of not only including certain variables but also their relative weighting and even the way they are coded to compute the risk of developing type 2 diabetes . only two scoring systems ( wilson and kahn [ cb ] ) included fasting glucose . this was somewhat unexpected because fasting glucose has a strong predictive value for diabetes ( 22 ) . ( 6 ) , fasting glucose was considered to be the best predictor , but no scoring system that included fasting glucose was provided , possibly because of the fact that the objective was to derive a clinically based scoring system . most values for each variable included in the scoring systems were derived from logistic or cox regression coefficients . still , it should be noticed that some scoring systems ( i.e. , swiss diabetes association ) include variables ( i.e. , familial history ) for which the scores were not based upon statistical analysis but on an educated proposal by the authors ( 7 ) . as a subject might shift from a low - risk to a high - risk category by one single scoring unit , care should be taken when such non evidence - based scores are applied . furthermore , the findrisc score has been used ( and in some cases modified ) by others ( 8,9 ) without any complementary statistical analysis or validation ; therefore , the results obtained by these modified , nonvalidated scores might be questionable . finally , many scoring systems did not take into account the nonlinear association between some variables ( i.e. , alcohol consumption ) and diabetes risk ; the reason might be that introducing nonlinearity complicates the scoring system , but no precise rationale could be obtained from the literature . the prevalence rates of participants at risk for developing type 2 diabetes varied almost 13-fold according to the scoring system used , leading to considerable differences in the number of subjects amenable to prevention measures in the corresponding swiss population . second , the thresholds used to define subjects at high risk also varied ( 3046% ) , as shown in table 1 . third , and as stated previously , the scoring systems were developed and validated in a given population , and applying them to a different population can lead to inconsistent results , as it has been underlined in a previous german study ( 16 ) . overall , our results suggest that the indiscriminate use of a nonvalidated scoring system might lead to considerable differences in the number of subjects to prevent , with a likely under- or overuse of the limited available preventive resources . indeed , we initially expected that two scoring systems detecting a low number of subjects at risk for diabetes would detect the same patients , but fig . likewise , even two scoring systems that included broadly the same variables ( i.e. , balkau and kahn [ c ] ) failed to detect the same participants . hence , our results indicate that different scoring systems detect different subjects at risk for developing type 2 diabetes and thus are not interchangeable . adequate validation of these scoring systems using prospective data is therefore necessary to select the best system applicable to the population under study . first , the predicting ability of the tested scores could not be achieved in the current study . second , the prevalence rates according to swiss diabetes association and findrisc may be over- or underestimated as a result of our lack of dietary data and second - degree familial history . of interest , sensitivity analyses showed that decreasing the threshold of these scores by two and three points led to a 50% increase in the number of subjects at risk for developing type 2 diabetes ( supplementary table 6 ) . these findings suggest that minor changes in the scoring system can lead to considerable changes in the number of subjects at risk for developing type 2 diabetes and that any risk - scoring system should be adequately validated before being applied in a given population . third , although the participation rate was similar to other epidemiological studies ( 23 ) , it was rather low ( 41% ) , which might limit the generalization of findings . indeed , the colaus study may not be representative of the swiss population , but seeing the great variability in the number of high - risk patients , this mistake may not be of great relevance . in addition , there was no sex or zip code distribution difference between the source population , the random sample , and the colaus participants . on the other hand , the colaus sample had more women and was slightly younger than the corresponding swiss population aged 3575 years ( supplementary table 7 ) . hence , it can be argued that although the colaus sample is not fully representative of the swiss population , the differences in population structure are relatively small . likewise , our population was at relatively low risk for diabetes , and it is unknown how this might influence the performance of some of the risk scores if applied in other populations or ethnic groups . still , most equations we used have been developed in european countries and should thus be generalizable to the european population . on the other hand , it has been shown that risk scores developed in the same country lead to different results ( 10 ) . thus and again , a precise validation within a given of any risk score should be conducted before its application in clinical or public health practice . fourth , diabetic subjects were excluded on the basis of fasting but not on 2-h plasma glucose ; hence , diabetic subjects by 2-h glucose ( but not by fasting glucose ) were retained in the analysis . nevertheless , because the number of subjects with type 2 diabetes ( by 2-h glucose ) is fixed , our results still indicate that the number of subjects at risk for developing type 2 diabetes varies considerably according to the risk score used . finally , this article only included leisure - time physical activity , and occupational physical activity was not considered . hence , it is possible that the risk of developing type 2 diabetes might be overestimated when using the equations that include physical activity . in summary , our results indicate that the prevalence of participants at risk for developing type 2 diabetes varies considerably according to the scoring system used . to adequately prevent type 2 diabetes

objectiveto compare in the swiss population the results of several scores estimating the risk of developing type 2 diabetes.research design and methodsthis was a single - center , cross - sectional study conducted between 2003 and 2006 in lausanne , switzerland . overall , 3,251 women and 2,937 men , aged 3575 years , were assessed , of which 5,760 ( 93% ) were free from diabetes and included in the current study . the risk of developing type 2 diabetes was assessed using seven different risk scores , including clinical data with or without biological data . participants were considered to be eligible for primary prevention according to the thresholds provided for each score . the results were then extrapolated to the swiss population of the same sex and age.resultsthe risk of developing type 2 diabetes increased with age in all scores . the prevalence of participants at high risk ranged between 1.6 and 24.9% in men and between 1.1 and 15.7% in women . extrapolated to the swiss population of similar age , the overall number of participants at risk , and thus susceptible to intervention , ranged between 46,708 and 636,841 . in addition , scores that included the same clinical variables led to a significantly different prevalence of participants at risk ( 4.2% [ 95% ci 3.45.0 ] vs. 12.8% [ 11.514.1 ] in men and 2.9% [ 2.43.6 ] vs. 6.0% [ 5.26.9 ] in women).conclusionsthe prevalence of participants at risk for developing type 2 diabetes varies considerably according to the scoring system used . to adequately prevent type 2 diabetes , risk - scoring systems must be validated for each population considered .

RESEARCH DESIGN AND METHODS Risk scores Recruitment Clinical data Biological analyses Diabetes Statistical analysis RESULTS Prevalence of subjects at risk for type 2 diabetes Comparison between scores CONCLUSIONS Supplementary Material

we performed a pubmed search and selected risk scores for their relative novelty and their applicability to the swiss population . overall , seven risk scores , including clinical ( c ) or clinical and biological variables ( cb ) were studied : 10-year risk scores from kahn et al . the characteristics of the studies , where the scores were developed , and the variables included in each score are summarized in supplementary tables 1 and 2 . from this point on , the scores will be referenced by the name of the first author , with a further differentiation by c or cb in the case of the kahn and balkau scores . we used the thresholds recommended by the authors to define participants at high risk of developing type 2 diabetes ( supplementary table 1 ) . these thresholds were defined differently according to the study : kahn ( 3 ) , wilson ( 4 ) , and balkau ( 6 ) used a probability , whereas the swiss diabetes association and findrisc used a score above a given number of points . because these data were not available in our study , the scoring system was adapted by reducing by one point the cutoff value for high - risk participants . the colaus study is a cross - sectional study in the caucasian population of lausanne , switzerland , a town of 117,161 inhabitants , of which 79,420 are of a swiss nationality . a complete list of lausanne inhabitants , aged 3575 years ( n = 56,694 ) the following inclusion criteria were applied : 1 ) provided written informed consent ; 2 ) was aged 3575 years ; 3 ) was willing to take part in the examination and donate blood samples ; and 4 ) was of caucasian origin , defined as having both parents and grandparents born in a restricted list of countries ( available from the authors ) . participation rate was 41% , and 6,188 caucasian participants ( 3,251 women and 2,937 men ) took part in the study . all participants attended the outpatient clinic of the university hospital of lausanne in the morning after an overnight fast ( minimum fasting time 8 h ) . a participant was considered to be physically active if he / she reported practicing at least 2 h of leisure - time physical activity per week . only corticosteroids , being of systemic or topical use , were considered for testing the scores . type 2 diabetes was defined in cases of diabetes without self - reported type 1 diabetes . of the initial 6,188 participants , 21 ( 0.3% ) had missing data for the variables of interest , 407 ( 6.6% ) had diabetes , and 655 had impaired fasting glucose ( 10.6% ) . diabetic participants were excluded , and the remaining 5,760 ( 93.1% ) participants were used in the analyses . the prevalence of participants at risk for developing type 2 diabetes according to each score was determined and expressed in percentages and 95% cis . the number of participants at risk in switzerland was then estimated for each score by applying the sex - specific and 10-year age - group specific prevalence obtained to the corresponding diabetes - free population numbers , obtained by averaging the population estimates between 2003 and 2006 , provided by the swiss federal statistical office ( www.statistique.admin.ch ) . to assess the number of subjects without diabetes in the swiss population , we assumed that the proportion of nondiabetic patients in our study was representative of the whole country . we performed a pubmed search and selected risk scores for their relative novelty and their applicability to the swiss population . overall , seven risk scores , including clinical ( c ) or clinical and biological variables ( cb ) were studied : 10-year risk scores from kahn et al . the characteristics of the studies , where the scores were developed , and the variables included in each score are summarized in supplementary tables 1 and 2 . from this point on , the scores will be referenced by the name of the first author , with a further differentiation by c or cb in the case of the kahn and balkau scores . we used the thresholds recommended by the authors to define participants at high risk of developing type 2 diabetes ( supplementary table 1 ) . these thresholds were defined differently according to the study : kahn ( 3 ) , wilson ( 4 ) , and balkau ( 6 ) used a probability , whereas the swiss diabetes association and findrisc used a score above a given number of points . because these data were not available in our study , the scoring system was adapted by reducing by one point the cutoff value for high - risk participants . the colaus study is a cross - sectional study in the caucasian population of lausanne , switzerland , a town of 117,161 inhabitants , of which 79,420 are of a swiss nationality . the population of lausanne can be considered as representative of the whole country because a considerable proportion is non - swiss or comes from other cantons ( political regions of switzerland ) . in 2006 , of 128,231 lausanne inhabitants , 38% were non - swiss , 30% came from other cantons ( including italian and german - speaking cantons ) , and only 32% were actually from the vaud canton ( 13 ) . a complete list of lausanne inhabitants , aged 3575 years ( n = 56,694 ) , was provided by the population registry of the city . the following inclusion criteria were applied : 1 ) provided written informed consent ; 2 ) was aged 3575 years ; 3 ) was willing to take part in the examination and donate blood samples ; and 4 ) was of caucasian origin , defined as having both parents and grandparents born in a restricted list of countries ( available from the authors ) . participation rate was 41% , and 6,188 caucasian participants ( 3,251 women and 2,937 men ) took part in the study . current smokers were defined as giving a positive answer to the statement i currently smoke , i do nt smoke anymore , and never smokers were defined as giving a positive answer to the statement i have never smoked . a participant was considered to be physically active if he / she reported practicing at least 2 h of leisure - time physical activity per week . only corticosteroids , being of systemic or topical use , were considered for testing the scores . type 2 diabetes was defined in cases of diabetes without self - reported type 1 diabetes . of the initial 6,188 participants , 21 ( 0.3% ) had missing data for the variables of interest , 407 ( 6.6% ) had diabetes , and 655 had impaired fasting glucose ( 10.6% ) . diabetic participants were excluded , and the remaining 5,760 ( 93.1% ) participants were used in the analyses . the prevalence of participants at risk for developing type 2 diabetes according to each score was determined and expressed in percentages and 95% cis . the number of participants at risk in switzerland was then estimated for each score by applying the sex - specific and 10-year age - group specific prevalence obtained to the corresponding diabetes - free population numbers , obtained by averaging the population estimates between 2003 and 2006 , provided by the swiss federal statistical office ( www.statistique.admin.ch ) . to assess the number of subjects without diabetes in the swiss population , we assumed that the proportion of nondiabetic patients in our study was representative of the whole country . the prevalence of participants at risk for developing type 2 diabetes is shown in fig . in men , the prevalence of participants at high risk of developing type 2 diabetes was the following : 1.6% ( 1.22.2 ) ( wilson ) ; 4.2% ( 3.45.0 ) ( balkau ) ; 12.8% ( 11.514.1 ) ( kahn [ c ] ) ; 13.5% ( 12.214.9 ) ( swiss diabetes association ) ; 13.7% ( 12.415.0 ) ( kahn [ cb ] ) ; 22.9% ( 21.324.5 ) ( findrisc ) ; and 24.9% ( 23.426.6 ) ( griffin ) . in women , the corresponding values were 1.1% ( 0.81.6 ) ( wilson ) ; 2.9% ( 2.43.6 ) ( balkau ) ; 6.0% ( 5.26.9 ) ( kahn [ c ] ) ; 11.1% ( 10.012.3 ) ( swiss diabetes association ) ; 6.1% ( 5.37.0 ) ( kahn [ cb ] ) ; 15.7% ( 14.517.1 ) ( findrisc ) ; and 10.7% ( 9.611.8 ) ( griffin ) . overall , men tended to present a higher risk of type 2 diabetes than women . extrapolated to the swiss population of the same age , the number of subjects at risk ranged from 46,708 to 636,841 , more than a 13-fold variation ( table 1 ) . restricting the analysis to participants aged < 65 years showed either slight increases ( wilson ) or decreases ( findrisc ; griffin ) in the prevalence of subjects at risk for developing type 2 diabetes ( supplementary table 4 ) . proportion of participants at high risk of developing type 2 diabetes according to each score , by sex . number of participants in the swiss population at high risk of developing type 2 diabetes according to each score , by sex and age - group c and cb are only specified in the case of various equations provided by the authors . we also checked whether the same participants were considered to be at risk according to the different scores . for this , we compared the participants at risk for type 2 diabetes according to the scores that led to the lowest prevalence ( wilson and balkau ) and also according to the scores that included the same clinical variables ( balkau and kahn [ c ] ) . of 78 participants at risk according to wilson et al . ( 4 ) , only 21 ( 26.9% ) also were considered at risk according to balkau . likewise , of 201 participants at risk according to balkau , only 145 ( 72% ) also were considered at risk according to kahn ( c ) . number of patients at high risk of developing type 2 diabetes according to three scores . the prevalence of participants at risk for developing type 2 diabetes is shown in fig . in men , the prevalence of participants at high risk of developing type 2 diabetes was the following : 1.6% ( 1.22.2 ) ( wilson ) ; 4.2% ( 3.45.0 ) ( balkau ) ; 12.8% ( 11.514.1 ) ( kahn [ c ] ) ; 13.5% ( 12.214.9 ) ( swiss diabetes association ) ; 13.7% ( 12.415.0 ) ( kahn [ cb ] ) ; 22.9% ( 21.324.5 ) ( findrisc ) ; and 24.9% ( 23.426.6 ) ( griffin ) . in women , the corresponding values were 1.1% ( 0.81.6 ) ( wilson ) ; 2.9% ( 2.43.6 ) ( balkau ) ; 6.0% ( 5.26.9 ) ( kahn [ c ] ) ; 11.1% ( 10.012.3 ) ( swiss diabetes association ) ; 6.1% ( 5.37.0 ) ( kahn [ cb ] ) ; 15.7% ( 14.517.1 ) ( findrisc ) ; and 10.7% ( 9.611.8 ) ( griffin ) . overall , men tended to present a higher risk of type 2 diabetes than women . extrapolated to the swiss population of the same age , the number of subjects at risk ranged from 46,708 to 636,841 , more than a 13-fold variation ( table 1 ) . restricting the analysis to participants aged < 65 years showed either slight increases ( wilson ) or decreases ( findrisc ; griffin ) in the prevalence of subjects at risk for developing type 2 diabetes ( supplementary table 4 ) . proportion of participants at high risk of developing type 2 diabetes according to each score , by sex . number of participants in the swiss population at high risk of developing type 2 diabetes according to each score , by sex and age - group c and cb are only specified in the case of various equations provided by the authors . we also checked whether the same participants were considered to be at risk according to the different scores . for this , we compared the participants at risk for type 2 diabetes according to the scores that led to the lowest prevalence ( wilson and balkau ) and also according to the scores that included the same clinical variables ( balkau and kahn [ c ] ) . ( 4 ) , only 21 ( 26.9% ) also were considered at risk according to balkau . likewise , of 201 participants at risk according to balkau , only 145 ( 72% ) also were considered at risk according to kahn ( c ) . number of patients at high risk of developing type 2 diabetes according to three scores . to our knowledge , this is one of the few studies that assessed the effect of differing type 2 diabetes risk scoring systems in a given population . in agreement with previous studies ( 10,16 ) , our results indicate that the prevalence of subjects at risk for developing type 2 diabetes varies considerably according to the scoring system used . this has a considerable impact in the number of subjects susceptible of benefiting from measures regarding the primary prevention of type 2 diabetes . the risk - scoring systems compared in this study shared several types of variables ( supplementary table 1 ) . for instance , all of them included a genetic background ( personal or family history ) , which can be explained by the association between certain genes and diabetes ( 17 ) , and most of them also included age , which has been shown to be related to the risk of diabetes . most scores also included obesity markers , such as bmi or waist circumference , as well as cardiovascular risk factors , such as hypertension and dyslipidemia , all of which are involved in the metabolic syndrome definition ( 18 ) . it should be noticed that in some studies nondrinkers and former drinkers were included in the same group ( 3 ) and that the nonlinear , u - shaped association between alcohol consumption and the risk of developing type 2 diabetes ( 20 ) was not considered . overall , these findings suggest that any generic scoring system that included clinical variables known to be related to type 2 diabetes ( age , obesity , cardiovascular risk factors , and lifestyle ) could be used to derive diabetes risk scores but that the relative weight of each variable might be different according to the population considered . , wilson and balkau ) gave the lowest prevalence rates of subjects at risk for type 2 diabetes , whereas the griffin score , which includes a linear relationship with age ( 5 ) , provided the greatest prevalence . considering how easy it is to collect age and the increasing prevalence of type 2 diabetes with age ( 18 ) , we can postulate that this variable should be included in any risk - scoring system . another possible explanation for the low prevalence rates of participants at risk using the wilson score is the fact that it includes a low hdl cholesterol level , whose prevalence was 36.9% in the original study . however , in the colaus study ( 14 ) , the prevalence of low hdl cholesterol was only 2.8% , which might lead to spurious results . overall , these findings further stress the importance of not only including certain variables but also their relative weighting and even the way they are coded to compute the risk of developing type 2 diabetes . ( 6 ) , fasting glucose was considered to be the best predictor , but no scoring system that included fasting glucose was provided , possibly because of the fact that the objective was to derive a clinically based scoring system . most values for each variable included in the scoring systems were derived from logistic or cox regression coefficients . , alcohol consumption ) and diabetes risk ; the reason might be that introducing nonlinearity complicates the scoring system , but no precise rationale could be obtained from the literature . the prevalence rates of participants at risk for developing type 2 diabetes varied almost 13-fold according to the scoring system used , leading to considerable differences in the number of subjects amenable to prevention measures in the corresponding swiss population . second , the thresholds used to define subjects at high risk also varied ( 3046% ) , as shown in table 1 . third , and as stated previously , the scoring systems were developed and validated in a given population , and applying them to a different population can lead to inconsistent results , as it has been underlined in a previous german study ( 16 ) . overall , our results suggest that the indiscriminate use of a nonvalidated scoring system might lead to considerable differences in the number of subjects to prevent , with a likely under- or overuse of the limited available preventive resources . indeed , we initially expected that two scoring systems detecting a low number of subjects at risk for diabetes would detect the same patients , but fig . likewise , even two scoring systems that included broadly the same variables ( i.e. hence , our results indicate that different scoring systems detect different subjects at risk for developing type 2 diabetes and thus are not interchangeable . adequate validation of these scoring systems using prospective data is therefore necessary to select the best system applicable to the population under study . first , the predicting ability of the tested scores could not be achieved in the current study . second , the prevalence rates according to swiss diabetes association and findrisc may be over- or underestimated as a result of our lack of dietary data and second - degree familial history . of interest , sensitivity analyses showed that decreasing the threshold of these scores by two and three points led to a 50% increase in the number of subjects at risk for developing type 2 diabetes ( supplementary table 6 ) . these findings suggest that minor changes in the scoring system can lead to considerable changes in the number of subjects at risk for developing type 2 diabetes and that any risk - scoring system should be adequately validated before being applied in a given population . indeed , the colaus study may not be representative of the swiss population , but seeing the great variability in the number of high - risk patients , this mistake may not be of great relevance . in addition , there was no sex or zip code distribution difference between the source population , the random sample , and the colaus participants . on the other hand , the colaus sample had more women and was slightly younger than the corresponding swiss population aged 3575 years ( supplementary table 7 ) . hence , it can be argued that although the colaus sample is not fully representative of the swiss population , the differences in population structure are relatively small . likewise , our population was at relatively low risk for diabetes , and it is unknown how this might influence the performance of some of the risk scores if applied in other populations or ethnic groups . on the other hand , it has been shown that risk scores developed in the same country lead to different results ( 10 ) . fourth , diabetic subjects were excluded on the basis of fasting but not on 2-h plasma glucose ; hence , diabetic subjects by 2-h glucose ( but not by fasting glucose ) were retained in the analysis . nevertheless , because the number of subjects with type 2 diabetes ( by 2-h glucose ) is fixed , our results still indicate that the number of subjects at risk for developing type 2 diabetes varies considerably according to the risk score used . hence , it is possible that the risk of developing type 2 diabetes might be overestimated when using the equations that include physical activity . in summary , our results indicate that the prevalence of participants at risk for developing type 2 diabetes varies considerably according to the scoring system used . to adequately prevent type 2 diabetes

prevention of -cell dysfunction is critical for avoiding the development and progression of type 2 diabetes ( 1 ) . assessing in vivo insulin secretion is complex because the release of insulin from the -cell is influenced by multiple factors including systemic insulin sensitivity , hepatic insulin extraction , plasma free fatty acid ( ffa ) concentrations , and glucolipid toxicity ( 24 ) . an important aspect of the regulation of insulin secretion in healthy adults with normal glucose tolerance is that a reduction in insulin sensitivity is generally accompanied by a compensatory increase in insulin secretion ( 5,6 ) . this relationship typically is curvilinear and has been referred to as the disposition index ( 5 ) . the disposition index was originally calculated from insulin sensitivity and acute insulin response during the intravenous glucose tolerance test ( ivgtt ) ( 5,6 ) . insulin sensitivity measured with the ivgtt is an aggregate of skeletal muscle , hepatic , and adipose insulin sensitivity . when the disposition index is calculated using the hyperinsulinemic - euglycemic clamp technique with supraphysiological insulin infusion rates ( 4080 mu m min ) , hepatic glucose production and lipolysis are almost completely suppressed , and 8090% of glucose is disposed in skeletal muscle ( 7 ) . therefore , when using the euglycemic insulin clamp to calculate the disposition index , two physiologically relevant tissues and two physiologically relevant processes , that is , hepatic insulin sensitivity ( 2 ) and adipose insulin sensitivity ( 8) , are not taken into account . adipose tissue should be considered in the interaction of insulin secretion and insulin action because fat tissue secretes factors ( e.g. , ffas and adipokines ) that contribute to multiorgan insulin resistance ( 8) . these factors may contribute not only to skeletal muscle and hepatic insulin resistance but also to impaired -cell function in insulin - resistant states , including impaired fasting glucose , impaired glucose tolerance , type 2 diabetes mellitus , and obesity ( 9,10 ) . because the fasting plasma ffa concentration is influenced by a feedback loop between adipose tissue and the pancreas ( 11 ) , a valid estimate of adipose insulin resistance can be obtained from the product of the basal rate of ffa turnover and the fasting insulin concentration ( 12,13 ) . relating adipose insulin resistance with glucose - stimulated insulin secretion ( gsis ) could yield a novel index of -cell function . in this study we examine the physiologic relevance of the adipose disposition index as it relates to blood glucose . forty - four obese individuals ( table 1 ) who had a stable weight ( < 2-kg change over the previous 6 months ) , were sedentary ( < 90 min / week ) , and were free of chronic disease participated in the study ( 14,15 ) . women were postmenopausal and were not receiving hormone replacement therapy . the diagnosis of type 2 diabetes was based on a standard 75-g oral glucose tolerance test ( ogtt ) : fasting plasma glucose > 126 mg / dl and/or 2-h glucose > 200 mg / dl . subjects were in otherwise good health without evidence of renal , hepatic , cardiovascular , or hematological disease as determined by history , physical examination , screening blood chemistries , urinalysis , and electrocardiography . the study protocol was approved by the institutional review board of the university of texas health science center at san antonio and the cleveland clinic . demographics , cardiometabolic risk , and basal rate of palmitate appearance , fasting plasma ffa concentration , and adipocyte insulin resistance disposition index details for nondiabetic and type 2 diabetic subjects normally distributed data are mean sem . disposition index ( di ) was calculated as : gsis 1/adipose insulin resistance ( plasma ffa and c palmitate ra ) , where gsis is ins / glucose area under the curve . di calculations for c palmitate ra were multiplied here by 1,000 for data presentation of first- and second - phase gsis . height was measured without shoes using a wall - mounted stadiometer , and weight was recorded on a digital platform scale with minimal clothing . total body fat and fat - free mass were measured by underwater weighing or use of h - water radioactivity , as previously described ( 14,15 ) . before metabolic testing , subjects were instructed to avoid strenuous physical activity for 24 h. all subjects also were instructed to consume weight - maintenance meals ( 55% carbohydrates , 30% fat , and 15% protein ) . palmitate turnover , an index of lipolysis , was assessed with an isotopically labeled palmitate infusion . after an overnight fast , a polyethylene catheter was inserted into an antecubital vein for the infusion of [ 1-c ] palmitate . a second catheter was inserted retrogradely into the distal portion of a vein on the contralateral hand for blood sampling . the hand was kept in a box warmed to 60c for the duration of the study to collect arterialized blood . a prime ( 2.5 uci ) and continuous ( 0.1 uci / min ) infusion of 1-c palmitate was prepared as previously described ( 14,15 ) and started at 8 a.m. briefly , [ 1-c ] palmitate was bound to human serum albumin and infused for the duration of the study . during the last 30 min of tracer equilibration , plasma samples were obtained at 30 , 20 , 10 , 5 , and 0 min for determination of plasma [ 1-c ] palmitate radioactivity and plasma ffa concentration . at 8 a.m. blood samples were placed on ice and immediately centrifuged and stored at 70c until subsequent analysis . adipose insulin resistance index ( adipose - ir ) was calculated as the product of the fasting plasma ffa fasting insulin and compared with the product of basal rate palmitate turnover fasting plasma insulin , as previously described ( 12,13 ) . at 8 a.m. on a different day , following a 10- to 12-h overnight fast , blood samples were collected from a superficial antecubital vein to determine fasting plasma glucose , triglyceride , ffa , total cholesterol , ldl , and hdl concentrations . hba1c also was measured in patients with type 2 diabetes to further characterize glycemic control . following collection of baseline samples , subjects orally consumed 75 g of glucose , and blood samples were obtained at 30 , 60 , 90 , and 120 min to determine plasma glucose and insulin concentrations . whole - body insulin action , which reflects the aggregate of skeletal muscle , hepatic , and adipose insulin sensitivity , was estimated using the matsuda index ( 16 ) . the homa of insulin resistance also was calculated as a more reflective surrogate of hepatic insulin resistance ( 17,18 ) . glucose and insulin total area under the curve ( auc ) during the ogtt were calculated using the trapezoidal method . first- and second - phase gsis , that is , the insulinogenic index , was calculated during the first 30 min and last 60 min of the ogtt as insulin auc divided by plasma glucose auc ( i.e. , i030/g030 and i060120/g60120 ) . the first- and second - phase adipose disposition indices were calculated as follows : i030/g030 adipose plasma ffa ir and i60120/g60120 adipose plasma ffa ir , plasma glucose was determined using the glucose oxidase method ( ysi 2300 stat plus ; ysi life sciences , yellow springs , oh ) . plasma insulin was measured using a radioimmunoassay ( millipore , billerica , ma ) . plasma triglyceride and cholesterol ( total , hdl , and ldl ) concentrations were analyzed using enzymatic methods with an automated platform ( roche modular diagnostics , indianapolis , in ) . plasma ffa concentration was measured with a colorimetric assay ( wako chemicals , richmond , va ) . basal ffa turnover was determined by dividing the [ 1-c ] palmitate infusate rate by the plasma [ 1-c ] palmitate specific activity . plasma ffa and specific activity were constant during the final 30 min of the tracer infusion . the hyperbolic relationship between insulin secretion and insulin sensitivity is characterized as insulin secretion = constant / insulin sensitivity . using log transformation , when viewed in this context , regression analysis can be applied to ogtt - derived indices of insulin secretion and insulin sensitivity to determine the regression coefficient . as indicated by kahn et al . ( 5 ) , a hyperbolic relationship between a measure of insulin secretion and insulin sensitivity would be represented by = 1 . thus a hyperbolic relationship can be satisfied if is approximately equal to 1 and the 95% confidence interval of excludes 0 . if all assumptions with linear regression modeling , y = x + e ( where e is the sampling error associated with the dependent random variable y ) are met , such that the variance of sampling error associated with the dependent random variable is constant ; if we assume that neither x nor y has measurement error , then orthogonal least squares regression will give a more accurate linear unbiased estimate of . however , if x or y is associated with random error , larger variances in either parameter will bias the regression coefficient and alter the slope ( i.e. , ) . further complicating these issues because the dependent variable ( insulin secretion ) and independent variable ( insulin sensitivity ) are measured with error , use of ordinary least squares regression would yield both a biased coefficient ( underestimate of ) and a biased standard error of coefficient estimate ( overestimate of ) ( 5 ) . ( 5 ) , we used perpendicular least squares properly weighted regression , a method that accounts for the error in both independent and dependent variables to accurately predict ( 21 ) . we also calculated 95% confidence intervals to establish whether the hyperbolic criteria have been met . we used the bootstrap method to calculate the standard error of the coefficient of estimate of , as described previously ( 22,23 ) . data were assessed using r ( version 2.4.0 ; the r foundation , vienna , austria ) . independent two - tailed t tests were used to compare nondiabetic and type 2 diabetic cohorts . pearson product moment correlation was used to examine the relationship between adipose disposition index and metabolic characteristics . data are expressed as mean sem or median ( interquartile range ) , as appropriate . forty - four obese individuals ( table 1 ) who had a stable weight ( < 2-kg change over the previous 6 months ) , were sedentary ( < 90 min / week ) , and were free of chronic disease participated in the study ( 14,15 ) . women were postmenopausal and were not receiving hormone replacement therapy . the diagnosis of type 2 diabetes was based on a standard 75-g oral glucose tolerance test ( ogtt ) : fasting plasma glucose > 126 mg / dl and/or 2-h glucose > 200 mg / dl . subjects were in otherwise good health without evidence of renal , hepatic , cardiovascular , or hematological disease as determined by history , physical examination , screening blood chemistries , urinalysis , and electrocardiography . the study protocol was approved by the institutional review board of the university of texas health science center at san antonio and the cleveland clinic . demographics , cardiometabolic risk , and basal rate of palmitate appearance , fasting plasma ffa concentration , and adipocyte insulin resistance disposition index details for nondiabetic and type 2 diabetic subjects normally distributed data are mean sem . disposition index ( di ) was calculated as : gsis 1/adipose insulin resistance ( plasma ffa and c palmitate ra ) , where gsis is ins / glucose area under the curve . di calculations for c palmitate ra were multiplied here by 1,000 for data presentation of first- and second - phase gsis . height was measured without shoes using a wall - mounted stadiometer , and weight was recorded on a digital platform scale with minimal clothing . total body fat and fat - free mass were measured by underwater weighing or use of h - water radioactivity , as previously described ( 14,15 ) . before metabolic testing , subjects were instructed to avoid strenuous physical activity for 24 h. all subjects also were instructed to consume weight - maintenance meals ( 55% carbohydrates , 30% fat , and 15% protein ) . palmitate turnover , an index of lipolysis , was assessed with an isotopically labeled palmitate infusion . after an overnight fast , a polyethylene catheter was inserted into an antecubital vein for the infusion of [ 1-c ] palmitate . a second catheter was inserted retrogradely into the distal portion of a vein on the contralateral hand for blood sampling . the hand was kept in a box warmed to 60c for the duration of the study to collect arterialized blood . a prime ( 2.5 uci ) and continuous ( 0.1 uci / min ) infusion of 1-c palmitate was prepared as previously described ( 14,15 ) and started at 8 a.m. briefly , [ 1-c ] palmitate was bound to human serum albumin and infused for the duration of the study . during the last 30 min of tracer equilibration , plasma samples were obtained at 30 , 20 , 10 , 5 , and 0 min for determination of plasma [ 1-c ] palmitate radioactivity and plasma ffa concentration . at 8 a.m. blood samples were placed on ice and immediately centrifuged and stored at 70c until subsequent analysis . adipose insulin resistance index ( adipose - ir ) was calculated as the product of the fasting plasma ffa fasting insulin and compared with the product of basal rate palmitate turnover fasting plasma insulin , as previously described ( 12,13 ) . at 8 a.m. on a different day , following a 10- to 12-h overnight fast , blood samples were collected from a superficial antecubital vein to determine fasting plasma glucose , triglyceride , ffa , total cholesterol , ldl , and hdl concentrations . hba1c also was measured in patients with type 2 diabetes to further characterize glycemic control . following collection of baseline samples , subjects orally consumed 75 g of glucose , and blood samples were obtained at 30 , 60 , 90 , and 120 min to determine plasma glucose and insulin concentrations . whole - body insulin action , which reflects the aggregate of skeletal muscle , hepatic , and adipose insulin sensitivity , was estimated using the matsuda index ( 16 ) . the homa of insulin resistance also was calculated as a more reflective surrogate of hepatic insulin resistance ( 17,18 ) . glucose and insulin total area under the curve ( auc ) during the ogtt were calculated using the trapezoidal method . first- and second - phase gsis , that is , the insulinogenic index , was calculated during the first 30 min and last 60 min of the ogtt as insulin auc divided by plasma glucose auc ( i.e. , i030/g030 and i060120/g60120 ) . the first- and second - phase adipose disposition indices were calculated as follows : i030/g030 adipose plasma ffa ir and i60120/g60120 adipose plasma ffa ir , respectively ( 19 ) . plasma glucose was determined using the glucose oxidase method ( ysi 2300 stat plus ; ysi life sciences , yellow springs , oh ) . plasma triglyceride and cholesterol ( total , hdl , and ldl ) concentrations were analyzed using enzymatic methods with an automated platform ( roche modular diagnostics , indianapolis , in ) . plasma ffa concentration was measured with a colorimetric assay ( wako chemicals , richmond , va ) . basal ffa turnover was determined by dividing the [ 1-c ] palmitate infusate rate by the plasma [ 1-c ] palmitate specific activity . plasma ffa and specific activity were constant during the final 30 min of the tracer infusion . the hyperbolic relationship between insulin secretion and insulin sensitivity is characterized as insulin secretion = constant / insulin sensitivity . using log transformation , when viewed in this context , regression analysis can be applied to ogtt - derived indices of insulin secretion and insulin sensitivity to determine the regression coefficient . as indicated by kahn et al . ( 5 ) , a hyperbolic relationship between a measure of insulin secretion and insulin sensitivity would be represented by = 1 . thus a hyperbolic relationship can be satisfied if is approximately equal to 1 and the 95% confidence interval of excludes 0 . if all assumptions with linear regression modeling , y = x + e ( where e is the sampling error associated with the dependent random variable y ) are met , such that the variance of sampling error associated with the dependent random variable is constant ; if we assume that neither x nor y has measurement error , then orthogonal least squares regression will give a more accurate linear unbiased estimate of . however , if x or y is associated with random error , larger variances in either parameter will bias the regression coefficient and alter the slope ( i.e. , ) . further complicating these issues because the dependent variable ( insulin secretion ) and independent variable ( insulin sensitivity ) are measured with error , use of ordinary least squares regression would yield both a biased coefficient ( underestimate of ) and a biased standard error of coefficient estimate ( overestimate of ) ( 5 ) . ( 5 ) , we used perpendicular least squares properly weighted regression , a method that accounts for the error in both independent and dependent variables to accurately predict ( 21 ) . we also calculated 95% confidence intervals to establish whether the hyperbolic criteria have been met . we used the bootstrap method to calculate the standard error of the coefficient of estimate of , as described previously ( 22,23 ) . data were assessed using r ( version 2.4.0 ; the r foundation , vienna , austria ) . nonnormally distributed data were log transformed for analysis . independent two - tailed t tests were used to compare nondiabetic and type 2 diabetic cohorts . pearson product moment correlation was used to examine the relationship between adipose disposition index and metabolic characteristics . data are expressed as mean sem or median ( interquartile range ) , as appropriate . individuals with type 2 diabetes were slightly younger and had a lower bmi compared with nondiabetic subjects ( p < 0.05 ; table 1 ) . although whole - body insulin sensitivity was comparable between groups , type 2 diabetic subjects had higher hepatic and adipose insulin resistance compared with nondiabetic subjects ( p < 0.05 ; table 1 ) . adipose tissue insulin resistance using the plasma ffa concentration was strongly correlated with the [ 1-c ] palmitate derived measures ( r = 0.80 ; p < 0.00001 ) , although this relationship was attenuated when plasma ffa and [ 1-c ] palmitate were analyzed without adjusting for insulin ( r = 0.45 ; p < 0.003 ) . as expected , gsis and the adipose disposition index were lower in type 2 diabetic compared with nondiabetic subjects ( p < 0.05 ; table 1 ) . both [ 1-c ] palmitate and plasma ffa derived adipose disposition indices ( i.e. , gsis adipose tissue insulin resistance ) satisfied the hyperbolic relationship whether calculated using the first phase ( 030 min ) or second phase ( 60120 min ) of gsis in nondiabetic and type 2 diabetic subjects ( fig . 1 and table 2 ) . following log transformation there was no difference in the slope of the disposition index between nondiabetic and type 2 diabetic subjects for plasma ffa ( first phase : p = 0.73 ; second phase : p = 0.88 ) and [ 1-c ] palmitate ( first phase : p = 0.93 ; second phase : p = 0.98 ) , suggesting that both groups exhibited a similar curvilinear relationship . importantly , in nondiabetic subjects the curve was shifted up and to the right compared with patients with type 2 diabetes for both first- and second - phase plasma ffa and [ 1-c ] palmitate calculations ( p < 0.001 ; data not shown ) . first- and second - phase adipose tissue disposition index using plasma ffa concentration was strongly correlated with the first- and second - phase adipose tissue disposition index derived from [ 1-c ] palmitate ( r = 0.86 , p < 0.000001 and r = 0.89 , p < 0.000001 , respectively ) . a similar correlation between the plasma ffa derived and [ 1-c ] palmitate derived adipose disposition indices was observed in men ( first phase : r = 0.80 , p < 0.00001 ; second phase : r = 0.89 , p < 0.000001 ) and women ( first phase : r = 0.88 , p < 0.00001 ; second phase : r = 0.90 , p < 0.000001 ) . both first- and second - phase adipose disposition indices derived from plasma ffa concentrations were closely correlated with fasting ( r = 0.87 , p < 0.00001 and r = 0.89 , p < 0.00001 , respectively ) and 2-h plasma glucose ( ogtt ) ( r = 0.86 , p < 0.00001 and r = 0.90 , p < 0.00001 ; fig . the first- and second - phase adipose tissue disposition indices using plasma ffa concentration also was significantly correlated with age ( r = 0.35 , p < 0.03 and r = 0.45 , p < 0.004 , respectively ) and bmi ( r = 0.42 , p < 0.007 and r = 0.39 , p < 0.01 , respectively ) . gsis adjusted for adipose insulin resistance ( ir ) ( adipose tissue disposition index ) . a : c palmitate calculated adipose disposition index : first phase . closed circles represent diabetic subjects . estimated regression coefficients ( ) and 95% cis using orthogonal slope for based on first- and second - phase gsis and adipose insulin sensitivity no between - group difference in slope parameters was detected , suggesting diabetic and nondiabetic groups had similar curvilinear relationships between insulin secretion and sensitivity . correlation between plasma ffa derived calculation of the adipose tissue disposition index versus fasting and 2-h plasma glucose concentration ( ogtt ) . individuals with type 2 diabetes were slightly younger and had a lower bmi compared with nondiabetic subjects ( p < 0.05 ; table 1 ) . although whole - body insulin sensitivity was comparable between groups , type 2 diabetic subjects had higher hepatic and adipose insulin resistance compared with nondiabetic subjects ( p < 0.05 ; table 1 ) . adipose tissue insulin resistance using the plasma ffa concentration was strongly correlated with the [ 1-c ] palmitate derived measures ( r = 0.80 ; p < 0.00001 ) , although this relationship was attenuated when plasma ffa and [ 1-c ] palmitate were analyzed without adjusting for insulin ( r = 0.45 ; p < 0.003 ) . as expected , gsis and the adipose disposition index were lower in type 2 diabetic compared with nondiabetic subjects ( p < 0.05 ; table 1 ) . both [ 1-c ] palmitate and plasma ffa derived adipose disposition indices ( i.e. , gsis adipose tissue insulin resistance ) satisfied the hyperbolic relationship whether calculated using the first phase ( 030 min ) or second phase ( 60120 min ) of gsis in nondiabetic and type 2 diabetic subjects ( fig . 1 and table 2 ) . following log transformation there was no difference in the slope of the disposition index between nondiabetic and type 2 diabetic subjects for plasma ffa ( first phase : p = 0.73 ; second phase : p = 0.88 ) and [ 1-c ] palmitate ( first phase : p = 0.93 ; second phase : p = 0.98 ) , suggesting that both groups exhibited a similar curvilinear relationship . importantly , in nondiabetic subjects the curve was shifted up and to the right compared with patients with type 2 diabetes for both first- and second - phase plasma ffa and [ 1-c ] palmitate calculations ( p < 0.001 ; data not shown ) . first- and second - phase adipose tissue disposition index using plasma ffa concentration was strongly correlated with the first- and second - phase adipose tissue disposition index derived from [ 1-c ] palmitate ( r = 0.86 , p < 0.000001 and r = 0.89 , p < 0.000001 , respectively ) . a similar correlation between the plasma ffa derived and [ 1-c ] palmitate derived adipose disposition indices was observed in men ( first phase : r = 0.80 , p < 0.00001 ; second phase : r = 0.89 , p < 0.000001 ) and women ( first phase : r = 0.88 , p < 0.00001 ; second phase : r = 0.90 , p < 0.000001 ) . both first- and second - phase adipose disposition indices derived from plasma ffa concentrations were closely correlated with fasting ( r = 0.87 , p < 0.00001 and r = 0.89 , p < 0.00001 , respectively ) and 2-h plasma glucose ( ogtt ) ( r = 0.86 , p < 0.00001 and r = 0.90 , p < 0.00001 ; fig . the first- and second - phase adipose tissue disposition indices using plasma ffa concentration also was significantly correlated with age ( r = 0.35 , p < 0.03 and r = 0.45 , p < 0.004 , respectively ) and bmi ( r = 0.42 , p < 0.007 and r = 0.39 , p < 0.01 , respectively ) . gsis adjusted for adipose insulin resistance ( ir ) ( adipose tissue disposition index ) . closed circles represent diabetic subjects . estimated regression coefficients ( ) and 95% cis using orthogonal slope for based on first- and second - phase gsis and adipose insulin sensitivity no between - group difference in slope parameters was detected , suggesting diabetic and nondiabetic groups had similar curvilinear relationships between insulin secretion and sensitivity . correlation between plasma ffa derived calculation of the adipose tissue disposition index versus fasting and 2-h plasma glucose concentration ( ogtt ) . elevated fasting plasma ffa concentrations and impaired suppression of plasma ffa by insulin plays an important role in not only the development of insulin resistance ( 8,14,24 ) but also -cell dysfunction ( 9,10 ) . the major finding from this study is that the plasma ffa derived adipose tissue disposition index , whether calculated using first- or second - phase gsis , approximates the hyperbolic relationship ( fig . 1 ) that is purported to maintain normal glucose concentrations and correlates strongly with [ 1-c ] palmitate kinetic assessments . the variability in slope parameters detected in our study ( table 2 ) are comparable with those reported using whole - body estimates of insulin resistance ( 23 ) and is consistent with work by ferrannini and mari ( 25 ) describing the utility of the hyperbolic relationship when scaling gsis to insulin resistance across the glucose tolerance spectrum . therefore , based on our modest sample size of obese nondiabetic and diabetic subjects , adjusting gsis for adipose insulin resistance seems to be a reasonable approach to understanding the integrated multitissue metabolic abnormalities associated with -cell dysfunction in obesity- and diabetes - related glucose intolerance . to maintain normoglycemia the pancreatic -cell must compensate for multiorgan insulin resistance with an appropriate increase in insulin secretion . our data are consistent with the view that the capacity of pancreatic -cells to secrete insulin for the prevailing degree of insulin resistance declines as glucose intolerance worsens in obese subjects , that is , diabetic subjects have a downward , left - shifted insulin curve ( 20 ) ( see fig . however , it should be acknowledged that , in vivo , the compensatory increase in pancreatic insulin secretion in response to insulin resistance may be unable to completely normalize circulating glucose concentrations . in pima indians and caucasians , stumvoll and colleagues ( 26 ) reported that the feedback between insulin resistance on pancreatic -cell demand involves a signal from circulating glucose , such that when gsis is divided by insulin resistance to depict the -cell demand for maintaining blood glucose concentrations ( as opposed to multiplying gsis by insulin resistance ) , glycemia increases for a given decrease in insulin sensitivity despite normal compensation of insulin secretion ( termed glucose allostasis ) . thus gsis compensation for insulin resistance is likely more complex than that provided by a single calculation of a whole - body insulin sensitivity derived disposition index , and additional models of -cell response are needed ( 25 ) . recent work suggested that alterations in hepatic disposition index may precede changes in peripheral disposition index in response to overfeeding and/or physical inactivity , suggesting that multiple organs influence compensatory insulin secretion ( 2,27,28 ) . the novel calculation presented herein justifies the use of the adipose disposition index to characterize the contribution of adipose insulin resistance to gsis in obese nondiabetic and obese diabetic subjects . future work is needed to examine the role of skeletal muscle , hepatic , and adipose disposition indices following lifestyle modification and/or bariatric surgery to better understand the complexity of human glucose regulation . ultimately , these findings may provide greater clinical insight for targeted treatment strategies that prevent / reverse type 2 diabetes . we recognize that using the plasma c - peptide concentration as a measure of prehepatic insulin secretion provides a more direct measure of gsis and -cell function , although work by our laboratory suggested that hepatic insulin extraction does not affect insulin - derived calculations of -cell function before or after lifestyle modification in a cohort of obese insulin - resistant adults similar to that used in the current study ( 29 ) . in addition , use of the ivgtt or hyperglycemic clamp may provide a more accurate assessment of insulin secretion compared with ogtt - derived measures ( 5,6 ) . however , exclusion of the gastrointestinal tract limits the physiologic understanding of in vivo -cell function . further , circulating glycerol may represent a better biomarker for lipolysis because plasma ffa can be re - esterified and/or taken up for storage , thereby limiting ffa as a lipolytic indicator . consistent with this notion , basal [ 1-c ] palmitate turnover was only modestly associated with plasma ffa in our study ( r = 0.45 ; p < 0.003 ) . therefore , despite the observation that groups in our study exhibited similar fasting plasma ffa concentrations , the higher basal [ 1-c ] palmitate turnover in diabetic compared with nondiabetic subjects suggest differences in ffa metabolism ( i.e. , production vs. storage ) were present . it is worth recognizing , however , that ffas are implicated in the development of -cell dysfunction , and ffas are released from adipocytes , which make them a reasonable marker of lipolysis ( 9,10,27,30 ) . both fasting and 2-h plasma glucose concentrations were closely associated with adipose disposition index , and part of this finding could be related to estimation of the disposition index including plasma glucose . however , the possibility of autocorrelation is lessened by the fact that first - phase gsis ( 030 min of insulin divided by glucose ) adjusted for adipose insulin resistance ( fasting ffa fasting insulin ) correlates strongly with 2-h plasma glucose . this time course difference in glucose and insulin pattern suggests that the adipose disposition index calculation has physiologic relevance in characterizing -cell function across the glucose tolerance continuum ( fig . the index also correlated with age and body mass , which further confirms that it has physiological relevance . in conclusion , gsis is essential for preventing the progression from normal glucose tolerance to type 2 diabetes ( 1,2,30 ) . our results suggest that adjusting gsis for adipose insulin resistance provides a reasonable index of -cell function in obese nondiabetic and obese diabetic subjects . because a high - fat diet and lipid infusion in humans impair gsis ( 27,31 ) , characterization of in vivo pancreatic -cell insulin secretion in relation to adipose tissue insulin resistance may provide additional mechanistic insight into the regulation of glucose homeostasis .

objectivethe hyperbolic relationship between insulin secretion and sensitivity has been used to assess in vivo -cell function ( i.e. , the disposition index ) . the disposition index emphasizes the importance of taking into account both skeletal muscle and hepatic insulin resistance to depict insulin secretion . however , we propose that adipose tissue insulin resistance also needs to be accounted for when characterizing glucose - stimulated insulin secretion ( gsis ) because elevated plasma free fatty acids ( ffas ) impair -cell function.research design and methodsto characterize the adipose disposition index , we used [ 1 - 14c ] palmitate infusion to determine basal ffa turnover rate / adipose insulin resistance and an oral glucose tolerance test to characterize the first ( i.e. , 030 min ) and second phase ( i.e. , 60120 min ) of gsis . we validated a simplified version of the tracer infusion calculation as the product of ( 1/plasma ffa concentration plasma insulin concentration ) gsis in 44 obese insulin - resistant subjects.resultsthe plasma ffa and palmitate tracer infusion calculations of the first- and second - phase disposition index were strongly correlated ( r = 0.86 , p < 0.000001 and r = 0.89 , p < 0.000001 , respectively ) . the first- and second - phase adipose disposition index derived from plasma ffa also was tightly associated with fasting hyperglycemia ( r = 0.87 , p < 0.00001 and r = 0.89 , p < 0.00001 , respectively ) and 2-h glucose concentrations ( r = 0.86 , p < 0.00001 and r = 0.90 , p < 0.00001).conclusionsadjusting gsis for adipose insulin resistance provides an index of -cell function in obese subjects across the glucose spectrum . plasma ffa derived calculations of -cell function may provide additional insight into the role of adipose tissue in glucose regulation .

Introduction Research Design and Methods Subjects Body Composition Control Period Palmitate Turnover Insulin Secretion Biochemical Analysis Determining Hyperbolic Relationships Statistical Analysis Results Metabolic Characteristics Insulin Secretion Conclusions

assessing in vivo insulin secretion is complex because the release of insulin from the -cell is influenced by multiple factors including systemic insulin sensitivity , hepatic insulin extraction , plasma free fatty acid ( ffa ) concentrations , and glucolipid toxicity ( 24 ) . an important aspect of the regulation of insulin secretion in healthy adults with normal glucose tolerance is that a reduction in insulin sensitivity is generally accompanied by a compensatory increase in insulin secretion ( 5,6 ) . this relationship typically is curvilinear and has been referred to as the disposition index ( 5 ) . the disposition index was originally calculated from insulin sensitivity and acute insulin response during the intravenous glucose tolerance test ( ivgtt ) ( 5,6 ) . when the disposition index is calculated using the hyperinsulinemic - euglycemic clamp technique with supraphysiological insulin infusion rates ( 4080 mu m min ) , hepatic glucose production and lipolysis are almost completely suppressed , and 8090% of glucose is disposed in skeletal muscle ( 7 ) . therefore , when using the euglycemic insulin clamp to calculate the disposition index , two physiologically relevant tissues and two physiologically relevant processes , that is , hepatic insulin sensitivity ( 2 ) and adipose insulin sensitivity ( 8) , are not taken into account . these factors may contribute not only to skeletal muscle and hepatic insulin resistance but also to impaired -cell function in insulin - resistant states , including impaired fasting glucose , impaired glucose tolerance , type 2 diabetes mellitus , and obesity ( 9,10 ) . because the fasting plasma ffa concentration is influenced by a feedback loop between adipose tissue and the pancreas ( 11 ) , a valid estimate of adipose insulin resistance can be obtained from the product of the basal rate of ffa turnover and the fasting insulin concentration ( 12,13 ) . relating adipose insulin resistance with glucose - stimulated insulin secretion ( gsis ) could yield a novel index of -cell function . in this study we examine the physiologic relevance of the adipose disposition index as it relates to blood glucose . the diagnosis of type 2 diabetes was based on a standard 75-g oral glucose tolerance test ( ogtt ) : fasting plasma glucose > 126 mg / dl and/or 2-h glucose > 200 mg / dl . demographics , cardiometabolic risk , and basal rate of palmitate appearance , fasting plasma ffa concentration , and adipocyte insulin resistance disposition index details for nondiabetic and type 2 diabetic subjects normally distributed data are mean sem . disposition index ( di ) was calculated as : gsis 1/adipose insulin resistance ( plasma ffa and c palmitate ra ) , where gsis is ins / glucose area under the curve . di calculations for c palmitate ra were multiplied here by 1,000 for data presentation of first- and second - phase gsis . a prime ( 2.5 uci ) and continuous ( 0.1 uci / min ) infusion of 1-c palmitate was prepared as previously described ( 14,15 ) and started at 8 a.m. briefly , [ 1-c ] palmitate was bound to human serum albumin and infused for the duration of the study . adipose insulin resistance index ( adipose - ir ) was calculated as the product of the fasting plasma ffa fasting insulin and compared with the product of basal rate palmitate turnover fasting plasma insulin , as previously described ( 12,13 ) . the homa of insulin resistance also was calculated as a more reflective surrogate of hepatic insulin resistance ( 17,18 ) . first- and second - phase gsis , that is , the insulinogenic index , was calculated during the first 30 min and last 60 min of the ogtt as insulin auc divided by plasma glucose auc ( i.e. the first- and second - phase adipose disposition indices were calculated as follows : i030/g030 adipose plasma ffa ir and i60120/g60120 adipose plasma ffa ir , plasma glucose was determined using the glucose oxidase method ( ysi 2300 stat plus ; ysi life sciences , yellow springs , oh ) . basal ffa turnover was determined by dividing the [ 1-c ] palmitate infusate rate by the plasma [ 1-c ] palmitate specific activity . plasma ffa and specific activity were constant during the final 30 min of the tracer infusion . the hyperbolic relationship between insulin secretion and insulin sensitivity is characterized as insulin secretion = constant / insulin sensitivity . ( 5 ) , a hyperbolic relationship between a measure of insulin secretion and insulin sensitivity would be represented by = 1 . however , if x or y is associated with random error , larger variances in either parameter will bias the regression coefficient and alter the slope ( i.e. pearson product moment correlation was used to examine the relationship between adipose disposition index and metabolic characteristics . the diagnosis of type 2 diabetes was based on a standard 75-g oral glucose tolerance test ( ogtt ) : fasting plasma glucose > 126 mg / dl and/or 2-h glucose > 200 mg / dl . demographics , cardiometabolic risk , and basal rate of palmitate appearance , fasting plasma ffa concentration , and adipocyte insulin resistance disposition index details for nondiabetic and type 2 diabetic subjects normally distributed data are mean sem . disposition index ( di ) was calculated as : gsis 1/adipose insulin resistance ( plasma ffa and c palmitate ra ) , where gsis is ins / glucose area under the curve . di calculations for c palmitate ra were multiplied here by 1,000 for data presentation of first- and second - phase gsis . a prime ( 2.5 uci ) and continuous ( 0.1 uci / min ) infusion of 1-c palmitate was prepared as previously described ( 14,15 ) and started at 8 a.m. briefly , [ 1-c ] palmitate was bound to human serum albumin and infused for the duration of the study . during the last 30 min of tracer equilibration , plasma samples were obtained at 30 , 20 , 10 , 5 , and 0 min for determination of plasma [ 1-c ] palmitate radioactivity and plasma ffa concentration . adipose insulin resistance index ( adipose - ir ) was calculated as the product of the fasting plasma ffa fasting insulin and compared with the product of basal rate palmitate turnover fasting plasma insulin , as previously described ( 12,13 ) . the homa of insulin resistance also was calculated as a more reflective surrogate of hepatic insulin resistance ( 17,18 ) . first- and second - phase gsis , that is , the insulinogenic index , was calculated during the first 30 min and last 60 min of the ogtt as insulin auc divided by plasma glucose auc ( i.e. the first- and second - phase adipose disposition indices were calculated as follows : i030/g030 adipose plasma ffa ir and i60120/g60120 adipose plasma ffa ir , respectively ( 19 ) . basal ffa turnover was determined by dividing the [ 1-c ] palmitate infusate rate by the plasma [ 1-c ] palmitate specific activity . plasma ffa and specific activity were constant during the final 30 min of the tracer infusion . the hyperbolic relationship between insulin secretion and insulin sensitivity is characterized as insulin secretion = constant / insulin sensitivity . ( 5 ) , a hyperbolic relationship between a measure of insulin secretion and insulin sensitivity would be represented by = 1 . however , if x or y is associated with random error , larger variances in either parameter will bias the regression coefficient and alter the slope ( i.e. pearson product moment correlation was used to examine the relationship between adipose disposition index and metabolic characteristics . although whole - body insulin sensitivity was comparable between groups , type 2 diabetic subjects had higher hepatic and adipose insulin resistance compared with nondiabetic subjects ( p < 0.05 ; table 1 ) . adipose tissue insulin resistance using the plasma ffa concentration was strongly correlated with the [ 1-c ] palmitate derived measures ( r = 0.80 ; p < 0.00001 ) , although this relationship was attenuated when plasma ffa and [ 1-c ] palmitate were analyzed without adjusting for insulin ( r = 0.45 ; p < 0.003 ) . as expected , gsis and the adipose disposition index were lower in type 2 diabetic compared with nondiabetic subjects ( p < 0.05 ; table 1 ) . both [ 1-c ] palmitate and plasma ffa derived adipose disposition indices ( i.e. , gsis adipose tissue insulin resistance ) satisfied the hyperbolic relationship whether calculated using the first phase ( 030 min ) or second phase ( 60120 min ) of gsis in nondiabetic and type 2 diabetic subjects ( fig . following log transformation there was no difference in the slope of the disposition index between nondiabetic and type 2 diabetic subjects for plasma ffa ( first phase : p = 0.73 ; second phase : p = 0.88 ) and [ 1-c ] palmitate ( first phase : p = 0.93 ; second phase : p = 0.98 ) , suggesting that both groups exhibited a similar curvilinear relationship . importantly , in nondiabetic subjects the curve was shifted up and to the right compared with patients with type 2 diabetes for both first- and second - phase plasma ffa and [ 1-c ] palmitate calculations ( p < 0.001 ; data not shown ) . first- and second - phase adipose tissue disposition index using plasma ffa concentration was strongly correlated with the first- and second - phase adipose tissue disposition index derived from [ 1-c ] palmitate ( r = 0.86 , p < 0.000001 and r = 0.89 , p < 0.000001 , respectively ) . a similar correlation between the plasma ffa derived and [ 1-c ] palmitate derived adipose disposition indices was observed in men ( first phase : r = 0.80 , p < 0.00001 ; second phase : r = 0.89 , p < 0.000001 ) and women ( first phase : r = 0.88 , p < 0.00001 ; second phase : r = 0.90 , p < 0.000001 ) . both first- and second - phase adipose disposition indices derived from plasma ffa concentrations were closely correlated with fasting ( r = 0.87 , p < 0.00001 and r = 0.89 , p < 0.00001 , respectively ) and 2-h plasma glucose ( ogtt ) ( r = 0.86 , p < 0.00001 and r = 0.90 , p < 0.00001 ; fig . the first- and second - phase adipose tissue disposition indices using plasma ffa concentration also was significantly correlated with age ( r = 0.35 , p < 0.03 and r = 0.45 , p < 0.004 , respectively ) and bmi ( r = 0.42 , p < 0.007 and r = 0.39 , p < 0.01 , respectively ) . gsis adjusted for adipose insulin resistance ( ir ) ( adipose tissue disposition index ) . estimated regression coefficients ( ) and 95% cis using orthogonal slope for based on first- and second - phase gsis and adipose insulin sensitivity no between - group difference in slope parameters was detected , suggesting diabetic and nondiabetic groups had similar curvilinear relationships between insulin secretion and sensitivity . correlation between plasma ffa derived calculation of the adipose tissue disposition index versus fasting and 2-h plasma glucose concentration ( ogtt ) . although whole - body insulin sensitivity was comparable between groups , type 2 diabetic subjects had higher hepatic and adipose insulin resistance compared with nondiabetic subjects ( p < 0.05 ; table 1 ) . adipose tissue insulin resistance using the plasma ffa concentration was strongly correlated with the [ 1-c ] palmitate derived measures ( r = 0.80 ; p < 0.00001 ) , although this relationship was attenuated when plasma ffa and [ 1-c ] palmitate were analyzed without adjusting for insulin ( r = 0.45 ; p < 0.003 ) . as expected , gsis and the adipose disposition index were lower in type 2 diabetic compared with nondiabetic subjects ( p < 0.05 ; table 1 ) . both [ 1-c ] palmitate and plasma ffa derived adipose disposition indices ( i.e. , gsis adipose tissue insulin resistance ) satisfied the hyperbolic relationship whether calculated using the first phase ( 030 min ) or second phase ( 60120 min ) of gsis in nondiabetic and type 2 diabetic subjects ( fig . following log transformation there was no difference in the slope of the disposition index between nondiabetic and type 2 diabetic subjects for plasma ffa ( first phase : p = 0.73 ; second phase : p = 0.88 ) and [ 1-c ] palmitate ( first phase : p = 0.93 ; second phase : p = 0.98 ) , suggesting that both groups exhibited a similar curvilinear relationship . importantly , in nondiabetic subjects the curve was shifted up and to the right compared with patients with type 2 diabetes for both first- and second - phase plasma ffa and [ 1-c ] palmitate calculations ( p < 0.001 ; data not shown ) . first- and second - phase adipose tissue disposition index using plasma ffa concentration was strongly correlated with the first- and second - phase adipose tissue disposition index derived from [ 1-c ] palmitate ( r = 0.86 , p < 0.000001 and r = 0.89 , p < 0.000001 , respectively ) . a similar correlation between the plasma ffa derived and [ 1-c ] palmitate derived adipose disposition indices was observed in men ( first phase : r = 0.80 , p < 0.00001 ; second phase : r = 0.89 , p < 0.000001 ) and women ( first phase : r = 0.88 , p < 0.00001 ; second phase : r = 0.90 , p < 0.000001 ) . both first- and second - phase adipose disposition indices derived from plasma ffa concentrations were closely correlated with fasting ( r = 0.87 , p < 0.00001 and r = 0.89 , p < 0.00001 , respectively ) and 2-h plasma glucose ( ogtt ) ( r = 0.86 , p < 0.00001 and r = 0.90 , p < 0.00001 ; fig . the first- and second - phase adipose tissue disposition indices using plasma ffa concentration also was significantly correlated with age ( r = 0.35 , p < 0.03 and r = 0.45 , p < 0.004 , respectively ) and bmi ( r = 0.42 , p < 0.007 and r = 0.39 , p < 0.01 , respectively ) . gsis adjusted for adipose insulin resistance ( ir ) ( adipose tissue disposition index ) . estimated regression coefficients ( ) and 95% cis using orthogonal slope for based on first- and second - phase gsis and adipose insulin sensitivity no between - group difference in slope parameters was detected , suggesting diabetic and nondiabetic groups had similar curvilinear relationships between insulin secretion and sensitivity . correlation between plasma ffa derived calculation of the adipose tissue disposition index versus fasting and 2-h plasma glucose concentration ( ogtt ) . the major finding from this study is that the plasma ffa derived adipose tissue disposition index , whether calculated using first- or second - phase gsis , approximates the hyperbolic relationship ( fig . the variability in slope parameters detected in our study ( table 2 ) are comparable with those reported using whole - body estimates of insulin resistance ( 23 ) and is consistent with work by ferrannini and mari ( 25 ) describing the utility of the hyperbolic relationship when scaling gsis to insulin resistance across the glucose tolerance spectrum . therefore , based on our modest sample size of obese nondiabetic and diabetic subjects , adjusting gsis for adipose insulin resistance seems to be a reasonable approach to understanding the integrated multitissue metabolic abnormalities associated with -cell dysfunction in obesity- and diabetes - related glucose intolerance . however , it should be acknowledged that , in vivo , the compensatory increase in pancreatic insulin secretion in response to insulin resistance may be unable to completely normalize circulating glucose concentrations . in pima indians and caucasians , stumvoll and colleagues ( 26 ) reported that the feedback between insulin resistance on pancreatic -cell demand involves a signal from circulating glucose , such that when gsis is divided by insulin resistance to depict the -cell demand for maintaining blood glucose concentrations ( as opposed to multiplying gsis by insulin resistance ) , glycemia increases for a given decrease in insulin sensitivity despite normal compensation of insulin secretion ( termed glucose allostasis ) . thus gsis compensation for insulin resistance is likely more complex than that provided by a single calculation of a whole - body insulin sensitivity derived disposition index , and additional models of -cell response are needed ( 25 ) . the novel calculation presented herein justifies the use of the adipose disposition index to characterize the contribution of adipose insulin resistance to gsis in obese nondiabetic and obese diabetic subjects . future work is needed to examine the role of skeletal muscle , hepatic , and adipose disposition indices following lifestyle modification and/or bariatric surgery to better understand the complexity of human glucose regulation . we recognize that using the plasma c - peptide concentration as a measure of prehepatic insulin secretion provides a more direct measure of gsis and -cell function , although work by our laboratory suggested that hepatic insulin extraction does not affect insulin - derived calculations of -cell function before or after lifestyle modification in a cohort of obese insulin - resistant adults similar to that used in the current study ( 29 ) . however , exclusion of the gastrointestinal tract limits the physiologic understanding of in vivo -cell function . consistent with this notion , basal [ 1-c ] palmitate turnover was only modestly associated with plasma ffa in our study ( r = 0.45 ; p < 0.003 ) . therefore , despite the observation that groups in our study exhibited similar fasting plasma ffa concentrations , the higher basal [ 1-c ] palmitate turnover in diabetic compared with nondiabetic subjects suggest differences in ffa metabolism ( i.e. both fasting and 2-h plasma glucose concentrations were closely associated with adipose disposition index , and part of this finding could be related to estimation of the disposition index including plasma glucose . however , the possibility of autocorrelation is lessened by the fact that first - phase gsis ( 030 min of insulin divided by glucose ) adjusted for adipose insulin resistance ( fasting ffa fasting insulin ) correlates strongly with 2-h plasma glucose . this time course difference in glucose and insulin pattern suggests that the adipose disposition index calculation has physiologic relevance in characterizing -cell function across the glucose tolerance continuum ( fig . our results suggest that adjusting gsis for adipose insulin resistance provides a reasonable index of -cell function in obese nondiabetic and obese diabetic subjects . because a high - fat diet and lipid infusion in humans impair gsis ( 27,31 ) , characterization of in vivo pancreatic -cell insulin secretion in relation to adipose tissue insulin resistance may provide additional mechanistic insight into the regulation of glucose homeostasis .

long - acting anticholinergic agents may be the most effective class of bronchodilators for copd,1,2,6,7 and although tiotropium bromide is the only long - acting anticholinergic bronchodilator marketed for copd , several others are in various stages of development , including glycopyrronium bromide . glycopyrronium bromide has been used for years , administered as injections or tablets , as an adjunct in peptic ulcer disorders , to diminish secretions and block cardiac vagal reflexes before surgery , and has now been developed as an inhaled agent for the treatment of copd . this short review provides an overview of the available clinical studies evaluating the safety and efficacy of inhaled glycopyrronium bromide in patients with moderate to severe copd . a series of systematic searches were carried out and last updated in august 2012 , using the database pubmed . the strategy was intended to be broad in order to maximize the capture of citations for peer - reviewed publications relevant to glycopyrronium bromide and copd . the pubmed searches were carried out using the following algorithm of mesh terms : glycopyrronium bromide , nva237 , long - acting antimuscarinic agents , indacaterol , salmeterol , formoterol , tiotropium bromide , aclidinium bromide , long - acting bronchodilators , and copd , and the searches were repeated with these terms in combination with forced expiratory volume in one second ( fev1 ) , hyperinflation , exercise capacity , dyspnea , health status , exacerbations , and adverse effects . the citation pool was further supplemented from manual assessment of the reference lists in identified reviews and clinical studies of aspects related to long - acting bronchodilators and copd and from other publications identified as being relevant for further review . all identified randomized studies published in peer - reviewed journals of glycopyrronium bromide for the treatment of copd are included in the present review . glycopyrronium bromide ( nva237 ) is a synthetic quaternary ammonium compound acting as a competitive antagonist by binding to muscarinic receptors in bronchial smooth muscle , thereby inhibiting acetylcholine - induced bronchoconstriction . glycopyrronium bromide binds to all five subtypes of the muscarinic receptor ( m1m5 ) , of which the m3 receptor located on airway smooth muscle is primarily involved in smooth muscle contraction . blockade of m2 receptors attenuates the feedback inhibition of acetylcholine production , and thereby may reduce the bronchodilating effects of an antimuscarinic agent . furthermore , blocking cardiac m2 receptors may result in an increase in heart rate . therefore , the optimal antimuscarinic agent for use in copd has high affinity for m1 and m3 receptors and low affinity for m2 receptors . like tiotropium bromide , glycopyrronium bromide has higher selectivity for m3 receptors than for m2 receptors , and like both tiotropium bromide and aclidinium bromide , dissociates more slowly from the m3 receptor than it does from the m2 receptor.8,9 therefore , glycopyrronium bromide provides bronchodilatation via m3 blockade long after its less desirable m2 effects , such as an increased heart rate.810 in vitro studies have indicated that glycopyrronium bromide may have a faster onset of action than tiotropium , and this has been confirmed in clinical studies.8,11 glycopyrronium bromide equilibrates four to five times faster with the m3 receptor than does tiotropium bromide and aclidinium bromide.8,11 this more rapid equilibration of glycopyrronium bromide with the m3 receptor is assumed to underlie its faster onset of action compared with that of tiotropium bromide,8 and probably also aclidinium bromide . early preclinical and clinical studies have suggested a very favorable safety profile for inhaled glycopyrronium bromide . sechaud et al10 investigated the pharmacokinetics of glycopyrronium bromide following single and repeated once - daily inhalation in mild to moderate copd patients , and reported that the pharmacokinetics of glycopyrronium bromide were consistent between doses and with limited systemic accumulation at steady state after repeated once - daily inhalation . the overall incidence of adverse events was similar in patients treated with glycopyrronium bromide and placebo , apart from a higher frequency of dry mouth in the groups treated with glycopyrronium bromide . no serious adverse events were reported , and no clinically relevant effects were observed on vital signs , electrocardiography , or physical examinations.10 in a randomized , double - blind , placebo - controlled , parallel - group study , in which 257 patients with moderate to severe copd were assigned to indacaterol plus glycopyrronium bromide , indacaterol , or placebo , van de maele et al12 investigated the change from baseline in 24-hour mean heart rate versus placebo . a total of 255 patients were included in the safety analysis , which showed no clinically significant differences between groups in the mean 24-hour heart rate or qtc interval , suggesting a cardiovascular safety profile of both long - acting bronchodilators similar to that of placebo . the issue of inhaled anticholinergics and risk of major cardiovascular events has been much debated , and a meta - analysis of previously published studies has reported an increased risk of cardiovascular death , myocardial infarction , and stroke in copd patients treated with inhaled anticholinergics,13 but conclusions regarding this point await the findings of ongoing prospective studies . in the clinical studies published so far , cardiac and vascular disorders have been reported at a similar frequency in the glycopyrronium bromide and placebo groups , although , as in other clinical trials of antimuscarinic agents in copd , patients with any of a number of concomitant diseases , including symptomatic prostatic hyperplasia and narrow - angle glaucoma , were not eligible for inclusion . verkindre et al14 published one of the first double - blind , placebo - controlled trials of glycopyrronium bromide in patients with copd . the study was conducted in 83 patients ( 78 patients completed the study ) with stable moderate to severe copd ( mean post - bronchodilator fev1 53% predicted ) . the patients were randomized to glycopyrronium bromide 12.5 , 25 , 50 or 100 g , placebo , or tiotropium bromide 18 g once daily ( open - label active comparator ) for 7 days . the primary endpoint was mean trough ( 2324 hours following dosing ) fev1 on day 7 . mean trough fev1 on days 1 and 7 was significantly higher with all active treatments versus placebo ( p < 0.05 ) . glycopyrronium bromide 50 g and 100 g and tiotropium bromide showed clinically relevant improvements versus placebo on day 1 ( differences 121 , 135 , and 112 ml , respectively , p < 0.0001 ) and day 7 ( differences 131 , 142 , and 127 ml , respectively , p < 0.0001 ) . no clinically significant difference in improvements in fev1 was observed between either dose of glycopyrronium bromide and tiotropium bromide , although a statistically significant difference in level of fev1 in favor of glycopyrronium bromide was reported . therefore , this study showed that glycopyrronium bromide once daily was well tolerated and demonstrated rapid and sustained 24-hour bronchodilatation , the latter being at least comparable with tiotropium bromide . in a randomized , placebo - controlled trial , vogelmeier et al15 studied the bronchodilator efficacy of two doses of glycopyrronium bromide ( 100 g and 200 g ) versus placebo in patients with moderate to severe copd ( mean post - bronchodilator fev1 53% predicted ) . enrolled patients were randomized to treatment with glycopyrronium bromide 100 g ( n = 92 ) , glycopyrronium bromide 200 g ( n = 98 ) , or placebo ( n = 91 ) for 28 days . the primary objective was to evaluate safety , but efficacy measures were included as secondary objectives . mean trough fev1 was significantly higher in patients receiving glycopyrronium bromide compared with those receiving placebo . for glycopyrronium bromide 100 g , the treatment differences from placebo were 131 ml and 161 ml , respectively , on days 1 and 28 ( p < 0.05 ) ; for glycopyrronium bromide 200 g , the treatment differences were 146 ml and 151 ml , respectively , on days 1 and 28 ( p < 0.05 ) . in line with this , peak fev1 , fev1 at all time points up to 24 hours after dosing , and fev1 area under the curve ( auc ) from 5 minutes to 5 hours after dosing were also significantly higher for both doses of glycopyrronium bromide compared with placebo . although this study did not include an active comparator , its reported findings support the bronchodilator efficacy of glycopyrronium bromide in patients with moderate to severe copd . in a small , randomized , double - blind , placebo - controlled , two - period crossover trial , fogarty et al16 studied the bronchodilatory effects of glycopyrronium bromide versus placebo in 33 patients with moderate copd ( mean post - bronchodilator fev1 70% predicted ) . only subjects with at least a 5% increase in fev1 following inhalation of ipratropium bromide were eligible for the study . patients were randomized to receive glycopyrronium bromide 50 g once daily followed by placebo or vice versa for 14 days ; the treatment periods were separated by a washout period of 714 days . the primary efficacy variable was the standardized fev1 auc between 0 and 24 hours after dosing on day 14 . the fev1 auc024 h values on day 14 were significantly higher for glycopyrronium bromide than for placebo ( p < 0.001 ) with a least squares mean difference of 163 ml between glycopyrronium bromide and placebo . the fev1 with glycopyrronium bromide was significantly higher ( p < 0.001 ) than for placebo for all aucs evaluated . on day 14 , significant treatment differences were found in mean fev1 auc05 h ( difference 198 ml , p < 0.001 ) , fev1 auc012 h ( difference 165 ml , p = 0.001 ) , and fev1 auc1224 h ( difference 161 ml , p < furthermore , all primary analyses for trough fev1 comparisons showed a clinically significant effect ( predefined threshold exceeded , bromide on day 7 ( 91 ml ) . the mean difference in peak fev1 ie , 120 ml ) at the 5% level , except for the treatment least squares mean difference in trough value for glycopyrronium values between glycopyrronium and placebo was 208 ml ( p < 0.001 ) on day 14 and 173 ml ( p = 0.011 ) on day 7 , respectively . therefore , this study supports the notion that glycopyrronium bromide provides significant and sustained 24-hour bronchodilatation in patients with copd , similar to that provided by tiotropium bromide.17 in one of the largest published clinical trials of glycopyrronium bromide ( the glycopyrronium bromide in copd airways clinical study 1 [ glow1 ] ) , durzu et al18 randomized patients with stable moderate to severe copd ( mean post - bronchodilator fev1 54% predicted ) to either glycopyrronium bromide 50 g once daily ( n = 552 ) or placebo ( n = 270 ) for 26 weeks . patients were not eligible for the study if they were participating in a supervised pulmonary rehabilitation program . least squares mean trough fev1 at week 12 was significantly higher in patients receiving glycopyrronium bromide ( 1.41 l ) versus placebo ( 1.30 l ) , with a treatment difference of 108 15 ml ( p < 0.001 ) . significant improvements in trough fev1 were observed at the end of day 1 and sustained through week 26 . fev1 was significantly improved in the glycopyrronium bromide group versus the placebo group throughout a 24-hour period on day 1 , at weeks 12 and 26 , and at all other scheduled visits and time points . in the glow 2 study , kerwin et al19 randomized patients with moderate to severe copd ( mean fev1 56% predicted ) to either glycopyrronium bromide 50 g once daily ( n = 525 ) , open - label tiotropium 18 g once daily ( n = 267 ) , or placebo ( n = 268 ) for 52 weeks . least squares mean trough fev1 at week 12 was significantly higher in patients receiving glycopyrronium bromide and tiotropium compared with that in patients receiving placebo , with a treatment difference of 97 ml ( p < 0.001 ) and 83 ml ( p trough fev1 at weeks 26 and 52 was significantly higher for patients treated with glycopyrronium bromide versus placebo and comparable with tiotropium . at these time points , the treatment difference , compared with placebo , in favor of glycopyrronium bromide , was 134 ml and 108 ml , respectively ( p < 0.001 ) , while the difference in favor of tiotropium was 84 ml and 89 ml , respectively ( p < 0.001 ) . on day 1 , the least squares mean fev1 treatment difference for glycopyrronium bromide / placebo and tiotropium bromide / placebo was 87 ml and 45 ml at 5 minutes , respectively , and 143 ml and 78 ml at 15 minutes , respectively ( p < 0.001 ) . the latter finding further supports the notion that glycopyrronium bromide has a faster onset of action than other inhaled antimuscarinic agents , including tiotropium bromide . similar to the studies of indacaterol and tiotropium by odonnell et al20,21 and a study of aclidinium bromide by maltais et al,22 a study by durzu et the inspiratory capacity was significantly higher in patients receiving glycopyrronium bromide than in those receiving placebo at all time points on day 1 and at weeks 12 and 26 ( all p < 0.001 ) . the glycopyrronium bromide / placebo difference for inspiratory capacity by the end of day 1 , week 12 , and week 26 was 104 ml , 97 ml , and 113 ml , respectively ( all p < 0.001 ) . these findings suggest that glycopyrronium bromide , like other long - acting bronchodilators , has a beneficial effect on dynamic hyperinflation in patients with copd . in line with this , the glow2 trial19 also revealed a significantly higher inspiratory capacity in the glycopyrronium bromide group than in the placebo group , but comparable with that in the tiotropium group . in the glow3 trial , beeh et al23 evaluated the effect of once - daily glycopyrronium bromide on exercise tolerance in patients with moderate to severe copd ( mean post - bronchodilator fev1 57% predicted ) . a total of 108 patients were randomized to glycopyrronium bromide 50 g once daily followed by placebo or vice versa for 3 weeks , with a 14-day washout period . the primary outcome measure was the effect of glycopyrronium bromide versus placebo on exercise tolerance after 3 weeks of treatment . exercise tolerance was measured by exercise endurance time during a submaximal constant - load cycle ergometry test . secondary outcome measures included inspiratory capacity at isotime ( refers to the last time point in the ergometry test at which each patient had a valid test result for both treatment periods ) and inspiratory capacity at rest . treatment with glycopyrronium bromide was significantly superior to placebo with respect to exercise endurance time at 3 weeks , and the least squares mean difference between treatment groups was 89 seconds ( p < 0.001 ) . at the same time point , glycopyrronium bromide also produced statistically significant treatment differences in inspiratory capacity at isotime ( p < 0.001 ) and inspiratory capacity at rest ( p < 0.05 ) . therefore , the effect of glycopyrronium bromide on exercise tolerance in patients with copd appears to be comparable with that of other long - acting bronchodilators.2022,24 during the 28-day treatment period in the study by vogelmeier et al,15 patients receiving either dose of glycopyrronium bromide ( 100 g or 200 g once daily ) used significantly less rescue medication than patients in the placebo group ( p < 0.05 ) ; the change from baseline in the number of puffs of rescue medication was 0.55 and 0.26 , respectively , in groups treated with glycopyrronium bromide 100 g and 200 g versus + 0.51 in the placebo group . in keeping with this , the glow1 study18 showed that use of rescue medication was significantly lower in patients receiving glycopyrronium bromide than in those on placebo ( p = 0.005 ) , with a between - group difference of 0.46 puffs per day . a reduction in the use of rescue medication was also observed in the glow2 trial by kerwin et al,19 with a between - group treatment difference of 0.37 puffs per day ( p = 0.039 ) and 0.63 puffs per day ( p = 0.003 ) , respectively , in the glycopyrronium bromide and tiotropium groups . in the glow1 study by durzu et al,18 patients receiving glycopyrronium bromide had a significantly greater transition dyspnea index focal score at week 26 ( 1.84 ) compared with patients receiving placebo ( 0.80 ) . the observed treatment difference of 1.04 exceeded the minimal clinically important difference of one point . furthermore , patients treated with glycopyrronium bromide were 1.7-fold more likely to have at least a one - point improvement in transition dyspnea index focal score compared with those on placebo ( p = 0.001 ) . furthermore , patients treated with glycopyrronium bromide had a significantly lower ( ie , better ) st george s respiratory questionnaire score at week 26 ( 39.50 ) than patients treated with placebo ( 42.31 ) . the treatment difference was 2.81 , which was statistically significant , but did not reach the minimal clinically important difference of 4 points . the percentage of patients achieving a 4 point reduction in st george s respiratory questionnaire score was significantly greater with glycopyrronium bromide than with placebo ( 56.8% and 46.3% , respectively , odds ratio 1.58 , p = 0.006 ) . similar findings with regard to transition dyspnea index focal score and st george s respiratory questionnaire score were reported from the glow2 trial,19 although the statistically significant treatment difference in transition dyspnea index focal score did not reach the minimal clinically important difference ( glycopyrronium bromide versus placebo 0.81 and tiotropium versus placebo 0.94 , respectively ) . in the glow1 trial,18 acute exacerbations of copd were in line with those in previously published studies,25 being classified as of moderate severity if they required treatment with systemic corticosteroids or an antibiotic and severe if they also required hospitalization . a previous history of acute exacerbations of copd was not an inclusion criterion ( 79% of the enrolled patients had no history of moderate or severe exacerbation in the year prior to screening ) . during the 26-week study period,18 93 of 532 patients ( 17.5% ) in the glycopyrronium bromide group had at least one moderate or severe copd exacerbation , compared with 63 of 260 patients ( 24.2% ) in the placebo group . glycopyrronium bromide significantly reduced the risk of copd exacerbations in terms of time to first moderate or severe copd exacerbation by 31% compared with placebo ( hazards ratio [ hr ] 0.69 , confidence interval [ ci ] 0.500.95 , p = 0.023 ) . furthermore , a significant reduction was also observed in the risk of a copd exacerbation leading to hospitalization in the glycopyrronium bromide group versus placebo ( hr 0.35 , ci 0.140.86 , p = 0.022 ) . a significant reduction was also observed in the percentage of hospitalizations due to an exacerbation of copd ( 1.7% and 4.2% , respectively , odds ratio 0.34 , p = 0.024 ) with glycopyrronium bromide versus placebo . in the glow2 trial,19 glycopyrronium significantly reduced the risk of exacerbations ( defined as in the glow1 trial ) in terms of time to first moderate or severe exacerbation by 34% compared with placebo ( hr 0.66 , 95% ci 0.520.85 , p = 0.001 ) over 52 , thereby demonstrating results comparable with tiotropium , which provided a 39% risk reduction versus placebo ( hr 0.61 , 95% ci 0.460.81 , p = 0.001 ) . further , a 35% reduction was observed in the rate of moderate to severe copd exacerbations in the glycopyrronium group compared with that in the placebo ( 0.54 versus 0.80 per year , relative risk 0.66 , ci 0.500.87 , p = 0.003 ) . glycopyrronium bromide ( nva237 ) is a synthetic quaternary ammonium compound acting as a competitive antagonist by binding to muscarinic receptors in bronchial smooth muscle , thereby inhibiting acetylcholine - induced bronchoconstriction . glycopyrronium bromide binds to all five subtypes of the muscarinic receptor ( m1m5 ) , of which the m3 receptor located on airway smooth muscle is primarily involved in smooth muscle contraction . blockade of m2 receptors attenuates the feedback inhibition of acetylcholine production , and thereby may reduce the bronchodilating effects of an antimuscarinic agent . furthermore , blocking cardiac m2 receptors may result in an increase in heart rate . therefore , the optimal antimuscarinic agent for use in copd has high affinity for m1 and m3 receptors and low affinity for m2 receptors . like tiotropium bromide , glycopyrronium bromide has higher selectivity for m3 receptors than for m2 receptors , and like both tiotropium bromide and aclidinium bromide , dissociates more slowly from the m3 receptor than it does from the m2 receptor.8,9 therefore , glycopyrronium bromide provides bronchodilatation via m3 blockade long after its less desirable m2 effects , such as an increased heart rate.810 in vitro studies have indicated that glycopyrronium bromide may have a faster onset of action than tiotropium , and this has been confirmed in clinical studies.8,11 glycopyrronium bromide equilibrates four to five times faster with the m3 receptor than does tiotropium bromide and aclidinium bromide.8,11 this more rapid equilibration of glycopyrronium bromide with the m3 receptor is assumed to underlie its faster onset of action compared with that of tiotropium bromide,8 and probably also aclidinium bromide . early preclinical and clinical studies have suggested a very favorable safety profile for inhaled glycopyrronium bromide . sechaud et al10 investigated the pharmacokinetics of glycopyrronium bromide following single and repeated once - daily inhalation in mild to moderate copd patients , and reported that the pharmacokinetics of glycopyrronium bromide were consistent between doses and with limited systemic accumulation at steady state after repeated once - daily inhalation . the overall incidence of adverse events was similar in patients treated with glycopyrronium bromide and placebo , apart from a higher frequency of dry mouth in the groups treated with glycopyrronium bromide . no serious adverse events were reported , and no clinically relevant effects were observed on vital signs , electrocardiography , or physical examinations.10 in a randomized , double - blind , placebo - controlled , parallel - group study , in which 257 patients with moderate to severe copd were assigned to indacaterol plus glycopyrronium bromide , indacaterol , or placebo , van de maele et al12 investigated the change from baseline in 24-hour mean heart rate versus placebo . a total of 255 patients were included in the safety analysis , which showed no clinically significant differences between groups in the mean 24-hour heart rate or qtc interval , suggesting a cardiovascular safety profile of both long - acting bronchodilators similar to that of placebo . the issue of inhaled anticholinergics and risk of major cardiovascular events has been much debated , and a meta - analysis of previously published studies has reported an increased risk of cardiovascular death , myocardial infarction , and stroke in copd patients treated with inhaled anticholinergics,13 but conclusions regarding this point await the findings of ongoing prospective studies . in the clinical studies published so far , cardiac and vascular disorders have been reported at a similar frequency in the glycopyrronium bromide and placebo groups , although , as in other clinical trials of antimuscarinic agents in copd , patients with any of a number of concomitant diseases , including symptomatic prostatic hyperplasia and narrow - angle glaucoma , were not eligible for inclusion . verkindre et al14 published one of the first double - blind , placebo - controlled trials of glycopyrronium bromide in patients with copd . the study was conducted in 83 patients ( 78 patients completed the study ) with stable moderate to severe copd ( mean post - bronchodilator fev1 53% predicted ) . the patients were randomized to glycopyrronium bromide 12.5 , 25 , 50 or 100 g , placebo , or tiotropium bromide 18 g once daily ( open - label active comparator ) for 7 days . the primary endpoint was mean trough ( 2324 hours following dosing ) fev1 on day 7 . mean trough fev1 on days 1 and 7 was significantly higher with all active treatments versus placebo ( p < 0.05 ) . glycopyrronium bromide 50 g and 100 g and tiotropium bromide showed clinically relevant improvements versus placebo on day 1 ( differences 121 , 135 , and 112 ml , respectively , p < 0.0001 ) and day 7 ( differences 131 , 142 , and 127 ml , respectively , p < 0.0001 ) . no clinically significant difference in improvements in fev1 was observed between either dose of glycopyrronium bromide and tiotropium bromide , although a statistically significant difference in level of fev1 in favor of glycopyrronium bromide was reported . therefore , this study showed that glycopyrronium bromide once daily was well tolerated and demonstrated rapid and sustained 24-hour bronchodilatation , the latter being at least comparable with tiotropium bromide . in a randomized , placebo - controlled trial , vogelmeier et al15 studied the bronchodilator efficacy of two doses of glycopyrronium bromide ( 100 g and 200 g ) versus placebo in patients with moderate to severe copd ( mean post - bronchodilator fev1 53% predicted ) . enrolled patients were randomized to treatment with glycopyrronium bromide 100 g ( n = 92 ) , glycopyrronium bromide 200 g ( n = 98 ) , or placebo ( n = 91 ) for 28 days . the primary objective was to evaluate safety , but efficacy measures were included as secondary objectives . mean trough fev1 was significantly higher in patients receiving glycopyrronium bromide compared with those receiving placebo . for glycopyrronium bromide 100 g , the treatment differences from placebo were 131 ml and 161 ml , respectively , on days 1 and 28 ( p < 0.05 ) ; for glycopyrronium bromide 200 g , the treatment differences were 146 ml and 151 ml , respectively , on days 1 and 28 ( p < 0.05 ) . in line with this , peak fev1 , fev1 at all time points up to 24 hours after dosing , and fev1 area under the curve ( auc ) from 5 minutes to 5 hours after dosing were also significantly higher for both doses of glycopyrronium bromide compared with placebo . although this study did not include an active comparator , its reported findings support the bronchodilator efficacy of glycopyrronium bromide in patients with moderate to severe copd . in a small , randomized , double - blind , placebo - controlled , two - period crossover trial , fogarty et al16 studied the bronchodilatory effects of glycopyrronium bromide versus placebo in 33 patients with moderate copd ( mean post - bronchodilator fev1 70% predicted ) . only subjects with at least a 5% increase in fev1 following inhalation of ipratropium bromide were eligible for the study . patients were randomized to receive glycopyrronium bromide 50 g once daily followed by placebo or vice versa for 14 days ; the treatment periods were separated by a washout period of 714 days . the primary efficacy variable was the standardized fev1 auc between 0 and 24 hours after dosing on day 14 . the fev1 auc024 h values on day 14 were significantly higher for glycopyrronium bromide than for placebo ( p < 0.001 ) with a least squares mean difference of 163 ml between glycopyrronium bromide and placebo . the fev1 with glycopyrronium bromide was significantly higher ( p < 0.001 ) than for placebo for all aucs evaluated . on day 14 , significant treatment differences were found in mean fev1 auc05 h ( difference 198 ml , p < 0.001 ) , fev1 auc012 h ( difference 165 ml , p = 0.001 ) , and fev1 auc1224 h ( difference 161 ml , p < 0.001 ) versus placebo . furthermore , all primary analyses for trough fev1 comparisons showed a clinically significant effect ( predefined threshold exceeded , bromide on day 7 ( 91 ml ) . the mean difference in peak fev1 ie , 120 ml ) at the 5% level , except for the treatment least squares mean difference in trough value for glycopyrronium values between glycopyrronium and placebo was 208 ml ( p < 0.001 ) on day 14 and 173 ml ( p = 0.011 ) on day 7 , respectively . therefore , this study supports the notion that glycopyrronium bromide provides significant and sustained 24-hour bronchodilatation in patients with copd , similar to that provided by tiotropium bromide.17 in one of the largest published clinical trials of glycopyrronium bromide ( the glycopyrronium bromide in copd airways clinical study 1 [ glow1 ] ) , durzu et al18 randomized patients with stable moderate to severe copd ( mean post - bronchodilator fev1 54% predicted ) to either glycopyrronium bromide 50 g once daily ( n = 552 ) or placebo ( n = 270 ) for 26 weeks . patients were not eligible for the study if they were participating in a supervised pulmonary rehabilitation program . least squares mean trough fev1 at week 12 was significantly higher in patients receiving glycopyrronium bromide ( 1.41 l ) versus placebo ( 1.30 l ) , with a treatment difference of 108 15 ml ( p < 0.001 ) . significant improvements in trough fev1 were observed at the end of day 1 and sustained through week 26 . fev1 was significantly improved in the glycopyrronium bromide group versus the placebo group throughout a 24-hour period on day 1 , at weeks 12 and 26 , and at all other scheduled visits and time points . in the glow 2 study , kerwin et al19 randomized patients with moderate to severe copd ( mean fev1 56% predicted ) to either glycopyrronium bromide 50 g once daily ( n = 525 ) , open - label tiotropium 18 g once daily ( n = 267 ) , or placebo ( n = 268 ) for 52 weeks . least squares mean trough fev1 at week 12 was significantly higher in patients receiving glycopyrronium bromide and tiotropium compared with that in patients receiving placebo , with a treatment difference of 97 ml ( p < 0.001 ) and 83 ml ( p trough fev1 at weeks 26 and 52 was significantly higher for patients treated with glycopyrronium bromide versus placebo and comparable with tiotropium . at these time points , the treatment difference , compared with placebo , in favor of glycopyrronium bromide , was 134 ml and 108 ml , respectively ( p < 0.001 ) , while the difference in favor of tiotropium was 84 ml and 89 ml , respectively ( p < 0.001 ) . on day 1 , the least squares mean fev1 treatment difference for glycopyrronium bromide / placebo and tiotropium bromide / placebo was 87 ml and 45 ml at 5 minutes , respectively , and 143 ml and 78 ml at 15 minutes , respectively ( p < 0.001 ) . the latter finding further supports the notion that glycopyrronium bromide has a faster onset of action than other inhaled antimuscarinic agents , including tiotropium bromide . similar to the studies of indacaterol and tiotropium by odonnell et al20,21 and a study of aclidinium bromide by maltais et al,22 a study by durzu et al18 revealed a significant treatment difference in inspiratory capacity . the inspiratory capacity was significantly higher in patients receiving glycopyrronium bromide than in those receiving placebo at all time points on day 1 and at weeks 12 and 26 ( all p < 0.001 ) . the glycopyrronium bromide / placebo difference for inspiratory capacity by the end of day 1 , week 12 , and week 26 was 104 ml , 97 ml , and 113 ml , respectively ( all p < 0.001 ) . these findings suggest that glycopyrronium bromide , like other long - acting bronchodilators , has a beneficial effect on dynamic hyperinflation in patients with copd . in line with this , the glow2 trial19 also revealed a significantly higher inspiratory capacity in the glycopyrronium bromide group than in the placebo group , but comparable with that in the tiotropium group . in the glow3 trial , beeh et al23 evaluated the effect of once - daily glycopyrronium bromide on exercise tolerance in patients with moderate to severe copd ( mean post - bronchodilator fev1 57% predicted ) . a total of 108 patients were randomized to glycopyrronium bromide 50 g once daily followed by placebo or vice versa for 3 weeks , with a 14-day washout period . the primary outcome measure was the effect of glycopyrronium bromide versus placebo on exercise tolerance after 3 weeks of treatment . exercise tolerance was measured by exercise endurance time during a submaximal constant - load cycle ergometry test . secondary outcome measures included inspiratory capacity at isotime ( refers to the last time point in the ergometry test at which each patient had a valid test result for both treatment periods ) and inspiratory capacity at rest . treatment with glycopyrronium bromide was significantly superior to placebo with respect to exercise endurance time at 3 weeks , and the least squares mean difference between treatment groups was 89 seconds ( p < 0.001 ) . at the same time point , glycopyrronium bromide also produced statistically significant treatment differences in inspiratory capacity at isotime ( p < 0.001 ) and inspiratory capacity at rest ( p < 0.05 ) . therefore , the effect of glycopyrronium bromide on exercise tolerance in patients with copd appears to be comparable with that of other long - acting bronchodilators.2022,24 during the 28-day treatment period in the study by vogelmeier et al,15 patients receiving either dose of glycopyrronium bromide ( 100 g or 200 g once daily ) used significantly less rescue medication than patients in the placebo group ( p < 0.05 ) ; the change from baseline in the number of puffs of rescue medication was 0.55 and 0.26 , respectively , in groups treated with glycopyrronium bromide 100 g and 200 g versus + 0.51 in the placebo group . in keeping with this , the glow1 study18 showed that use of rescue medication was significantly lower in patients receiving glycopyrronium bromide than in those on placebo ( p = 0.005 ) , with a between - group difference of 0.46 puffs per day . a reduction in the use of rescue medication was also observed in the glow2 trial by kerwin et al,19 with a between - group treatment difference of 0.37 puffs per day ( p = 0.039 ) and 0.63 puffs per day ( p = 0.003 ) , respectively , in the glycopyrronium bromide and tiotropium groups . in the glow1 study by durzu et al,18 patients receiving glycopyrronium bromide had a significantly greater transition dyspnea index focal score at week 26 ( 1.84 ) compared with patients receiving placebo ( 0.80 ) . the observed treatment difference of 1.04 exceeded the minimal clinically important difference of one point . furthermore , patients treated with glycopyrronium bromide were 1.7-fold more likely to have at least a one - point improvement in transition dyspnea index focal score compared with those on placebo ( p = 0.001 ) . furthermore , patients treated with glycopyrronium bromide had a significantly lower ( ie , better ) st george s respiratory questionnaire score at week 26 ( 39.50 ) than patients treated with placebo ( 42.31 ) . the treatment difference was 2.81 , which was statistically significant , but did not reach the minimal clinically important difference of 4 points . the percentage of patients achieving a 4 point reduction in st george s respiratory questionnaire score was significantly greater with glycopyrronium bromide than with placebo ( 56.8% and 46.3% , respectively , odds ratio 1.58 , p = 0.006 ) . similar findings with regard to transition dyspnea index focal score and st george s respiratory questionnaire score were reported from the glow2 trial,19 although the statistically significant treatment difference in transition dyspnea index focal score did not reach the minimal clinically important difference ( glycopyrronium bromide versus placebo 0.81 and tiotropium versus placebo 0.94 , respectively ) . in the glow1 trial,18 acute exacerbations of copd were in line with those in previously published studies,25 being classified as of moderate severity if they required treatment with systemic corticosteroids or an antibiotic and severe if they also required hospitalization . a previous history of acute exacerbations of copd was not an inclusion criterion ( 79% of the enrolled patients had no history of moderate or severe exacerbation in the year prior to screening ) . time to first moderate or severe copd exacerbation was a secondary outcome measure . during the 26-week study period,18 93 of 532 patients ( 17.5% ) in the glycopyrronium bromide group had at least one moderate or severe copd exacerbation , compared with 63 of 260 patients ( 24.2% ) in the placebo group . glycopyrronium bromide significantly reduced the risk of copd exacerbations in terms of time to first moderate or severe copd exacerbation by 31% compared with placebo ( hazards ratio [ hr ] 0.69 , confidence interval [ ci ] 0.500.95 , p = 0.023 ) . furthermore , a significant reduction was also observed in the risk of a copd exacerbation leading to hospitalization in the glycopyrronium bromide group versus placebo ( hr 0.35 , ci 0.140.86 , p = 0.022 ) . a significant reduction was also observed in the percentage of hospitalizations due to an exacerbation of copd ( 1.7% and 4.2% , respectively , odds ratio 0.34 , p = 0.024 ) with glycopyrronium bromide versus placebo . in the glow2 trial,19 glycopyrronium significantly reduced the risk of exacerbations ( defined as in the glow1 trial ) in terms of time to first moderate or severe exacerbation by 34% compared with placebo ( hr 0.66 , 95% ci 0.520.85 , p = 0.001 ) over 52 , thereby demonstrating results comparable with tiotropium , which provided a 39% risk reduction versus placebo ( hr 0.61 , 95% ci 0.460.81 , p = 0.001 ) . further , a 35% reduction was observed in the rate of moderate to severe copd exacerbations in the glycopyrronium group compared with that in the placebo ( 0.54 versus 0.80 per year , relative risk 0.66 , ci 0.500.87 , p = 0.003 ) . verkindre et al14 published one of the first double - blind , placebo - controlled trials of glycopyrronium bromide in patients with copd . the study was conducted in 83 patients ( 78 patients completed the study ) with stable moderate to severe copd ( mean post - bronchodilator fev1 53% predicted ) . the patients were randomized to glycopyrronium bromide 12.5 , 25 , 50 or 100 g , placebo , or tiotropium bromide 18 g once daily ( open - label active comparator ) for 7 days . the primary endpoint was mean trough ( 2324 hours following dosing ) fev1 on day 7 . mean trough fev1 on days 1 and 7 was significantly higher with all active treatments versus placebo ( p < 0.05 ) . glycopyrronium bromide 50 g and 100 g and tiotropium bromide showed clinically relevant improvements versus placebo on day 1 ( differences 121 , 135 , and 112 ml , respectively , p < 0.0001 ) and day 7 ( differences 131 , 142 , and 127 ml , respectively , p < 0.0001 ) . no clinically significant difference in improvements in fev1 was observed between either dose of glycopyrronium bromide and tiotropium bromide , although a statistically significant difference in level of fev1 in favor of glycopyrronium bromide was reported . therefore , this study showed that glycopyrronium bromide once daily was well tolerated and demonstrated rapid and sustained 24-hour bronchodilatation , the latter being at least comparable with tiotropium bromide . in a randomized , placebo - controlled trial , vogelmeier et al15 studied the bronchodilator efficacy of two doses of glycopyrronium bromide ( 100 g and 200 g ) versus placebo in patients with moderate to severe copd ( mean post - bronchodilator fev1 53% predicted ) . enrolled patients were randomized to treatment with glycopyrronium bromide 100 g ( n = 92 ) , glycopyrronium bromide 200 g ( n = 98 ) , or placebo ( n = 91 ) for 28 days . the primary objective was to evaluate safety , but efficacy measures were included as secondary objectives . mean trough fev1 was significantly higher in patients receiving glycopyrronium bromide compared with those receiving placebo . for glycopyrronium bromide 100 g , the treatment differences from placebo were 131 ml and 161 ml , respectively , on days 1 and 28 ( p < 0.05 ) ; for glycopyrronium bromide 200 g , the treatment differences were 146 ml and 151 ml , respectively , on days 1 and 28 ( p < 0.05 ) . in line with this , peak fev1 , fev1 at all time points up to 24 hours after dosing , and fev1 area under the curve ( auc ) from 5 minutes to 5 hours after dosing were also significantly higher for both doses of glycopyrronium bromide compared with placebo . although this study did not include an active comparator , its reported findings support the bronchodilator efficacy of glycopyrronium bromide in patients with moderate to severe copd . in a small , randomized , double - blind , placebo - controlled , two - period crossover trial , fogarty et al16 studied the bronchodilatory effects of glycopyrronium bromide versus placebo in 33 patients with moderate copd ( mean post - bronchodilator fev1 70% predicted ) . only subjects with at least a 5% increase in fev1 following inhalation of ipratropium bromide were eligible for the study . patients were randomized to receive glycopyrronium bromide 50 g once daily followed by placebo or vice versa for 14 days ; the treatment periods were separated by a washout period of 714 days . the primary efficacy variable was the standardized fev1 auc between 0 and 24 hours after dosing on day 14 . the fev1 auc024 h values on day 14 were significantly higher for glycopyrronium bromide than for placebo ( p < 0.001 ) with a least squares mean difference of 163 ml between glycopyrronium bromide and placebo . the fev1 with glycopyrronium bromide was significantly higher ( p < 0.001 ) than for placebo for all aucs evaluated . on day 14 , significant treatment differences were found in mean fev1 auc05 h ( difference 198 ml , p < 0.001 ) , fev1 auc012 h ( difference 165 ml , p = 0.001 ) , and fev1 auc1224 h ( difference 161 ml , p < 0.001 ) versus placebo . furthermore , all primary analyses for trough fev1 comparisons showed a clinically significant effect ( predefined threshold exceeded , bromide on day 7 ( 91 ml ) . the mean difference in peak fev1 ie , 120 ml ) at the 5% level , except for the treatment least squares mean difference in trough value for glycopyrronium values between glycopyrronium and placebo was 208 ml ( p < 0.001 ) on day 14 and 173 ml ( p = 0.011 ) on day 7 , respectively . therefore , this study supports the notion that glycopyrronium bromide provides significant and sustained 24-hour bronchodilatation in patients with copd , similar to that provided by tiotropium bromide.17 in one of the largest published clinical trials of glycopyrronium bromide ( the glycopyrronium bromide in copd airways clinical study 1 [ glow1 ] ) , durzu et al18 randomized patients with stable moderate to severe copd ( mean post - bronchodilator fev1 54% predicted ) to either glycopyrronium bromide 50 g once daily ( n = 552 ) or placebo ( n = 270 ) for 26 weeks . patients were not eligible for the study if they were participating in a supervised pulmonary rehabilitation program . least squares mean trough fev1 at week 12 was significantly higher in patients receiving glycopyrronium bromide ( 1.41 l ) versus placebo ( 1.30 l ) , with a treatment difference of 108 15 ml ( p < 0.001 ) . significant improvements in trough fev1 were observed at the end of day 1 and sustained through week 26 . fev1 was significantly improved in the glycopyrronium bromide group versus the placebo group throughout a 24-hour period on day 1 , at weeks 12 and 26 , and at all other scheduled visits and time points . in the glow 2 study , kerwin et al19 randomized patients with moderate to severe copd ( mean fev1 56% predicted ) to either glycopyrronium bromide 50 g once daily ( n = 525 ) , open - label tiotropium 18 g once daily ( n = 267 ) , or placebo ( n = 268 ) for 52 weeks . least squares mean trough fev1 at week 12 was significantly higher in patients receiving glycopyrronium bromide and tiotropium compared with that in patients receiving placebo , with a treatment difference of 97 ml ( p < 0.001 ) and 83 ml ( p trough fev1 at weeks 26 and 52 was significantly higher for patients treated with glycopyrronium bromide versus placebo and comparable with tiotropium . at these time points , the treatment difference , compared with placebo , in favor of glycopyrronium bromide , was 134 ml and 108 ml , respectively ( p < 0.001 ) , while the difference in favor of tiotropium was 84 ml and 89 ml , respectively ( p < 0.001 ) . on day 1 , the least squares mean fev1 treatment difference for glycopyrronium bromide / placebo and tiotropium bromide / placebo was 87 ml and 45 ml at 5 minutes , respectively , and 143 ml and 78 ml at 15 minutes , respectively ( p < 0.001 ) . the latter finding further supports the notion that glycopyrronium bromide has a faster onset of action than other inhaled antimuscarinic agents , including tiotropium bromide . similar to the studies of indacaterol and tiotropium by odonnell et al20,21 and a study of aclidinium bromide by maltais et al,22 a study by durzu et al18 revealed a significant treatment difference in inspiratory capacity . the inspiratory capacity was significantly higher in patients receiving glycopyrronium bromide than in those receiving placebo at all time points on day 1 and at weeks 12 and 26 ( all p < 0.001 ) . the glycopyrronium bromide / placebo difference for inspiratory capacity by the end of day 1 , week 12 , and week 26 was 104 ml , 97 ml , and 113 ml , respectively ( all p < 0.001 ) . these findings suggest that glycopyrronium bromide , like other long - acting bronchodilators , has a beneficial effect on dynamic hyperinflation in patients with copd . in line with this , the glow2 trial19 also revealed a significantly higher inspiratory capacity in the glycopyrronium bromide group than in the placebo group , but comparable with that in the tiotropium group . in the glow3 trial , beeh et al23 evaluated the effect of once - daily glycopyrronium bromide on exercise tolerance in patients with moderate to severe copd ( mean post - bronchodilator fev1 57% predicted ) . a total of 108 patients were randomized to glycopyrronium bromide 50 g once daily followed by placebo or vice versa for 3 weeks , with a 14-day washout period . the primary outcome measure was the effect of glycopyrronium bromide versus placebo on exercise tolerance after 3 weeks of treatment . exercise tolerance was measured by exercise endurance time during a submaximal constant - load cycle ergometry test . secondary outcome measures included inspiratory capacity at isotime ( refers to the last time point in the ergometry test at which each patient had a valid test result for both treatment periods ) and inspiratory capacity at rest . treatment with glycopyrronium bromide was significantly superior to placebo with respect to exercise endurance time at 3 weeks , and the least squares mean difference between treatment groups was 89 seconds ( p < 0.001 ) . at the same time point , glycopyrronium bromide also produced statistically significant treatment differences in inspiratory capacity at isotime ( p < 0.001 ) and inspiratory capacity at rest ( p < 0.05 ) . therefore , the effect of glycopyrronium bromide on exercise tolerance in patients with copd appears to be comparable with that of other long - acting bronchodilators.2022,24 during the 28-day treatment period in the study by vogelmeier et al,15 patients receiving either dose of glycopyrronium bromide ( 100 g or 200 g once daily ) used significantly less rescue medication than patients in the placebo group ( p < 0.05 ) ; the change from baseline in the number of puffs of rescue medication was 0.55 and 0.26 , respectively , in groups treated with glycopyrronium bromide 100 g and 200 g versus + 0.51 in the placebo group . in keeping with this , the glow1 study18 showed that use of rescue medication was significantly lower in patients receiving glycopyrronium bromide than in those on placebo ( p = 0.005 ) , with a between - group difference of 0.46 puffs per day . a reduction in the use of rescue medication was also observed in the glow2 trial by kerwin et al,19 with a between - group treatment difference of 0.37 puffs per day ( p = 0.039 ) and 0.63 puffs per day ( p = 0.003 ) , respectively , in the glycopyrronium bromide and tiotropium groups . in the glow1 study by durzu et al,18 patients receiving glycopyrronium bromide had a significantly greater transition dyspnea index focal score at week 26 ( 1.84 ) compared with patients receiving placebo ( 0.80 ) . the observed treatment difference of 1.04 exceeded the minimal clinically important difference of one point . furthermore , patients treated with glycopyrronium bromide were 1.7-fold more likely to have at least a one - point improvement in transition dyspnea index focal score compared with those on placebo ( p = 0.001 ) . furthermore , patients treated with glycopyrronium bromide had a significantly lower ( ie , better ) st george s respiratory questionnaire score at week 26 ( 39.50 ) than patients treated with placebo ( 42.31 ) . the treatment difference was 2.81 , which was statistically significant , but did not reach the minimal clinically important difference of 4 points . the percentage of patients achieving a 4 point reduction in st george s respiratory questionnaire score was significantly greater with glycopyrronium bromide than with placebo ( 56.8% and 46.3% , respectively , odds ratio 1.58 , p = 0.006 ) . similar findings with regard to transition dyspnea index focal score and st george s respiratory questionnaire score were reported from the glow2 trial,19 although the statistically significant treatment difference in transition dyspnea index focal score did not reach the minimal clinically important difference ( glycopyrronium bromide versus placebo 0.81 and tiotropium versus placebo 0.94 , respectively ) . in the glow1 trial,18 acute exacerbations of copd were in line with those in previously published studies,25 being classified as of moderate severity if they required treatment with systemic corticosteroids or an antibiotic and severe if they also required hospitalization . a previous history of acute exacerbations of copd was not an inclusion criterion ( 79% of the enrolled patients had no history of moderate or severe exacerbation in the year prior to screening ) . time to first moderate or severe copd exacerbation was a secondary outcome measure . during the 26-week study period,18 93 of 532 patients ( 17.5% ) in the glycopyrronium bromide group had at least one moderate or severe copd exacerbation , compared with 63 of 260 patients ( 24.2% ) in the placebo group . glycopyrronium bromide significantly reduced the risk of copd exacerbations in terms of time to first moderate or severe copd exacerbation by 31% compared with placebo ( hazards ratio [ hr ] 0.69 , confidence interval [ ci ] 0.500.95 , p = 0.023 ) . furthermore , a significant reduction was also observed in the risk of a copd exacerbation leading to hospitalization in the glycopyrronium bromide group versus placebo ( hr 0.35 , ci 0.140.86 , p = 0.022 ) . a significant reduction was also observed in the percentage of hospitalizations due to an exacerbation of copd ( 1.7% and 4.2% , respectively , odds ratio 0.34 , p = 0.024 ) with glycopyrronium bromide versus placebo . in the glow2 trial,19 glycopyrronium significantly reduced the risk of exacerbations ( defined as in the glow1 trial ) in terms of time to first moderate or severe exacerbation by 34% compared with placebo ( hr 0.66 , 95% ci 0.520.85 , p = 0.001 ) over 52 , thereby demonstrating results comparable with tiotropium , which provided a 39% risk reduction versus placebo ( hr 0.61 , 95% ci 0.460.81 , p = 0.001 ) . further , a 35% reduction was observed in the rate of moderate to severe copd exacerbations in the glycopyrronium group compared with that in the placebo ( 0.54 versus 0.80 per year , relative risk 0.66 , ci 0.500.87 , p = 0.003 ) . based on the available evidence , glycopyrronium bromide ( nva237 ) is a safe and well tolerated long - acting anticholinergic bronchodilator with a fast onset of action . in patients suffering from copd , glycopyrronium bromide has clinically important effects on fev1 , use of relief medication , daytime dyspnea scores , and probably also on health status . furthermore , treatment with glycopyrronium bromide improves exercise tolerance in patients with moderate to severe copd , as assessed by exercise endurance time and inspiratory capacity , suggesting a beneficial effect on dynamic hyperinflation , comparable with that of other long - acting bronchodilators , including tiotropium bromide , indacaterol , and aclidinium bromide . glycopyrronium bromide has been shown to reduce the rate of exacerbations in patients with moderate to severe copd , but long - term controlled trials with exacerbation rate as the primary outcome variable have not been published yet . in conclusion , glycopyrronium bromide may have a potentially significant role in the future management of moderate to severe copd .

background : long - acting bronchodilators are central in the pharmacological management of patients with chronic obstructive pulmonary disease ( copd ) . the aim of this systematic review is to provide an overview of the studies evaluating the safety and clinical efficacy of inhaled glycopyrronium bromide , a novel long - acting muscarinic antagonist , in patients with copd.methods:this study was performed as a systematic literature review.results:inhaled glycopyrronium bromide seems to be a safe and well tolerated long - acting muscarinic antagonist with a fast onset of action . in patients suffering from moderate to severe copd , glycopyrronium bromide has clinically important effects on level of forced expiratory volume in one second , use of relief medication , percentage of days with no use of rescue medication , daytime dyspnea scores , and probably also on health status . furthermore , in this group of patients , glycopyrronium bromide has beneficial effects on dynamic hyperinflation and exercise tolerance . glycopyrronium bromide has been shown to reduce the rate of exacerbations in patients with moderate to severe copd , but long - term controlled trials with exacerbation rate as the primary outcome variable have not been published yet.conclusion:once-daily inhaled glycopyrronium bromide has characteristics important for use in copd , including a fast onset of action , sustained 24-hour bronchodilatation , and improvement in exercise tolerance , and therefore appears to have the potential for a significant role in the future management of copd .

Introduction Materials and methods Results Activity profile and safety of glycopyrronium bromide Clinical studies of glycopyrronium bromide FEV Hyperinflation and exercise capacity Health status, symptom relief, and use of rescue medication Exacerbations Conclusion

long - acting anticholinergic agents may be the most effective class of bronchodilators for copd,1,2,6,7 and although tiotropium bromide is the only long - acting anticholinergic bronchodilator marketed for copd , several others are in various stages of development , including glycopyrronium bromide . this short review provides an overview of the available clinical studies evaluating the safety and efficacy of inhaled glycopyrronium bromide in patients with moderate to severe copd . the pubmed searches were carried out using the following algorithm of mesh terms : glycopyrronium bromide , nva237 , long - acting antimuscarinic agents , indacaterol , salmeterol , formoterol , tiotropium bromide , aclidinium bromide , long - acting bronchodilators , and copd , and the searches were repeated with these terms in combination with forced expiratory volume in one second ( fev1 ) , hyperinflation , exercise capacity , dyspnea , health status , exacerbations , and adverse effects . the citation pool was further supplemented from manual assessment of the reference lists in identified reviews and clinical studies of aspects related to long - acting bronchodilators and copd and from other publications identified as being relevant for further review . like tiotropium bromide , glycopyrronium bromide has higher selectivity for m3 receptors than for m2 receptors , and like both tiotropium bromide and aclidinium bromide , dissociates more slowly from the m3 receptor than it does from the m2 receptor.8,9 therefore , glycopyrronium bromide provides bronchodilatation via m3 blockade long after its less desirable m2 effects , such as an increased heart rate.810 in vitro studies have indicated that glycopyrronium bromide may have a faster onset of action than tiotropium , and this has been confirmed in clinical studies.8,11 glycopyrronium bromide equilibrates four to five times faster with the m3 receptor than does tiotropium bromide and aclidinium bromide.8,11 this more rapid equilibration of glycopyrronium bromide with the m3 receptor is assumed to underlie its faster onset of action compared with that of tiotropium bromide,8 and probably also aclidinium bromide . no serious adverse events were reported , and no clinically relevant effects were observed on vital signs , electrocardiography , or physical examinations.10 in a randomized , double - blind , placebo - controlled , parallel - group study , in which 257 patients with moderate to severe copd were assigned to indacaterol plus glycopyrronium bromide , indacaterol , or placebo , van de maele et al12 investigated the change from baseline in 24-hour mean heart rate versus placebo . in the clinical studies published so far , cardiac and vascular disorders have been reported at a similar frequency in the glycopyrronium bromide and placebo groups , although , as in other clinical trials of antimuscarinic agents in copd , patients with any of a number of concomitant diseases , including symptomatic prostatic hyperplasia and narrow - angle glaucoma , were not eligible for inclusion . verkindre et al14 published one of the first double - blind , placebo - controlled trials of glycopyrronium bromide in patients with copd . therefore , this study showed that glycopyrronium bromide once daily was well tolerated and demonstrated rapid and sustained 24-hour bronchodilatation , the latter being at least comparable with tiotropium bromide . in a randomized , placebo - controlled trial , vogelmeier et al15 studied the bronchodilator efficacy of two doses of glycopyrronium bromide ( 100 g and 200 g ) versus placebo in patients with moderate to severe copd ( mean post - bronchodilator fev1 53% predicted ) . although this study did not include an active comparator , its reported findings support the bronchodilator efficacy of glycopyrronium bromide in patients with moderate to severe copd . therefore , this study supports the notion that glycopyrronium bromide provides significant and sustained 24-hour bronchodilatation in patients with copd , similar to that provided by tiotropium bromide.17 in one of the largest published clinical trials of glycopyrronium bromide ( the glycopyrronium bromide in copd airways clinical study 1 [ glow1 ] ) , durzu et al18 randomized patients with stable moderate to severe copd ( mean post - bronchodilator fev1 54% predicted ) to either glycopyrronium bromide 50 g once daily ( n = 552 ) or placebo ( n = 270 ) for 26 weeks . in the glow 2 study , kerwin et al19 randomized patients with moderate to severe copd ( mean fev1 56% predicted ) to either glycopyrronium bromide 50 g once daily ( n = 525 ) , open - label tiotropium 18 g once daily ( n = 267 ) , or placebo ( n = 268 ) for 52 weeks . these findings suggest that glycopyrronium bromide , like other long - acting bronchodilators , has a beneficial effect on dynamic hyperinflation in patients with copd . in the glow3 trial , beeh et al23 evaluated the effect of once - daily glycopyrronium bromide on exercise tolerance in patients with moderate to severe copd ( mean post - bronchodilator fev1 57% predicted ) . therefore , the effect of glycopyrronium bromide on exercise tolerance in patients with copd appears to be comparable with that of other long - acting bronchodilators.2022,24 during the 28-day treatment period in the study by vogelmeier et al,15 patients receiving either dose of glycopyrronium bromide ( 100 g or 200 g once daily ) used significantly less rescue medication than patients in the placebo group ( p < 0.05 ) ; the change from baseline in the number of puffs of rescue medication was 0.55 and 0.26 , respectively , in groups treated with glycopyrronium bromide 100 g and 200 g versus + 0.51 in the placebo group . in keeping with this , the glow1 study18 showed that use of rescue medication was significantly lower in patients receiving glycopyrronium bromide than in those on placebo ( p = 0.005 ) , with a between - group difference of 0.46 puffs per day . a reduction in the use of rescue medication was also observed in the glow2 trial by kerwin et al,19 with a between - group treatment difference of 0.37 puffs per day ( p = 0.039 ) and 0.63 puffs per day ( p = 0.003 ) , respectively , in the glycopyrronium bromide and tiotropium groups . further , a 35% reduction was observed in the rate of moderate to severe copd exacerbations in the glycopyrronium group compared with that in the placebo ( 0.54 versus 0.80 per year , relative risk 0.66 , ci 0.500.87 , p = 0.003 ) . like tiotropium bromide , glycopyrronium bromide has higher selectivity for m3 receptors than for m2 receptors , and like both tiotropium bromide and aclidinium bromide , dissociates more slowly from the m3 receptor than it does from the m2 receptor.8,9 therefore , glycopyrronium bromide provides bronchodilatation via m3 blockade long after its less desirable m2 effects , such as an increased heart rate.810 in vitro studies have indicated that glycopyrronium bromide may have a faster onset of action than tiotropium , and this has been confirmed in clinical studies.8,11 glycopyrronium bromide equilibrates four to five times faster with the m3 receptor than does tiotropium bromide and aclidinium bromide.8,11 this more rapid equilibration of glycopyrronium bromide with the m3 receptor is assumed to underlie its faster onset of action compared with that of tiotropium bromide,8 and probably also aclidinium bromide . no serious adverse events were reported , and no clinically relevant effects were observed on vital signs , electrocardiography , or physical examinations.10 in a randomized , double - blind , placebo - controlled , parallel - group study , in which 257 patients with moderate to severe copd were assigned to indacaterol plus glycopyrronium bromide , indacaterol , or placebo , van de maele et al12 investigated the change from baseline in 24-hour mean heart rate versus placebo . a total of 255 patients were included in the safety analysis , which showed no clinically significant differences between groups in the mean 24-hour heart rate or qtc interval , suggesting a cardiovascular safety profile of both long - acting bronchodilators similar to that of placebo . in the clinical studies published so far , cardiac and vascular disorders have been reported at a similar frequency in the glycopyrronium bromide and placebo groups , although , as in other clinical trials of antimuscarinic agents in copd , patients with any of a number of concomitant diseases , including symptomatic prostatic hyperplasia and narrow - angle glaucoma , were not eligible for inclusion . verkindre et al14 published one of the first double - blind , placebo - controlled trials of glycopyrronium bromide in patients with copd . therefore , this study showed that glycopyrronium bromide once daily was well tolerated and demonstrated rapid and sustained 24-hour bronchodilatation , the latter being at least comparable with tiotropium bromide . in a randomized , placebo - controlled trial , vogelmeier et al15 studied the bronchodilator efficacy of two doses of glycopyrronium bromide ( 100 g and 200 g ) versus placebo in patients with moderate to severe copd ( mean post - bronchodilator fev1 53% predicted ) . although this study did not include an active comparator , its reported findings support the bronchodilator efficacy of glycopyrronium bromide in patients with moderate to severe copd . therefore , this study supports the notion that glycopyrronium bromide provides significant and sustained 24-hour bronchodilatation in patients with copd , similar to that provided by tiotropium bromide.17 in one of the largest published clinical trials of glycopyrronium bromide ( the glycopyrronium bromide in copd airways clinical study 1 [ glow1 ] ) , durzu et al18 randomized patients with stable moderate to severe copd ( mean post - bronchodilator fev1 54% predicted ) to either glycopyrronium bromide 50 g once daily ( n = 552 ) or placebo ( n = 270 ) for 26 weeks . in the glow 2 study , kerwin et al19 randomized patients with moderate to severe copd ( mean fev1 56% predicted ) to either glycopyrronium bromide 50 g once daily ( n = 525 ) , open - label tiotropium 18 g once daily ( n = 267 ) , or placebo ( n = 268 ) for 52 weeks . these findings suggest that glycopyrronium bromide , like other long - acting bronchodilators , has a beneficial effect on dynamic hyperinflation in patients with copd . in the glow3 trial , beeh et al23 evaluated the effect of once - daily glycopyrronium bromide on exercise tolerance in patients with moderate to severe copd ( mean post - bronchodilator fev1 57% predicted ) . therefore , the effect of glycopyrronium bromide on exercise tolerance in patients with copd appears to be comparable with that of other long - acting bronchodilators.2022,24 during the 28-day treatment period in the study by vogelmeier et al,15 patients receiving either dose of glycopyrronium bromide ( 100 g or 200 g once daily ) used significantly less rescue medication than patients in the placebo group ( p < 0.05 ) ; the change from baseline in the number of puffs of rescue medication was 0.55 and 0.26 , respectively , in groups treated with glycopyrronium bromide 100 g and 200 g versus + 0.51 in the placebo group . in keeping with this , the glow1 study18 showed that use of rescue medication was significantly lower in patients receiving glycopyrronium bromide than in those on placebo ( p = 0.005 ) , with a between - group difference of 0.46 puffs per day . a reduction in the use of rescue medication was also observed in the glow2 trial by kerwin et al,19 with a between - group treatment difference of 0.37 puffs per day ( p = 0.039 ) and 0.63 puffs per day ( p = 0.003 ) , respectively , in the glycopyrronium bromide and tiotropium groups . a significant reduction was also observed in the percentage of hospitalizations due to an exacerbation of copd ( 1.7% and 4.2% , respectively , odds ratio 0.34 , p = 0.024 ) with glycopyrronium bromide versus placebo . further , a 35% reduction was observed in the rate of moderate to severe copd exacerbations in the glycopyrronium group compared with that in the placebo ( 0.54 versus 0.80 per year , relative risk 0.66 , ci 0.500.87 , p = 0.003 ) . verkindre et al14 published one of the first double - blind , placebo - controlled trials of glycopyrronium bromide in patients with copd . in a randomized , placebo - controlled trial , vogelmeier et al15 studied the bronchodilator efficacy of two doses of glycopyrronium bromide ( 100 g and 200 g ) versus placebo in patients with moderate to severe copd ( mean post - bronchodilator fev1 53% predicted ) . although this study did not include an active comparator , its reported findings support the bronchodilator efficacy of glycopyrronium bromide in patients with moderate to severe copd . therefore , this study supports the notion that glycopyrronium bromide provides significant and sustained 24-hour bronchodilatation in patients with copd , similar to that provided by tiotropium bromide.17 in one of the largest published clinical trials of glycopyrronium bromide ( the glycopyrronium bromide in copd airways clinical study 1 [ glow1 ] ) , durzu et al18 randomized patients with stable moderate to severe copd ( mean post - bronchodilator fev1 54% predicted ) to either glycopyrronium bromide 50 g once daily ( n = 552 ) or placebo ( n = 270 ) for 26 weeks . in the glow 2 study , kerwin et al19 randomized patients with moderate to severe copd ( mean fev1 56% predicted ) to either glycopyrronium bromide 50 g once daily ( n = 525 ) , open - label tiotropium 18 g once daily ( n = 267 ) , or placebo ( n = 268 ) for 52 weeks . these findings suggest that glycopyrronium bromide , like other long - acting bronchodilators , has a beneficial effect on dynamic hyperinflation in patients with copd . in the glow3 trial , beeh et al23 evaluated the effect of once - daily glycopyrronium bromide on exercise tolerance in patients with moderate to severe copd ( mean post - bronchodilator fev1 57% predicted ) . therefore , the effect of glycopyrronium bromide on exercise tolerance in patients with copd appears to be comparable with that of other long - acting bronchodilators.2022,24 during the 28-day treatment period in the study by vogelmeier et al,15 patients receiving either dose of glycopyrronium bromide ( 100 g or 200 g once daily ) used significantly less rescue medication than patients in the placebo group ( p < 0.05 ) ; the change from baseline in the number of puffs of rescue medication was 0.55 and 0.26 , respectively , in groups treated with glycopyrronium bromide 100 g and 200 g versus + 0.51 in the placebo group . in keeping with this , the glow1 study18 showed that use of rescue medication was significantly lower in patients receiving glycopyrronium bromide than in those on placebo ( p = 0.005 ) , with a between - group difference of 0.46 puffs per day . a reduction in the use of rescue medication was also observed in the glow2 trial by kerwin et al,19 with a between - group treatment difference of 0.37 puffs per day ( p = 0.039 ) and 0.63 puffs per day ( p = 0.003 ) , respectively , in the glycopyrronium bromide and tiotropium groups . furthermore , a significant reduction was also observed in the risk of a copd exacerbation leading to hospitalization in the glycopyrronium bromide group versus placebo ( hr 0.35 , ci 0.140.86 , p = 0.022 ) . further , a 35% reduction was observed in the rate of moderate to severe copd exacerbations in the glycopyrronium group compared with that in the placebo ( 0.54 versus 0.80 per year , relative risk 0.66 , ci 0.500.87 , p = 0.003 ) . based on the available evidence , glycopyrronium bromide ( nva237 ) is a safe and well tolerated long - acting anticholinergic bronchodilator with a fast onset of action . in patients suffering from copd , glycopyrronium bromide has clinically important effects on fev1 , use of relief medication , daytime dyspnea scores , and probably also on health status . furthermore , treatment with glycopyrronium bromide improves exercise tolerance in patients with moderate to severe copd , as assessed by exercise endurance time and inspiratory capacity , suggesting a beneficial effect on dynamic hyperinflation , comparable with that of other long - acting bronchodilators , including tiotropium bromide , indacaterol , and aclidinium bromide . glycopyrronium bromide has been shown to reduce the rate of exacerbations in patients with moderate to severe copd , but long - term controlled trials with exacerbation rate as the primary outcome variable have not been published yet . in conclusion , glycopyrronium bromide may have a potentially significant role in the future management of moderate to severe copd .

diet composition importantly influences metabolic responses whose dysregulation leads to obesity , diabetes and cancer . but how exactly different diets modulate underlying biological functions remains unclear . in order to appropriately face the dietary change , the organism needs to regulate metabolic flexibility , which is the capacity to continuously adjust use of energy substrates ( primarily glucose and fatty acids ) based on energy supply and demand , and to achieve stability through physiological and behavioral adaptation to the new diet . , a high - fat diet ( hfd ) will not only provide high levels of energy , but will also cause a set of allostatic modifications influencing both energy intake and storage so as to ultimately maintain the newly acquired weight . this is likely one of the reasons why fighting against obesity is a very difficult and challenging task . thus , understanding the biological mechanisms regulating metabolic flexibility might lead to better therapeutic strategies tackling metabolic disorders . hfd consumption plays a significant role in the development of insulin and leptin resistance in the central nervous system ( cns ) . in addition , recent studies have demonstrated that nutrients are not only a source of calories , but also work as intracellular signals capable of modifying the activity of specific molecular cascades . however , little is known about the underlying cns mechanisms that regulate metabolic flexibility in response to dietary changes . endocannabinoid signaling through the cannabinoid type-1 ( cb1 ) receptor plays a key role in energy balance regulation and , importantly , its physiological functions are influenced by the diet and especially by the consumption of fat . within the rodent hypothalamus , cb1 receptor mrna this structure has been long associated with the regulation of food intake and body weight . vmn neurons , particularly those expressing the steroidogenic factor 1 ( sf1 , also named nr5a1 ; ) are critically involved in the regulation of energy balance and exert this function by projecting both locally , within the vmn , as well as to other hypothalamic and extra - hypothalamic sites . in particular , recent studies have shown that sf1-positive neurons project to proopiomelanocortin ( pomc ) neurons in the hypothalamic arcuate nucleus ( arc ) and to a number of autonomic centers , thus regulating sympathetic nervous system ( sns ) outflow . the adipocyte - secreted hormone leptin directly activates sf1-positive neurons in the vmn and this action is required for normal body weight regulation , since deletion of the leptin receptor in sf1 neurons favors diet - induced obesity ( dio ) . additionally , the expression of the phosphatidyl inositol 3-kinase ( pi3k , p110alpha ) , a downstream molecular target of leptin , in sf1-positive neurons is required for the anorectic action of leptin and the appropriate regulation of energy expenditure in response to an hfd . finally , selective inactivation of the suppressor of cytokine signaling 3 ( socs3 ) , a negative regulator of leptin signaling , in sf1 neurons facilitates leptin - induced phosphorylation of signal transducer and activator of transcription 3 ( stat3 ) in this neuronal population , leading to enhanced anorectic and weight - reducing effects of exogenous leptin . intra - vmn administration of the endocannabinoid anandamide stimulates food intake in rats and treatment of hypothalamic slice preparations with a specific cb1 receptor agonist inhibits the electrical activity of sf1-positive neurons . however , the physiological relevance of cb1 receptors in the vmn in the regulation of metabolic homeostasis remains to be established . here we used mice carrying a deletion of the cb1 receptor gene in sf1-positive neurons ( sf1-cb1-ko ) and studied their behavioral and metabolic responses to a normocaloric standard chow and to a 40% hfd , respectively . these studies reveal that cb1 receptors in vmn neurons exert a diet - dependent bidirectional role in regulating energy balance and metabolic responses to leptin , thereby representing a molecular switch for correct metabolic flexibility . all experiments were conducted in strict compliance with the european union recommendations ( 2010/63/eu ) and were approved by the french ministry of agriculture and fisheries ( animal experimentation authorization n 3309004 ) and the local ethical committee of the university of bordeaux ( project authorization n 5012062a ) . conditional mutant mice lacking cb1 receptor gene in sf1-positive cells ( cb1 ) hereafter called sf1-cb1-ko and their sf1-cre negative , wild - type littermates ( cb1 ) , hereafter called sf1-cb1-wt , were generated , maintained and genotyped as previously described in ref . . all mice used in the study were male littermates and they were on a c57bl/6n genetic background ( at least seven backcrossings ) . effective cre - mediated deletion of cb1 in the vmn was assessed by in situ hybridization ( see below ) . 7-week - old male mice were individually housed in a thermo - regulated animal facility ( 22 c 2 c ) , with a 12 h/12 h light / dark cycle ( light on at 1 a.m. , light off at 1 p.m. ) . animals had ad libitum access to water and a standard rodent diet ( standard rodent diet a03 , 3.2 kcal / g , safe , france ) unless otherwise specified . for high - fat diet ( hfd ) studies , 7-weeks old male mice were placed on an hfd with 40% of calories from fat ( 40% hfd , research diets , 4.54 kcal / g , new brunswick , nj ) . feed efficiency was calculated as body weight gained over caloric intake 100 . at the end of the study , mice were sacrificed by cervical dislocation or anesthetized to undergo perfusion , and tissues collected for further molecular and biochemical analysis . assessment of lean and fat mass in 15-weeks old conscious male mice was performed using a nuclear echo magnetic resonance imaging whole - body composition analyzer ( echo mri 900 ; echomedical systems , houston , tx , usa ) . hoppers containing chow were removed from the cages 1 h before the administration of leptin [ depending on studies , 5 mg / kg or 2.5 mg / kg , ip ; mouse recombinant leptin obtained from dr . parlow , national hormone and pituitary program ( torrance , ca ) ] or its vehicle ( phosphate buffer saline , pbs ) , which occurred 4 h before the onset of the dark phase , when food hoppers were returned to the cages , as in ref . . the leptin food intake studies were performed using a within subjects design in which mice received both vehicle and leptin in counterbalanced order . for the gtt , 1617 weeks old male mice underwent an overnight fast and were injected ip with 2 g / kg of d - glucose ( sigma aldrich , st louis , mo ) . for the itt , animals underwent a 6 h fast and were injected ip with 0.5 u / kg of insulin ( humulin , lilly , france ) . blood samples were collected from the tail vein and glucose was measured using glucose sticks ( onetouch , vita , france ) . indirect calorimetry , in - cage locomotor activity and gas exchange analysis were carried out in calorimetric chambers ( tse systems , bad homburg , germany ) after 72 h of acclimatization , as previously described in ref . . vo2 values reported in figures were expressed per animal . unless specified , studies were carried out at 22 c . to evaluate metabolic changes in response to cold ( 15 c ) or to an acute injection of leptin , recordings were respectively carried out in chow - fed male mice exposed for 24 h to 15 c starting at the onset of the dark or in male mice maintained at 22 c and injected with vehicle or leptin ( 2.5 mg / kg ) in the light phase , 4 h before the onset of the dark . for the studies using the [ f]- fluorodeoxyglucose ( fdg ) , chow - fed 16-week - old male sf1-cb1-ko and their wt littermates were analyzed in three different sessions . in the first session , mice were placed at 24 c and then treated ip with a vehicle solution and a reference ct scan with pet images was recorded as in ref . to properly evaluate subsequent tracer absorption in the bat . mice were placed in a cold room ( 6 c ) for 3 h , then slightly anesthetized with sevoflurane , injected with [ f]-fdg ( 15 mbq ) and placed back in the cold room for 1 h to evaluate the tracer 's absorption . five days later , the experiment was repeated in the same animals treated with the 3-adrenergic receptor agonist cl 316,243 and maintained at 24 c . [ f]-fdg accumulation data on pet images are expressed as standard absorption values ( suv ) , representing the radioactivity per gram of tissue , divided by the radioactivity dose injected per gram of animal . qpcr assays on epididymal white adipose tissue ( wat ) , adrenals , testis , pituitary and hypothalamus ( defined caudally by the mammillary bodies , rostrally by the optic chiasm , laterally by the optic tract , and superiorly by the apex of the hypothalamic third ventricle ) were performed with a lightcycler 480 real - time pcr system ( roche diagnostics , meylan , france ) . for qpcr studies done after leptin treatment , chow - fed male mice were sacrificed 30 min after the administration of the hormone ( 2.5 mg / kg , ip ) . primers sequences are reported in supplementary table 1 in the supplementary information . for determination of the reference genes , chow - fed male sf1-cb1-ko and their wt littermates received either leptin ( 2.5 mg / kg , ip ) or its vehicle 4 h before the onset of the dark and 1 h later were anesthetized with 0.1 ml pentobarbital and perfused transcardially with ice - cold pbs and 4% paraformaldehyde ( pfa ) . in a different series of experiments , male sf1-cb1-ko and their wt littermates maintained on 40% hfd for 2 weeks received leptin ( 5 mg / kg , ip ) or its vehicle as described above and 1 h later were anesthetized and perfused transcardially . brains were collected and maintained in a 4% pfa solution for 24 h at 4 c and subsequently transferred to a 30% sucrose solution for one week at 4 c . brains were then cut with a cryostat ( 30 m , cm 1950 , leica ) and sections stored at 20 c in antifreeze solution until further histological processing . sections were washed in pbs ( ph = 7.4 ) for 1 h before being treated with a solution of 1% naoh ( 1 m ) , 1% h2o2 in pbs for 20 min , washed in pbs , and then incubated for 10 min in a 0.3% glycine solution . sections were then placed in a 20% sds solution for 10 min , washed in pbs , and then incubated in 8% goat serum ( dako , trappes , france ) , 0.4% triton x 100 ( sigma aldrich , st louis , mo ) solution for 2 h. sections were incubated overnight at 4 c in a solution containing rabbit serum phospho - stat3 tyr 705 ( 1:500 , catalog # 9131 , cell signaling technologies , beverly , ma ) , 4% goat serum and 0.4% triton x 100 . the following day , sections were washed in a 1% goat serum , 0.02% triton x 100 solution , then incubated in a 4% goat serum solution containing 1:250 biotinylated goat anti - rabbit secondary antibody for 1 h , then washed in a 1% goat serum solution , followed by 1 h incubation in an avidin - biotin complex solution ( vectastain elite abc kit , vector laboratories , burlingame , ca ) . sections were incubated in a diaminobenzidine tetrahydrochloride ( dab ; vectastain peroxidase substrate kit , vector laboratories ) solution . sections were finally washed , dehydrated and mounted on superfrost slides , then visualized at the microscope ( dm5000 , leica ) . all sections containing the vmn were rostro - caudally ( from bregma 1.22 mm to bregma 1.82 mm ) analyzed to examine distribution of phospho - stat3 staining . the number of phospho - stat3 positive cells was counted by an observer blind to the genotype or treatment using a grid reticule ( 520 520 m ) under a 20 microscope objective ( leitz aristoplan , leitz wetzlar , germany ) . proteins from epididymal wat were extracted and quantified and western blots carried out as in ref . . membranes were incubated with the following primary antibodies : 3-adrenergic receptor ( 1:2000 , catalog # 15688 , millipore , molsheim , france ) , adipose triglyceride lipase ( atgl , 1:1000 , catalog # 2439 , cell signaling ) , hormone - sensitive lipase ( hsl , 1:1000 , catalog # 4107 , cell signaling ) , phospho - hsl ser 563 ( 1:1000 , catalog # 4139 , cell signaling ) , phospho - hsl ser 660 ( 1:1000 , catalog # 4126 , cell signaling ) and -actin ( 1:4000 , catalog # 4967 , cell signaling ) . -actin was used as loading control . no statistical methods were used to predetermine sample sizes , but they are similar to those reported previously . in some experiments ( pet analysis , leptin food intake studies and leptin effect on rq ) , the same animals were also their own controls . in experiments with different treatment conditions ( ihc following leptin or vehicle administration ) mice were randomly assigned to the treatment . for multiple group designs , data were analyzed by two - way or repeated measure anova followed by fisher lsd post - hoc test . for designs with only two groups , statistical validity was assessed with unpaired t test or mann whitney u test . fish analysis revealed a specific strong decrease of cb1 receptor mrna expression in the vmn of sf1-cb1-ko mice as compared to sf1-cb1-wt littermates ( figure 1a ) . reduction in cb1 mrna expression was present throughout the vmn , and particularly in the dorsomedial part of the nucleus , where leptin receptors are localized . importantly , mrna expression of the vesicular transporter of glutamate 2 receptor ( vglut2 ) did not differ between genotypes , excluding the possibility of a defective cytoarchitecture of the vmn in sf1-cb1-ko mice ( figure 1a ) . as expected , no appreciable reduction of cb1 receptor mrna was observed in other brain regions ( data not shown ) . sf1 is also present in the gonads , the adrenals and the pituitary , leaving open the possibility that cre - mediated recombination of cb1 might have occurred in these organs . however , cb1 receptor mrna expression was not altered in the testis , the adrenals or the pituitary of sf1-cb1-ko mice when compared with sf1-cb1-wt littermates ( figure 1b ) , suggesting that the expression of the cb1 receptor was preserved in these organs . thus , sf1-cb1-ko mice carry a specific deletion of cb1 receptor in the vmn , thereby representing an ideal tool to study the roles of the endocannabinoid signaling in neurons located in this hypothalamic nucleus . as measured from 7 until 15 weeks of age , cb1 deletion in sf1-positive neurons did not affect body weight in male mice maintained on a normocaloric , standard chow ( figure 2a ) . even at younger age ( 4 weeks old ) , body weight was comparable between genotypes [ sf1-cb1-wt ( n = 8) : 11.6 0.7 g vs. sf1-cb1-ko : 11.2 0.7 g ( n = 14 ) , p = 0.68 ; t(20 ) = 0.4093 ] . however , body composition analysis carried out at 15 weeks of age revealed a significant decrease in fat mass in sf1-cb1-ko mice as compared with their wt littermates ( figure 2b ) , associated with a slight , non - significant increase in lean mass ( figure 2c ) . no differences were found in either cumulative food intake ( figure 2d ) or weekly food intake during weeks 715 of age ( sf1-cb1-wt : 5.2 0.1 g vs. sf1-cb1-ko : 5.4 0.1 g , n = 7 , repeated measures anova , genotype effect : p = 0.65 ; f(1 ) = 0.214 ) . having found a decrease in fat mass in chow - fed sf1-cb1-ko mice , we studied glucose responses to glucose- or insulin - tolerance test ( gtt or itt , respectively ) . while fasting glucose levels were not different between genotypes ( figure 2e , values at time 0 ) , sf1-cb1-ko mice had better glucose tolerance ( figure 2e , f ) and increased insulin sensitivity ( figure 2g , h ) as compared with their wt littermates . thus , in chow , deletion of cb1 receptors in vmn neurons decreases adiposity and improves glucose metabolism . since there were no obvious alterations in food intake that could explain the decreased adiposity of sf1-cb1-ko mice , we assessed whether this phenomenon could be due to changes in locomotor activity or in the use of energy substrates . chow - fed sf1-cb1-ko and wt littermates had similar activity ( figure 3a ) and vo2 consumption ( figure 3b ) at 22 c ambient temperature , but sf1-cb1-ko mice showed decreased respiratory quotient ( rq , figure 3c ) , suggesting a higher consumption rate of lipids as energy source . activation of the sns favors the oxidation of lipids rather than carbohydrates and induces lipolysis in the white adipose tissue ( wat ) . several sns functions are negatively affected by cb1 receptor signaling , whilst excitation of neurons of the vmn , particularly those located in the dorsomedial part of the nucleus , induces sns activity . to assess the impact of vmn cb1 receptors on sns activity under chow , we physiologically and pharmacologically induced sns activity , by exposing sf1-cb1-ko and their sf1-cb1-wt littermates to cold or by administering a 3-adrenergic receptor agonist , respectively . changes in in vivo lipid oxidation and in the metabolic activity of the brown adipose tissue ( bat ) were used as an indirect measure of sns activity . rq differences between chow - fed sf1-cb1-ko and sf1-cb1-wt littermates were maintained when mice were exposed to 15 c ambient temperature for 24 h and became particularly evident during the light phase of the diurnal cycle ( figure 3d ) . additionally , positron emission tomography ( pet ) analysis showed that bat f - fdg accumulation was greater in sf1-cb1-ko than in their sf1-cb1-wt littermates following short - term ( 4 h ) exposure to 6 c or the acute administration of the 3-adrenergic receptor agonist cl 316,223 ( figure 3e g ) . these data thus strongly suggest that vmn cb1 receptors control sns activity . to then determine whether the enhanced sns activity found in sf1-cb1-ko mice was associated with increased lipolysis , we studied the expression of the 3-adrenergic receptor and the activity of lipolytic markers in the epididymal wat . as compared with sf1-cb1-wt littermates , sf1-cb1-ko mice had increased 3-adrenergic receptor protein expression in the wat ( figure 3h , i ) . this was associated with increased phosphorylation of the lipolytic enzyme hormone - sensitive lipase ( hsl ) on ser 660 and 563 ( figure 3h , i ) , two phosphorylation sites induced by the activation of 3-adrenergic receptor in the wat . conversely , protein expression of the adipose triglyceride lipase ( atgl ) , another enzyme participating in lipolysis , was similar between genotypes ( figure 3h , i ) . a close relationship exists between leptin and cb1 receptor signaling in the regulation of energy balance . knowing that sf1-positive neurons critically mediate the actions of leptin on metabolism , we investigated the effects of the hormone in chow - fed sf1-cb1-ko and sf1-cb1-wt littermates . hypothalamic mrna expression of the leptin receptor and of its downstream targets stat3 and socs3 was similar between chow - fed sf1-cb1-wt and sf1-cb1-ko mice ( supplementary figure 1 ) , and both genotypes responded to the dose of 5 mg / kg of leptin with a comparable 24 h decrease in food intake [ sf1-cb1-wt ( n = 9 ) : 25.2 5.9% fi decrease vs. sf1-cb1-ko ( n = 8) : 25.5 2.6% fi decrease , p = 0.96 ; t(15 ) = 0.0474 ] . however , the administration of a lower dose of the hormone ( 2.5 mg / kg ) decreased 24 h food intake in sf1-cb1-ko mice only ( figure 4a ) and led to a strong increase in stat3 phosphorylation in the dorsomedial part of the vmn of sf1-cb1-ko but not sf1-cb1-wt mice ( figure 4b , c ) . whilst no phospho - stat3 labeling was found in the vmn of vehicle - treated mice ( figure 4b , c ) moreover , at metabolic level , leptin at 2.5 mg / kg rapidly increased in vivo lipid oxidation in sf1-cb1-ko but not in sf1-cb1-wt littermates ( figure 4d ) . leptin is known to activate neuronal circuits in the medio - basal hypothalamus leading to increased sns outflow to the wat and to stimulate hsl in this tissue . analysis of the expression of key lipolytic genes in the wat of chow - fed sf1-cb1-ko and sf1-cb1-wt littermates after the administration of leptin at 2.5 mg / kg showed increased mrna expression of the 3-adrenergic receptor , hsl , and atgl in sf1-cb1-ko only ( figure 4e ) . western blot studies confirmed that the acute administration of the hormone significantly increased protein expression of the 3-adrenergic receptor and induced hsl phosphorylation on ser 660 and 563 in the wat of sf1-cb1-ko mice as compared with sf1-cb1-wt littermates ( figure 4f , g ) . overall , these findings imply that cb1 receptors in vmn neurons specifically modulate the sensitivity of the vmn to the action of leptin and determine leptin 's ability to decrease food intake and to stimulate sns activity and lipolysis in the wat . deletion of the cb1 gene , either complete or limited to the principal forebrain neurons and sns , protects from dio . in particular , deletion of the leptin receptor in these neurons favors body weight gain during exposure to an hfd . to evaluate the specific role of cb1 receptors in the vmn in dio , male sf1-cb1-ko and sf1-cb1-wt littermates were maintained for 8 weeks on a mild hfd ( 40% of calories from fat ) . surprisingly , sf1-cb1-ko mice gained significantly more weight than their sf1-cb1-wt controls ( figure 5a ) , resulting in a significant increase in fat mass ( figure 5b ) , while lean mass was similar between genotypes ( figure 5c ) . to determine whether the increased adiposity had a detrimental effect on glucose metabolism , gtt and itt hfd - fed sf1-cb1-ko mice showed an overall worsened glucose tolerance in response to the acute administration of glucose during the gtt ( figure 5d ) , whilst no differences were observed between genotypes in the response to the itt ( figure 5e ) . thus , despite the mild lean phenotype of mutant mice under chow , sf1-cb1-ko mice are paradoxically more prone to the deleterious effects of an hfd . hfd - fed sf1-cb1-ko mice had a significant increase in cumulative food intake as compared with hfd - fed sf1-cb1-wt littermates ( figure 6a ) . this increase was the result of a slight increase in daily food intake , present during both the dark and the light phase of the diurnal cycle ( data not shown ) . we therefore went on to assess the mrna expression levels of several hypothalamic neuropeptides involved in the regulation of food intake and found that only pomc mrna expression was significantly decreased in the hypothalamus of obese sf1-cb1-ko mice ( figure 6b ) . to further determine whether mechanisms other than the observed hyperphagia might have participated to the sf1-cb1-ko obese - prone phenotype , we studied changes in energy expenditure , locomotor activity and use of fuel substrates . no significant differences in feed efficiency or vo2 consumption were observed between obese sf1-cb1-ko mice and sf1-cb1-wt littermates ( figure 6c , d ) . in - cage activity was also similar between genotypes ( figure 6e ) . however , use of substrates was different , with hfd - fed sf1-cb1-ko mice having a significant decrease in lipid oxidation , as revealed by the increased rq ( figure 6f ) , possibly favoring fat accumulation . accordingly , sf1-cb1-ko mice had an almost 50% decrease in the mrna expression of the 3-adrenergic receptor in the wat as compared with control littermates ( figure 6 g ) . subsequent protein analysis confirmed the significant reduction in 3-adrenergic receptor in the wat of hfd - fed sf1-cb1-ko mice only ( figure 6h , i ) , together with a decreased hsl phosphorylation at ser 660 and ser 563 ( figure 6h , i ) . thus , during chronic hfd exposure , lack of cb1 receptors in sf1-positive neurons leads to body weight gain and obesity by inducing hyperphagia and decreased use of lipid substrates , likely through decreased sns outflow to the wat . resistance to the appetite - suppressant action of leptin and decreased hypothalamic leptin signaling are features commonly associated with dio . however , once obesity is established it is difficult to determine whether hypothalamic alterations in leptin signaling are the cause or the consequence of the increased adiposity . to determine whether decreased leptin signaling might have a causal role in the sf1-cb1-ko obese - prone phenotype described during chronic hfd exposure , we investigated leptin - induced stat3 phosphorylation in the vmn of sf1-cb1-wt and sf1-cb1-ko mice maintained on hfd for only 2 weeks before the acute administration of leptin ( 5 mg / kg ) . animals were matched by body weight ( sf1-cb1-wt , 29.1 1 g vs. sf1-cb1-ko , 28.8 2.1 g , n = 4 ; p = 0.90 , t(6 ) = 0.1279 ) and by body composition ( fat mass : sf1-cb1-wt , 5.1 0.5 g vs. sf1-cb1-ko , 5.5 0.8 g ; p = 0.68 , t(6 ) = 0.4267 ; lean mass : sf1-cb1-wt , 23.8 0.7 g vs. sf1-cb1-ko , 23.2 1.2 g , n = 4 ; p = 0.65 , t(6 ) = 0.4636 ) in order to study the action of leptin in the absence of any possible confounding effect due to differences in adiposity . under these conditions , sf1-cb1-ko mice displayed a significantly decreased leptin - induced phospho - stat3 response in the vmn as compared with sf1-cb1-wt littermates ( figure 7a , b ) . thus , exposure to hfd blunts molecular leptin sensitivity in the vmn of sf1-cb1-ko before they overtly gain weight . what we eat critically influences the biological mechanisms regulating energy balance . besides obviously providing different amounts of energy , diets with different nutrient composition affect a plethora of metabolic responses , and specific nutrients can work as signals able to modulate well - identified intracellular pathways however , less is known about how diet - induced metabolic changes are integrated and coordinated at the cns level . the present study demonstrates the critical role of cb1 receptors in the vmn in regulating metabolic flexibility and in determining behavioral and metabolic responses to leptin . during consumption of a standard chow , lack of cb1 in sf1-positive neurons heightens sns outflow and lipolysis , thus decreasing adiposity , while improving glucose tolerance and insulin sensitivity . conversely , during exposure to an hfd , deletion of the cb1 gene from the vmn blunts peripheral use of lipid substrates and causes hyperphagia , body weight gain and glucose intolerance . importantly , the diet - dependent , bidirectional regulation of energy balance observed when cb1 is deleted from vmn neurons is closely intertwined with the effects of leptin . thus , an enhanced sensitivity to the anorectic and metabolic action of the hormone is observed in mutant mice under chow , whereas lack of cb1 in the vmn of animals fed with an hfd for only 2 weeks determines the appearance of molecular leptin resistance in the vmn . when exposed to a diet with a prevalent carbohydrate content , such as the regular chow routinely used in animal studies , sf1-cb1-ko mice did not show changes in body weight or food intake , but had a small , significant decrease in fat mass . this was associated with improved peripheral glucose metabolism and resulted from enhanced sns activity , leading to increased metabolism in the bat , lipolysis in the wat and in vivo lipid oxidation . overall , these findings demonstrate that cb1 receptors in the vmn affect peripheral metabolic responses by modulating vmn - sns outflow . studies in the 80s had initially proposed that vmn neurons exert their metabolic functions on the periphery by modulating the activity of the sns . for instance , vmn lesions reduce sns outflow in a number of tissues , including the wat and the bat . conversely , administration of leptin directly into the vmn produces elevations of plasma epinephrine and norepinephrine . however , although evidence has clearly suggested that excitation of vmn neurons evokes sns outflow , the anatomical and mechanistic bases underlying vmn - mediated autonomic functions have remained for a long time unclear . only very recent studies have finally demonstrated the neuroanatomical connection between the vmn and the sns . in particular , it has been shown that sf1-positive neurons of the dorsomedial subdivision of the vmn project to autonomic centers of both the hypothalamus and hindbrain . taking into account that sf1-positive neurons are largely glutamatergic and that cb1 receptor agonists inhibit the firing of action potentials of sf1-positive neurons , deletion of cb1 on this neuronal population is expected to facilitate sf1-positive neurons activation and to increase sns activity . the relationship between leptin and hypothalamic endocannabinoid signaling was established more than a decade ago , but the functional consequences of this interaction have been only partially addressed . sf1-positive neurons express leptin receptors and are the target of the action of leptin . our data now demonstrate that leptin 's ability to regulate food intake and peripheral lipid metabolism depend upon cb1 receptors expressed on sf1-positive neurons . in particular leptin engages , under the modulatory control of endocannabinoid signaling , the above - mentioned vmn - sns output to exert its metabolic actions . in 2008 , buettner and colleagues demonstrated that direct administration of leptin in the rat medio - basal hypothalamus stimulated wat lipolysis by increasing sns outflow . however , that study did not provide any information about the exact neuronal population or the cellular signaling pathways involved in determining the effects of leptin on the wat . our present findings reveal that such population is the sf1-positive neurons , which regulate sns outflow via endocannabinoid signaling . our data also reveal that lack of cb1 in sf1-positive neurons facilitates molecular leptin sensitivity specifically within the vmn , as assessed by leptin - induced phospho - stat3 expression , the gold standard method used to study hypothalamic leptin signaling . this finding could be explained by taking into account that sf1-positive neurons form a local circuit with extensive axonal fibers and terminals particularly in the dorsomedial part of the vmn . thus , this local circuit is the likely anatomical substrate used by the endocannabinoid signaling to fine - tune vmn responses to leptin . as opposed to what was observed in chow , when sf1-cb1-ko mice were chronically exposed to a mild hfd , they gained more weight and fat mass than their wt littermates . this was due to the presence of hyperphagia , likely due to decreased hypothalamic expression of pomc . this molecular change is of interest , particularly considering that vmn neurons provide excitatory input to pomc neurons in the arc , thus facilitating the activation of this anorexigenic neuronal pathway . moreover , obese sf1-cb1-ko mice showed decreased in vivo lipid oxidation and lipolysis in the wat . these metabolic changes were related to reduced sns outflow , as suggested by the decreased activity of sns - dependent lipolytic markers and 3-adrenergic receptor expression in the wat . interestingly , long - term ( over 10 years ) changes in body weight in humans are inversely correlated with 3-adrenergic receptor induced lipolysis in white adipocytes , so that subjects with low basal 3-adrenergic receptor function gain weight , while those with high 3-adrenergic receptor function are protected from weight gain , implying a critical role for adipocyte 3-adrenergic receptor activity in body weight regulation . leptin receptor signaling , as assessed by stat3 phosphorylation , was impaired in the vmn of sf1-cb1-ko mice consuming an hfd , even before the occurrence of changes in body weight and adiposity , thus suggesting that cb1 receptors on vmn neurons may actually protect from the development of hfd - induced leptin resistance . moreover , in view of the link among central leptin action , modulation of sns outflow and wat lipolysis , the present findings hint that decreased wat 3-adrenergic receptor expression and activity found in obese sf1-cb1-ko mice might be the result of the decreased leptin signaling described in the vmn of hfd - fed sf1-cb1-ko mice before they phenotypically differ from hfd - fed sf1-cb1-wt . the molecular , behavioral and metabolic changes found in sf1-cb1-ko mice on hfd , are likely the result of blunted activity of vmn neurons projecting to autonomic centers regulating the sns , to the arc and to the vmn itself . thus , while in chow endocannabinoid signaling on vmn neurons favors body weight gain , in hfd it protects from dio . the reason for this opposite , diet - dependent effect may reside in the ability of nutrients to directly affect both vmn neurons activity and endocannabinoid signaling . indeed , vmn neurons use both glucose and long - chain fatty acids as signaling molecules to alter their activity , whilst endocannabinoid - mediated neuronal function and plasticity are affected by dietary fat content . lastly , future studies will have to clarify the molecular cascade downstream the cb1 receptor . recent evidence suggests that modulation of the cellular fuel sensor amp - activated protein kinase ( ampk ) directly in the vmn regulates sns outflow and bat function . thus , ampk may be a likely downstream target of cb1 receptors in vmn neurons . taken together , these data demonstrate that cb1 receptors in the vmn determine metabolic and molecular adaptations to different environmental dietary conditions . in order to do so , cb1 receptors in vmn neurons orchestrate peripheral use of substrates and fine - tune sensitivity ( or resistance ) to the actions of leptin .

metabolic flexibility allows rapid adaptation to dietary change , however , little is known about the cns mechanisms regulating this process . neurons in the hypothalamic ventromedial nucleus ( vmn ) participate in energy balance and are the target of the metabolically relevant hormone leptin . cannabinoid type-1 ( cb1 ) receptors are expressed in vmn neurons , but the specific contribution of endocannabinoid signaling in this neuronal population to energy balance regulation is unknown . here we demonstrate that vmn cb1 receptors regulate metabolic flexibility and actions of leptin . in chow - fed mice , conditional deletion of cb1 in vmn neurons ( expressing the steroidogenic factor 1 , sf1 ) decreases adiposity by increasing sympathetic activity and lipolysis , and facilitates metabolic effects of leptin . conversely , under high - fat diet , lack of cb1 in vmn neurons produces leptin resistance , blunts peripheral use of lipid substrates and increases adiposity . thus , cb1 receptors in vmn neurons provide a molecular switch adapting the organism to dietary change .

Introduction Material and methods Results Discussion

in order to appropriately face the dietary change , the organism needs to regulate metabolic flexibility , which is the capacity to continuously adjust use of energy substrates ( primarily glucose and fatty acids ) based on energy supply and demand , and to achieve stability through physiological and behavioral adaptation to the new diet . , a high - fat diet ( hfd ) will not only provide high levels of energy , but will also cause a set of allostatic modifications influencing both energy intake and storage so as to ultimately maintain the newly acquired weight . thus , understanding the biological mechanisms regulating metabolic flexibility might lead to better therapeutic strategies tackling metabolic disorders . however , little is known about the underlying cns mechanisms that regulate metabolic flexibility in response to dietary changes . endocannabinoid signaling through the cannabinoid type-1 ( cb1 ) receptor plays a key role in energy balance regulation and , importantly , its physiological functions are influenced by the diet and especially by the consumption of fat . vmn neurons , particularly those expressing the steroidogenic factor 1 ( sf1 , also named nr5a1 ; ) are critically involved in the regulation of energy balance and exert this function by projecting both locally , within the vmn , as well as to other hypothalamic and extra - hypothalamic sites . in particular , recent studies have shown that sf1-positive neurons project to proopiomelanocortin ( pomc ) neurons in the hypothalamic arcuate nucleus ( arc ) and to a number of autonomic centers , thus regulating sympathetic nervous system ( sns ) outflow . the adipocyte - secreted hormone leptin directly activates sf1-positive neurons in the vmn and this action is required for normal body weight regulation , since deletion of the leptin receptor in sf1 neurons favors diet - induced obesity ( dio ) . additionally , the expression of the phosphatidyl inositol 3-kinase ( pi3k , p110alpha ) , a downstream molecular target of leptin , in sf1-positive neurons is required for the anorectic action of leptin and the appropriate regulation of energy expenditure in response to an hfd . finally , selective inactivation of the suppressor of cytokine signaling 3 ( socs3 ) , a negative regulator of leptin signaling , in sf1 neurons facilitates leptin - induced phosphorylation of signal transducer and activator of transcription 3 ( stat3 ) in this neuronal population , leading to enhanced anorectic and weight - reducing effects of exogenous leptin . however , the physiological relevance of cb1 receptors in the vmn in the regulation of metabolic homeostasis remains to be established . here we used mice carrying a deletion of the cb1 receptor gene in sf1-positive neurons ( sf1-cb1-ko ) and studied their behavioral and metabolic responses to a normocaloric standard chow and to a 40% hfd , respectively . these studies reveal that cb1 receptors in vmn neurons exert a diet - dependent bidirectional role in regulating energy balance and metabolic responses to leptin , thereby representing a molecular switch for correct metabolic flexibility . effective cre - mediated deletion of cb1 in the vmn was assessed by in situ hybridization ( see below ) . for high - fat diet ( hfd ) studies , 7-weeks old male mice were placed on an hfd with 40% of calories from fat ( 40% hfd , research diets , 4.54 kcal / g , new brunswick , nj ) . at the end of the study , mice were sacrificed by cervical dislocation or anesthetized to undergo perfusion , and tissues collected for further molecular and biochemical analysis . to evaluate metabolic changes in response to cold ( 15 c ) or to an acute injection of leptin , recordings were respectively carried out in chow - fed male mice exposed for 24 h to 15 c starting at the onset of the dark or in male mice maintained at 22 c and injected with vehicle or leptin ( 2.5 mg / kg ) in the light phase , 4 h before the onset of the dark . qpcr assays on epididymal white adipose tissue ( wat ) , adrenals , testis , pituitary and hypothalamus ( defined caudally by the mammillary bodies , rostrally by the optic chiasm , laterally by the optic tract , and superiorly by the apex of the hypothalamic third ventricle ) were performed with a lightcycler 480 real - time pcr system ( roche diagnostics , meylan , france ) . for qpcr studies done after leptin treatment , chow - fed male mice were sacrificed 30 min after the administration of the hormone ( 2.5 mg / kg , ip ) . for determination of the reference genes , chow - fed male sf1-cb1-ko and their wt littermates received either leptin ( 2.5 mg / kg , ip ) or its vehicle 4 h before the onset of the dark and 1 h later were anesthetized with 0.1 ml pentobarbital and perfused transcardially with ice - cold pbs and 4% paraformaldehyde ( pfa ) . fish analysis revealed a specific strong decrease of cb1 receptor mrna expression in the vmn of sf1-cb1-ko mice as compared to sf1-cb1-wt littermates ( figure 1a ) . reduction in cb1 mrna expression was present throughout the vmn , and particularly in the dorsomedial part of the nucleus , where leptin receptors are localized . however , cb1 receptor mrna expression was not altered in the testis , the adrenals or the pituitary of sf1-cb1-ko mice when compared with sf1-cb1-wt littermates ( figure 1b ) , suggesting that the expression of the cb1 receptor was preserved in these organs . thus , sf1-cb1-ko mice carry a specific deletion of cb1 receptor in the vmn , thereby representing an ideal tool to study the roles of the endocannabinoid signaling in neurons located in this hypothalamic nucleus . having found a decrease in fat mass in chow - fed sf1-cb1-ko mice , we studied glucose responses to glucose- or insulin - tolerance test ( gtt or itt , respectively ) . thus , in chow , deletion of cb1 receptors in vmn neurons decreases adiposity and improves glucose metabolism . since there were no obvious alterations in food intake that could explain the decreased adiposity of sf1-cb1-ko mice , we assessed whether this phenomenon could be due to changes in locomotor activity or in the use of energy substrates . chow - fed sf1-cb1-ko and wt littermates had similar activity ( figure 3a ) and vo2 consumption ( figure 3b ) at 22 c ambient temperature , but sf1-cb1-ko mice showed decreased respiratory quotient ( rq , figure 3c ) , suggesting a higher consumption rate of lipids as energy source . activation of the sns favors the oxidation of lipids rather than carbohydrates and induces lipolysis in the white adipose tissue ( wat ) . several sns functions are negatively affected by cb1 receptor signaling , whilst excitation of neurons of the vmn , particularly those located in the dorsomedial part of the nucleus , induces sns activity . changes in in vivo lipid oxidation and in the metabolic activity of the brown adipose tissue ( bat ) were used as an indirect measure of sns activity . rq differences between chow - fed sf1-cb1-ko and sf1-cb1-wt littermates were maintained when mice were exposed to 15 c ambient temperature for 24 h and became particularly evident during the light phase of the diurnal cycle ( figure 3d ) . these data thus strongly suggest that vmn cb1 receptors control sns activity . to then determine whether the enhanced sns activity found in sf1-cb1-ko mice was associated with increased lipolysis , we studied the expression of the 3-adrenergic receptor and the activity of lipolytic markers in the epididymal wat . conversely , protein expression of the adipose triglyceride lipase ( atgl ) , another enzyme participating in lipolysis , was similar between genotypes ( figure 3h , i ) . a close relationship exists between leptin and cb1 receptor signaling in the regulation of energy balance . knowing that sf1-positive neurons critically mediate the actions of leptin on metabolism , we investigated the effects of the hormone in chow - fed sf1-cb1-ko and sf1-cb1-wt littermates . hypothalamic mrna expression of the leptin receptor and of its downstream targets stat3 and socs3 was similar between chow - fed sf1-cb1-wt and sf1-cb1-ko mice ( supplementary figure 1 ) , and both genotypes responded to the dose of 5 mg / kg of leptin with a comparable 24 h decrease in food intake [ sf1-cb1-wt ( n = 9 ) : 25.2 5.9% fi decrease vs. sf1-cb1-ko ( n = 8) : 25.5 2.6% fi decrease , p = 0.96 ; t(15 ) = 0.0474 ] . however , the administration of a lower dose of the hormone ( 2.5 mg / kg ) decreased 24 h food intake in sf1-cb1-ko mice only ( figure 4a ) and led to a strong increase in stat3 phosphorylation in the dorsomedial part of the vmn of sf1-cb1-ko but not sf1-cb1-wt mice ( figure 4b , c ) . leptin is known to activate neuronal circuits in the medio - basal hypothalamus leading to increased sns outflow to the wat and to stimulate hsl in this tissue . analysis of the expression of key lipolytic genes in the wat of chow - fed sf1-cb1-ko and sf1-cb1-wt littermates after the administration of leptin at 2.5 mg / kg showed increased mrna expression of the 3-adrenergic receptor , hsl , and atgl in sf1-cb1-ko only ( figure 4e ) . western blot studies confirmed that the acute administration of the hormone significantly increased protein expression of the 3-adrenergic receptor and induced hsl phosphorylation on ser 660 and 563 in the wat of sf1-cb1-ko mice as compared with sf1-cb1-wt littermates ( figure 4f , g ) . overall , these findings imply that cb1 receptors in vmn neurons specifically modulate the sensitivity of the vmn to the action of leptin and determine leptin 's ability to decrease food intake and to stimulate sns activity and lipolysis in the wat . deletion of the cb1 gene , either complete or limited to the principal forebrain neurons and sns , protects from dio . in particular , deletion of the leptin receptor in these neurons favors body weight gain during exposure to an hfd . to evaluate the specific role of cb1 receptors in the vmn in dio , male sf1-cb1-ko and sf1-cb1-wt littermates were maintained for 8 weeks on a mild hfd ( 40% of calories from fat ) . thus , despite the mild lean phenotype of mutant mice under chow , sf1-cb1-ko mice are paradoxically more prone to the deleterious effects of an hfd . to further determine whether mechanisms other than the observed hyperphagia might have participated to the sf1-cb1-ko obese - prone phenotype , we studied changes in energy expenditure , locomotor activity and use of fuel substrates . however , use of substrates was different , with hfd - fed sf1-cb1-ko mice having a significant decrease in lipid oxidation , as revealed by the increased rq ( figure 6f ) , possibly favoring fat accumulation . accordingly , sf1-cb1-ko mice had an almost 50% decrease in the mrna expression of the 3-adrenergic receptor in the wat as compared with control littermates ( figure 6 g ) . thus , during chronic hfd exposure , lack of cb1 receptors in sf1-positive neurons leads to body weight gain and obesity by inducing hyperphagia and decreased use of lipid substrates , likely through decreased sns outflow to the wat . however , once obesity is established it is difficult to determine whether hypothalamic alterations in leptin signaling are the cause or the consequence of the increased adiposity . to determine whether decreased leptin signaling might have a causal role in the sf1-cb1-ko obese - prone phenotype described during chronic hfd exposure , we investigated leptin - induced stat3 phosphorylation in the vmn of sf1-cb1-wt and sf1-cb1-ko mice maintained on hfd for only 2 weeks before the acute administration of leptin ( 5 mg / kg ) . animals were matched by body weight ( sf1-cb1-wt , 29.1 1 g vs. sf1-cb1-ko , 28.8 2.1 g , n = 4 ; p = 0.90 , t(6 ) = 0.1279 ) and by body composition ( fat mass : sf1-cb1-wt , 5.1 0.5 g vs. sf1-cb1-ko , 5.5 0.8 g ; p = 0.68 , t(6 ) = 0.4267 ; lean mass : sf1-cb1-wt , 23.8 0.7 g vs. sf1-cb1-ko , 23.2 1.2 g , n = 4 ; p = 0.65 , t(6 ) = 0.4636 ) in order to study the action of leptin in the absence of any possible confounding effect due to differences in adiposity . thus , exposure to hfd blunts molecular leptin sensitivity in the vmn of sf1-cb1-ko before they overtly gain weight . what we eat critically influences the biological mechanisms regulating energy balance . besides obviously providing different amounts of energy , diets with different nutrient composition affect a plethora of metabolic responses , and specific nutrients can work as signals able to modulate well - identified intracellular pathways however , less is known about how diet - induced metabolic changes are integrated and coordinated at the cns level . the present study demonstrates the critical role of cb1 receptors in the vmn in regulating metabolic flexibility and in determining behavioral and metabolic responses to leptin . during consumption of a standard chow , lack of cb1 in sf1-positive neurons heightens sns outflow and lipolysis , thus decreasing adiposity , while improving glucose tolerance and insulin sensitivity . conversely , during exposure to an hfd , deletion of the cb1 gene from the vmn blunts peripheral use of lipid substrates and causes hyperphagia , body weight gain and glucose intolerance . importantly , the diet - dependent , bidirectional regulation of energy balance observed when cb1 is deleted from vmn neurons is closely intertwined with the effects of leptin . thus , an enhanced sensitivity to the anorectic and metabolic action of the hormone is observed in mutant mice under chow , whereas lack of cb1 in the vmn of animals fed with an hfd for only 2 weeks determines the appearance of molecular leptin resistance in the vmn . overall , these findings demonstrate that cb1 receptors in the vmn affect peripheral metabolic responses by modulating vmn - sns outflow . studies in the 80s had initially proposed that vmn neurons exert their metabolic functions on the periphery by modulating the activity of the sns . however , although evidence has clearly suggested that excitation of vmn neurons evokes sns outflow , the anatomical and mechanistic bases underlying vmn - mediated autonomic functions have remained for a long time unclear . taking into account that sf1-positive neurons are largely glutamatergic and that cb1 receptor agonists inhibit the firing of action potentials of sf1-positive neurons , deletion of cb1 on this neuronal population is expected to facilitate sf1-positive neurons activation and to increase sns activity . the relationship between leptin and hypothalamic endocannabinoid signaling was established more than a decade ago , but the functional consequences of this interaction have been only partially addressed . sf1-positive neurons express leptin receptors and are the target of the action of leptin . in particular leptin engages , under the modulatory control of endocannabinoid signaling , the above - mentioned vmn - sns output to exert its metabolic actions . in 2008 , buettner and colleagues demonstrated that direct administration of leptin in the rat medio - basal hypothalamus stimulated wat lipolysis by increasing sns outflow . however , that study did not provide any information about the exact neuronal population or the cellular signaling pathways involved in determining the effects of leptin on the wat . our present findings reveal that such population is the sf1-positive neurons , which regulate sns outflow via endocannabinoid signaling . our data also reveal that lack of cb1 in sf1-positive neurons facilitates molecular leptin sensitivity specifically within the vmn , as assessed by leptin - induced phospho - stat3 expression , the gold standard method used to study hypothalamic leptin signaling . this molecular change is of interest , particularly considering that vmn neurons provide excitatory input to pomc neurons in the arc , thus facilitating the activation of this anorexigenic neuronal pathway . leptin receptor signaling , as assessed by stat3 phosphorylation , was impaired in the vmn of sf1-cb1-ko mice consuming an hfd , even before the occurrence of changes in body weight and adiposity , thus suggesting that cb1 receptors on vmn neurons may actually protect from the development of hfd - induced leptin resistance . moreover , in view of the link among central leptin action , modulation of sns outflow and wat lipolysis , the present findings hint that decreased wat 3-adrenergic receptor expression and activity found in obese sf1-cb1-ko mice might be the result of the decreased leptin signaling described in the vmn of hfd - fed sf1-cb1-ko mice before they phenotypically differ from hfd - fed sf1-cb1-wt . thus , while in chow endocannabinoid signaling on vmn neurons favors body weight gain , in hfd it protects from dio . the reason for this opposite , diet - dependent effect may reside in the ability of nutrients to directly affect both vmn neurons activity and endocannabinoid signaling . thus , ampk may be a likely downstream target of cb1 receptors in vmn neurons . taken together , these data demonstrate that cb1 receptors in the vmn determine metabolic and molecular adaptations to different environmental dietary conditions . in order to do so , cb1 receptors in vmn neurons orchestrate peripheral use of substrates and fine - tune sensitivity ( or resistance ) to the actions of leptin .

helium nanodroplets ( hen ) have attracted strong interest as superfluid nanocryostats for the efficient preparation of tailored molecules and clusters at ultracold temperatures . the spectroscopy of atoms on / in helium nanodroplets gives insight into the nature of interactions between a nanosized superfluid and single atoms . moreover , the special properties of helium nanodroplets enable the formation of tailored molecules and offer a possibility to study complexes that can not , or only with great expense , be produced in the gas phase or in molecular beam experiments . an example is the preparation of alkali ( ak ) metal dimers and trimers in their weakly bound high - spin configuration ( triplet and quartet states , respectively ) or even high spin clusters at low temperatures ( 0.37 k ) . the lowest electronic transitions ( d1 and d2 lines ) in ak atoms on the surface of helium nanodroplets are well characterized . the d1 and d2 lines of li atoms on the surface of hen are an exception among the ak hen systems because their main absorption peak is lower in energy than the free atom transition . this red - shift has its origin in bound bound transitions from the ground state into the two 2p( ) states . while excitation spectra of rydberg states for na , rb , and cs have been reported , less work has been done on the characterization of k hen and li hen . besides the d1 and d2 lines , only the 5p 4s transition in k hen and the 3s 2s transition in li hen have been investigated . for the assignment of excited states of ak atoms on hen , we use the pseudodiatomic model ( hund s case the symmetry of the atomic system is broken and only the projection of the atomic orbital angular momentum l onto the intermolecular axis ( i.e. , ) , which is defined by the connection between the droplet center and the ak nucleus , remains a conserved quantity . in the case of li , spin orbit ( so ) splitting can be neglected ( e.g. , so splitting for the 2p state : 0.34 cm ) . for two - photon excitations , transitions from the li hen 2s( ) ground state into , , and molecular substates are allowed . the fragility of systems formed by helium nanodroplets with molecules on their surface enhances the formation of weakly bound complexes because the binding energy , which is gained during the formation of the molecule , is released into the droplet . this excess energy could destroy the complex formed by the molecule and the droplet . hence , the weakly bound alkali dimers in the triplet manifold have a higher probability of survival than the singlet molecules , and the helium droplets act as a filter for ak dimers in the triplet manifold . triplet bands of na2 , k2 , rb2 , and cs2 have been studied in detail . for li2 on hen , only the 1g 1u+ transition , which is a bound - free transition , has been reported so far . the orientation of the ground state li dimers for the two different spin configurations ( g+ and u+ state ) with respect to the droplet surface has been investigated theoretically for small hen ( for n up to 30 ) , where it was found that the singlet molecule is perpendicular and the triplet one is oriented parallel to the droplet s surface . in contrast to the li2hen system , triplet bands of free li2 molecules have been studied extensively experimentally as well as theoretically . the most accurate experimental data have been recorded by perturbation facilitated optical optical double resonance ( pfoodr ) spectroscopy . this allows a detailed comparison between molecules on hen and free molecules , which reveals insight into the interaction between the superfluid droplet and the molecule on its surface . in the first part of this article , we report on the investigation of the two - photon 3p 2s and 3d 2s transitions in li hen by resonance enhanced multiphoton ionization time - of - flight ( rempi - tof ) spectroscopy . pseudodiatomic molecular substates are identified on the basis of the observed abundance of exciplexes because the exciplex formation process depends on of the molecular substate . calculations based on a pairwise additive approach support our assignment . in the second part of this article , we report on the investigation of the 2g 1u+ transition of the li dimer on hen . vibronic states could be resolved and the low mass of the li atom in combination with the rempi - tof technique allows to study the band for all three li isotopomers , li2 , li2 , and lili , as well as an investigation of their interaction with the droplet . a detailed description of the experimental setup can be found in refs ( 33 ) and ( 34 ) . high purity helium gas is cooled down to a temperature of t0 = 15 k and expanded through a d0 = 5 m nozzle under a pressure of p0 = 60 bar into vacuum . these conditions lead to a subcritical supersonic jet expansion of the he gas and consequently to the formation of helium droplets , where the droplet size follows a logarithmic normal distribution . the source conditions lead to a distribution maximum ( np0,t0 ) at n60,15 = 6000 he atoms , which corresponds to a droplet radius of r60,15 = 40 . after production , the helium droplet beam is guided into another vacuum chamber where it is doped with li by passing through a resistively heated pickup cell . the process depends on the vapor pressure within the pickup cell , which is controlled by the cell temperature . at the used temperature of t 350 c , li atoms as well as li dimers are present on the droplets . the excitation spectra have been obtained by resonance enhanced multiphoton ionization time - of - flight ( rempi - tof ) spectroscopy . a tunable pulsed dye laser ( lambda physik fl 3002 ) is used to access li hen and li2hen transitions . to cover the spectral range , the li2hen dimer spectrum has been obtained by resonant two - photon ionization , using coumarin 2 ( 0.3 mj ) and stilbene 3 ( 0.5 mj ) . hen complexes have been excited via a two - photon scheme ( without a resonant intermediate state ) with dcm ( 5.2 mj ) and ionized with a fraction ( 0.6 mj ) of the xecl pump laser radiation ( radiant dyes rd - exc-200 xecl laser , 26 ns pulse duration , 100 hz ) . in this section , we discuss the two - photon 3p 2s and 3d 2s transitions in li hen . because of the small size of the li ion core , states with l > 0 lie energetically very close together in the lithium atom ( these states have a core nonpenetrating character ) . only the ns states are significantly shifted with respect to the hydrogenic term energy . note that because of the l < n condition for the orbital angular momentum quantum number l the 3d state investigated here is a special case among the nd li the two - photon excitation spectrum of the 3p 2s and 3d 2s transitions in li the ion yield for li he ( red , 9 , smoothed signal only ) , li he ( green ) , li he2 ( blue ) , and li he3 ( black ) is monitored as a function of the laser wavenumber , which is multiplied by two to account for the absorption of two photons . the vertical lines represent the positions of the free atom 3p and 3d states . the bare li atom signal is not shown because the high photon density also excites the strongly repulsive blue wing of the 2p( ) state ( one - photon transition ) and ionizes desorbed li 2p atoms , causing a large background signal . the li hem ( m > 0 ) spectra are free from this background signal . the resonance with the intermediate 2p( ) state enhances the two - photon transition , similar to the two - photon transition studied for rb hen in ref ( 18 ) . two - photon excitation spectrum of li hen . the spectrum is obtained by monitoring the li he ( red , 9 , smoothed signal only ) , li he ( green ) , li he2 ( blue ) , and li he3 ( black ) ion yield as a function of the excitation laser wavenumber ( multiplied by two ) with a time - of - flight mass spectrometer . the 3p and 3d free atom states at 30 925 cm and 31 283 cm are shown as vertical black lines . 2s transition in ref ( 24 ) only bare lithium ions were detected , the excitation of states with > 0 leads to the formation of exciplexes ( ak*hem ) . hem ion signals show the same structure as has been observed for heavier ak atoms and can be explained within the pseudodiatomic model . in this picture , the 3p state splits into a and component ( neglecting spin orbit effects ) . it is known that upon excitation of the ak hen system into states a large fraction of the atoms desorb without attached helium atoms . in contrast , the ionization via states with > 0 is always accompanied by an abundance of ak hem ( m > 0 ) complexes in the time - of - flight mass spectra . hence , we attribute the pronounced peak at ( 31 016 2 ) cm , full width at half - maximum ( fwhm ) = ( 43 3 ) cm ( as monitored on the li he2 mass ) to the 3p( ) state . we call this approach for the assignment of excited states based on the exciplex formation process the exciplex approach in the following . in contrast , the signal in between the 3p( ) and the 3d line is only visible in the li he ion signal ; hence , we attribute this part of the spectrum to the onset of the 3p( ) state . the structure of the signal , which correlates to the 3d molecular substates , is similar to the signal obtained for the rb the state related to the peak at ( 31 278 5 ) cm , fwhm = ( 52 7 ) cm ( as monitored on the li it is remarkable that this state appears almost unshifted with respect to the free atom transition at 31 283 cm . ground state potential of 10 cm , which has to be taken into account when excitation spectra are compared to free atom transitions , the 3d( ) potential has a binding character and its minimum lies about ( 15 7 ) cm below the asymptotic 3d energy . this demonstrates a weak attractive interaction of the li atom in the 3d( ) state with the droplet . hen 4p 3s and 4d 3s transitions , which compare to the presented li hen transitions . therein , increased exciplex formation upon excitations into states associated with high was observed close by the free na atom lines . in the li he ion signal , the blue shoulder of the 3d( ) peak extends to much higher wavenumbers than in the li he2 detected spectrum , and it is followed by a second peak at ( 31 536 10 ) cm , which we attribute either to a part of the 3d ( ) or ( ) state . to simulate the spectra , we use the orsay trento density functional as implemented in the code by f. dalfovo , with modifications by k. k. lehmann and r. schmied . the interaction between the helium droplet and the alkali atom is introduced by a pairwise additive ( pwa ) approach . to obtain the radial density profile of the ground state , the lithium helium potential of patil is used . for the excited states , the curves of pascale represent the set of li*he interactions of the electronic states relevant for this work . an effective coordinate zli is introduced as the distance between the center of mass of the droplet and the lithium atom . by convoluting the density with the above pair potentials , we obtain the pseudodiatomic potentials shown in figure 2 . we accounted for the correct projection of the angular momentum on the helium lithium axis by means of wigner rotation matrices , following ref ( 13 ) and transforming the hamiltonian in a diagonal shape . orbit coupling can be neglected , as the interaction with the droplet is much larger . in the frozen droplet picture , assuming a fast desorption of the lithium atom upon excitation , franck condon factors ( fcfs ) are calculated with the help of bcont 2.2 by leroy for bound - free transitions of the pseudodiatomic molecule . the majority of the li hen systems are in their vibronic ground state ( = 0 ) before the excitation . calculated pseudodiatomic potential energy curves for a li the li atomic states are used as a label on the right side for large distances zli . depending on the angular momentum the curve splits into ( = 0 ) states ( solid line ) , ( = 1 ) states ( dashed ) , and ( = 2 ) states ( dashed dotted ) . note the different ordinate scales . it can be seen that our approach overestimates the blue shift of the excited states . such large blue shifts on the order of several thousand wavenumbers have already been observed for calculations of excited states of na hen in ref ( 17 ) , where it was shown that the pwa approach works best at states with low principal quantum number n. in addition to the pwa approach , for na hen a perturbative configuration interaction ( pci ) approach was used , where the helium dopant interaction is introduced by a perturbation rather than a pair potential . the pci model shows a better agreement with the experimental spectra than the pwa approach ; unfortunately , this model is not yet parametrized for lithium . although our potential energy curves ( see figure 2 ) for the 2s( ) , 2p(, ) , and 3s( ) reproduce the results of other calculations , the spectrum for the 3p and 3d states is far off the experimental one . however , the calculated theoretical spectrum for the transition from the ground state into the 3s( ) state has a maximum at 27 880 cm , which matches the experimental peak maximum ( 27 820 cm ) very well as can be seen from figure 4 in ref ( 24 ) . the difference between theory and experiment for the 3p and 3d spectra is expected to some extent due to several approximations in the pwa approach : ( i ) the ground state helium density is assumed to be maintained during the excitation ( frozen droplet approximation ) , ( ii ) interactions between 3p and 3d states are not included , but in the case of li the states lie relatively close , and ( iii ) the approach rests on the potentials calculated by pascale . despite the fact that pascale s potentials have been leading to numerous fruitful results , they might be not as accurate as modern calculations . condon factors ( scaled to one ) as obtained with the pwa approach as described in the text . full lines represent the molecular substates arising from the 3d state , and dashed lines represent the 3p substates . time - of - flight mass spectrum as obtained while the laser is scanned across the two - photon transitions , which is shown in figure 1 . peaks in steps of 4 amu are related to li ions with attached helium atoms . in addition to li dimers , which are formed on the droplets , also complexes of li , li2 , and residual water , which is present in the vacuum chamber , are observed in the mass spectrum . the above discussion reveals that a more sophisticated approach is needed to describe higher excited states of li hen accurately . different approaches have been presented recently where the best results for li hen have been obtained with the methods developed by the barranco group . however , the energetic ordering as described above for the 3p( ) , 3p( ) , 3d( ) , 3d( ) , and 3d( ) states is reproduced by our calculations . according to the pwa approach , the 3p( ) state lies energetically lower than the 3p( ) state , and the 3d( ) state is the energetically lowest among the three 3d molecular substates , which is in agreement with our assignment within the exciplex approach . despite the differences between calculation and experiment , we think that the expected energetic ordering of excited states confirms our interpretation within the exciplex approach . for the 3s 2s transition , an isotope shift for the two different li isotopes has been observed . as can be seen from the excitation spectrum in figure 1 , we do not observe a significant isotope effect within the accuracy of our data . figure 4 shows the time - of - flight mass spectrum as obtained , while the laser is scanned across the two - photon transitions ( figure 1 ) . the highest signal is detected at the mass - to - charge ratio m / z corresponding to bare li and li ions . the intensity of the peaks reflects the natural abundance of the two li isotopes ( li , 7.4% ; li , 92.6% ) . these two li mass peaks are followed by peaks originating from li he , li he2 , and li he3 in steps of 4 amu ( he ) . because of the high photon density and the presence of the 308 nm light , li dimers ( li2 , lili , and li2 ) are also observed in the mass spectrum . at higher m / z ratios ( for m / z > 16 the signal is multiplied by a factor of 20 ) , two prominent peaks are observed . these peaks are related to residual water , which was present in our chamber during the experiment . we attribute the peaks at m / z = 24 and m / z = 31 to the products of an oxidative hydrolysis of lioh and li(lioh ) , respectively , which are produced upon a reaction with water in the helium droplet . because of the resonant excitation of an intermediate state , the reaction may be enhanced by the present laser photons . this could explain the absence of a prominent peak corresponding to the weakly bound li h2o ( m / z = 25 ) van der waals complex , which is in contrast to the findings in ref ( 49 ) . we find no evidence for the formation of li clusters ( i.e. , lim>2 ) at our source conditions . this is in agreement with other experiments as discussed in ref ( 5 ) . the absence of these complexes is related to the high formation energies of lithium clusters , which are significantly higher than those of the heavier ak clusters and have been investigated in detail on / in helium nanodroplets . we think that the production of li clusters will require larger helium droplets ( n > 10 ) produced by source conditions with lower temperatures in the supercritical expansion regime . the excitation spectrum of li dimers in the range between 21 250 and 24 000 cm is shown in figure 5a . the observed vibronic transitions are identified as 2g ( ) 1u+ ( = 0 ) . the ion yield for m / z = 12 ( li2 ) , 13 ( lili ) , and 14 ( li2 ) the composition of the molecules on the droplets is 85.71% li2 , 13.71% lili , and 0.55% li2 . because of the low abundance of li2 , the ion signal obtained for li2 is very weak and was smoothed and multiplied by a factor of 10 . the spectrum comprises a series of progressive vibrational bands , which can be followed , in the case of li2 , from = 0 up to = 11 . a large fraction of the molecules fragments into li and li upon ionization , which is observed when monitoring li for the same excitation spectrum ( not shown ) . = 0 ) triplet transitions of li dimers on the surface of helium nanodroplets ( a ) . the spectra of all three isotopomers li2 ( red ) , lili ( green ) , and li2 ( blue ) are shown . condon factors are shown ( in arbitrary units ) as vertical bars and scaled to the = 1 peak ( b ) . the vertical bars in figure 5b represent the transition for j = 0 rotational states starting from = 0 in the 1u+ ground state as calculated with the potentials given in ref ( 32 ) by using the level 8.0 program of leroy . the height of the bars reflects the calculated franck condon ( fc ) distribution . note that the use of a pulsed laser with relatively high pulse energy ( 0.3 mj ) , which is needed to ionize sufficient molecules in a one - color r2pi experiment , is not well suited for the determination of transition probabilities . despite this disadvantage , the trend of the peak intensities reflects the trend in the calculated fc factors . the only exception is the relatively low intensity of the = 0 state when compared to the fc factor , which is most probably caused by saturation effects . hence , we scale the fc factors to the = 1 peak ( the fcf for = 0 is beyond the scale in figure 5b ) . as can be seen in figure 5 , the origin of the rising edge of each peak matches the calculated values . this represents a situation where a molecule is located on a two - dimensional superfluid . the vibronic bound bound transitions with vibrationally resolved states typically consist of triangular shaped peaks , which have been analyzed for na2 in detail recently . the rising edge of the peaks has its origin approximately ( within a few wavenumbers ) at the vibrational level of the free molecule . a small droplet induced shift on the order of a few wavenumbers was reported for na2 on helium droplets ( e.g. , 2.8 cm for the 1u+ 1g+ transition and + 5 cm for the 1g+ 1u+ transition . ) within the accuracy of our data , we can not exclude such a slight shift for the observed li2hen transition . this broad wing is caused by the interaction of the excited electron of the molecule with the helium droplet , i.e. , the excitation of phonons ( phonon - wing ) . according to bovino et al . , the binding energies of li2 1g+ and li2 1u+ to a helium cluster of 30 atoms are of similar value around 50 cm , but the positions at the surface differ significantly : while the intermolecular axis of the li2 1g+ is oriented perpendicular to the surface , li2 1u+ lies flat on the surface . for sodium dimers , the singlet molecule spectra exhibit vibrational bands with narrow zero phonon lines and wide phonon - wings separated by a gap . in contrast , triplet spectra of na2 show a sharp rise at the onset of a vibrational band followed by a triangularly shaped phonon - wing without gap . the gapless appearance of a phonon - wing was attributed to a stronger coupling of vibrational motion of the dimer molecule to excitation modes of the helium droplet . the recorded spectrum was fitted with a sum of asymmetric two - sigma functions1we identify the position of the recorded peaks at the wavelength where the second derivative of eq 1 has a maximum for each set of fit parameters . this wavelength corresponds to the onset of the rising edge within a few wavenumbers ( depending on the signal - to - noise ratio ) and hence to the origin of the vibrational band . the determined positions lie within an interval of 0 to + 4 cm for li2 ( = 18 ) , 5 to + 4 cm for lili ( = 18 ) , and 0 to + 11 cm for li2 ( = 14 ) around each calculated value . higher vibrational states and the li2hen spectrum have a larger uncertainty due to the relatively low signal . the fit procedure does not reproduce accurately the calculated position of the origin of the = 0 level for li2 ( the wavenumber for lili differs by 12 cm from the calculated value ) . in this case , we identify the peak position with the onset of the rising edge at 21 707 cm ( 10 cm below the calculated value ) . the spectra in the upper panel in figure 5 appear to be superimposed by a large nonresonant background signal , which reaches from 21 250 to 23 000 cm . at the applied laser pulse energy , resonance enhanced two - photon ionization may be expected . according to ref ( 57 ) , the ionization potential of free li2 lies at te = 41 496 4 cm , and another li2 state , 2g+ , may enhance the broad two - photon ionization signal . experimental high resolution data and theoretical calculations are available for the 2g state . this allows a detailed comparison between the spectroscopy of free molecules and molecules on the droplet . the most accurate experimental data have been obtained by perturbation facilitated optical optical double resonance ( pfoodr ) spectroscopy , where for the 2g state all three isotopomers of the li dimer have been investigated . in order to use helium nanodroplets in future experiments as a matrix for the electronic spectroscopy of tailored molecules on their surface , we are interested in the accuracy of spectroscopic constants , which can be extracted from their spectra . therefore , we determine the molecular constants for the sake of comparison with the literature values of free molecules . in table 1 , we summarize the molecular constants as obtained from a least - squares fit of the observed band positions to the standard expression2the obtained results are compared to experimental results from xie and field ( for li2 ) and li et al . the parameters for lili are calculated from the li2 parameter by a multiplication with the isotope factor = ( / ) , where and are the reduced masses of li2 and lili , respectively . although born oppenheimer breakdown effects are significant in li2 , they can be neglected within the accuracy of our results . a direct determination of te from our data is not possible since we only have access to the triplet molecule . hence , we calculate te from t in eq 2 by adding a constant offset of 8213.8 , 8215.2 , and 8216.4 cm for li2 , lili , and li2 , respectively , which corresponds to the absolute energy of the 1u+( = 0 ) state . the molecular parameters listed in table 1 demonstrate that parameters deduced from lithium molecules on the surface of helium nanodroplets agree well with free molecule parameters ( within the 2 uncertainty interval ) . only vibronic transitions where the rising edge could be clearly identified are included in the fit procedure li2 , = 08 ; lili , = 08 ; li2 , = 04 . parameters are given in cm , and one standard deviation uncertainties are given in parentheses . we have shown that two - photon spectroscopy of li hen offers a possibility to access li hen rydberg states without uv lasers . we were able to assign the 3p 2s and 3d 2s transition of li atoms on helium nanodroplets by using two different methods . within the pseudodiatomic model , the states split into the 3p ( , ) and 3d ( ,, ) different molecular substates . the first approach ( exciplex approach ) used for the assignment exploits the fact that the exciplex formation process depends on the quantum number of the intermediate state . in the second part of the discussion of li hen hen potentials are generated by using a pairwise additive approach and assuming a frozen helium density that interacts with the excited dopant . condon factors of the bound free transitions overestimate the blue shift of the transitions and reveal the necessity of more sophisticated theoretical models for the accurate description of excited li hen states , which are capable of including quantum effects , which are non - negligible for lithium , as well as configuration interactions of the energetically close p and d states . as the lightest of the alkali metals , recording the blue or red shifts of rydberg states on droplets with respect to the free atom energies may provide further insight into the shielding effects of the helium droplet as obtained from our recent rydberg ritz analysis . in addition to the spectroscopy of li hen , we show for the first time a spectrum of a bound bound transition of li2 molecules on the surface of hen . the observed bands are identified as 2g ( ) 1u+ ( = 0 ) transitions . the rempi - tof technique allows to study all three isotopomers ( li2 , lili , and li2 ) of the li dimer simultaneously . the spectrum comprises a series of vibronic transitions , broadened by the influence of the droplet . the vibronic transitions could be followed from = 0 to = 11 for the li2 isotope . our investigation of li2 shows that despite the interaction between the molecule and the droplet , helium droplet isolation spectroscopy can serve as a tool to test ab initio molecular potentials of electronically excited states of species on the droplet surface . in future experiments , we plan to use our insights gained from the li2hen experiments presented in this article and combine the li atom with alkaline - earth atoms , a complex that can not be formed easily in the gas phase . helium droplet isolation spectroscopy will provide a versatile tool to study these species and to test the corresponding ab initio potentials .

we report on the spectroscopic investigation of lithium atoms and lithium dimers in their triplet manifold on the surface of helium nanodroplets ( hen ) . we present the excitation spectrum of the 3p 2s and 3d 2s two - photon transitions for single li atoms on hen . the atoms are excited from the 2s( ) ground state into , , and pseudodiatomic molecular substates . excitation spectra are recorded by resonance enhanced multiphoton ionization time - of - flight ( rempi - tof ) mass spectroscopy , which allows an investigation of the exciplex ( li*hem , m = 13 ) formation process in the li hen system . electronic states are shifted and broadened with respect to free atom states , which is explained within the pseudodiatomic model . the assignment is assisted by theoretical calculations , which are based on the orsay trento density functional where the interaction between the helium droplet and the lithium atom is introduced by a pairwise additive approach . when a droplet is doped with more than one alkali atom , the fragility of the alkali hen systems leads preferably to the formation of high - spin molecules on the droplets . we use this property of helium nanodroplets for the preparation of li dimers in their triplet ground state ( 13u+ ) . the excitation spectrum of the 23g( = 011 ) 13u+( = 0 ) transition is presented . the interaction between the molecule and the droplet manifests in a broadening of the transitions with a characteristic asymmetric form . the broadening extends to the blue side of each vibronic level , which is caused by the simultaneous excitation of the molecule and vibrations of the droplet ( phonons ) . the two isotopes of li form 6li2 and 7li2 as well as isotope mixed 6li7li molecules on the droplet surface . by using rempi - tof mass spectroscopy , isotope - dependent effects could be studied .

Introduction Experimental Section Results Summary and Conclusions

helium nanodroplets ( hen ) have attracted strong interest as superfluid nanocryostats for the efficient preparation of tailored molecules and clusters at ultracold temperatures . moreover , the special properties of helium nanodroplets enable the formation of tailored molecules and offer a possibility to study complexes that can not , or only with great expense , be produced in the gas phase or in molecular beam experiments . an example is the preparation of alkali ( ak ) metal dimers and trimers in their weakly bound high - spin configuration ( triplet and quartet states , respectively ) or even high spin clusters at low temperatures ( 0.37 k ) . the lowest electronic transitions ( d1 and d2 lines ) in ak atoms on the surface of helium nanodroplets are well characterized . the d1 and d2 lines of li atoms on the surface of hen are an exception among the ak hen systems because their main absorption peak is lower in energy than the free atom transition . this red - shift has its origin in bound bound transitions from the ground state into the two 2p( ) states . while excitation spectra of rydberg states for na , rb , and cs have been reported , less work has been done on the characterization of k hen and li hen . for the assignment of excited states of ak atoms on hen , we use the pseudodiatomic model ( hund s case the symmetry of the atomic system is broken and only the projection of the atomic orbital angular momentum l onto the intermolecular axis ( i.e. , ) , which is defined by the connection between the droplet center and the ak nucleus , remains a conserved quantity . for two - photon excitations , transitions from the li hen 2s( ) ground state into , , and molecular substates are allowed . the fragility of systems formed by helium nanodroplets with molecules on their surface enhances the formation of weakly bound complexes because the binding energy , which is gained during the formation of the molecule , is released into the droplet . this excess energy could destroy the complex formed by the molecule and the droplet . hence , the weakly bound alkali dimers in the triplet manifold have a higher probability of survival than the singlet molecules , and the helium droplets act as a filter for ak dimers in the triplet manifold . the orientation of the ground state li dimers for the two different spin configurations ( g+ and u+ state ) with respect to the droplet surface has been investigated theoretically for small hen ( for n up to 30 ) , where it was found that the singlet molecule is perpendicular and the triplet one is oriented parallel to the droplet s surface . this allows a detailed comparison between molecules on hen and free molecules , which reveals insight into the interaction between the superfluid droplet and the molecule on its surface . in the first part of this article , we report on the investigation of the two - photon 3p 2s and 3d 2s transitions in li hen by resonance enhanced multiphoton ionization time - of - flight ( rempi - tof ) spectroscopy . pseudodiatomic molecular substates are identified on the basis of the observed abundance of exciplexes because the exciplex formation process depends on of the molecular substate . in the second part of this article , we report on the investigation of the 2g 1u+ transition of the li dimer on hen . vibronic states could be resolved and the low mass of the li atom in combination with the rempi - tof technique allows to study the band for all three li isotopomers , li2 , li2 , and lili , as well as an investigation of their interaction with the droplet . these conditions lead to a subcritical supersonic jet expansion of the he gas and consequently to the formation of helium droplets , where the droplet size follows a logarithmic normal distribution . after production , the helium droplet beam is guided into another vacuum chamber where it is doped with li by passing through a resistively heated pickup cell . the process depends on the vapor pressure within the pickup cell , which is controlled by the cell temperature . at the used temperature of t 350 c , li atoms as well as li dimers are present on the droplets . the excitation spectra have been obtained by resonance enhanced multiphoton ionization time - of - flight ( rempi - tof ) spectroscopy . hen complexes have been excited via a two - photon scheme ( without a resonant intermediate state ) with dcm ( 5.2 mj ) and ionized with a fraction ( 0.6 mj ) of the xecl pump laser radiation ( radiant dyes rd - exc-200 xecl laser , 26 ns pulse duration , 100 hz ) . in this section , we discuss the two - photon 3p 2s and 3d 2s transitions in li hen . because of the small size of the li ion core , states with l > 0 lie energetically very close together in the lithium atom ( these states have a core nonpenetrating character ) . only the ns states are significantly shifted with respect to the hydrogenic term energy . note that because of the l < n condition for the orbital angular momentum quantum number l the 3d state investigated here is a special case among the nd li the two - photon excitation spectrum of the 3p 2s and 3d 2s transitions in li the ion yield for li he ( red , 9 , smoothed signal only ) , li he ( green ) , li he2 ( blue ) , and li he3 ( black ) is monitored as a function of the laser wavenumber , which is multiplied by two to account for the absorption of two photons . two - photon excitation spectrum of li hen . the spectrum is obtained by monitoring the li he ( red , 9 , smoothed signal only ) , li he ( green ) , li he2 ( blue ) , and li he3 ( black ) ion yield as a function of the excitation laser wavenumber ( multiplied by two ) with a time - of - flight mass spectrometer . the 3p and 3d free atom states at 30 925 cm and 31 283 cm are shown as vertical black lines . 2s transition in ref ( 24 ) only bare lithium ions were detected , the excitation of states with > 0 leads to the formation of exciplexes ( ak*hem ) . hem ion signals show the same structure as has been observed for heavier ak atoms and can be explained within the pseudodiatomic model . it is known that upon excitation of the ak hen system into states a large fraction of the atoms desorb without attached helium atoms . in contrast , the ionization via states with > 0 is always accompanied by an abundance of ak hem ( m > 0 ) complexes in the time - of - flight mass spectra . we call this approach for the assignment of excited states based on the exciplex formation process the exciplex approach in the following . in contrast , the signal in between the 3p( ) and the 3d line is only visible in the li he ion signal ; hence , we attribute this part of the spectrum to the onset of the 3p( ) state . the structure of the signal , which correlates to the 3d molecular substates , is similar to the signal obtained for the rb the state related to the peak at ( 31 278 5 ) cm , fwhm = ( 52 7 ) cm ( as monitored on the li it is remarkable that this state appears almost unshifted with respect to the free atom transition at 31 283 cm . ground state potential of 10 cm , which has to be taken into account when excitation spectra are compared to free atom transitions , the 3d( ) potential has a binding character and its minimum lies about ( 15 7 ) cm below the asymptotic 3d energy . this demonstrates a weak attractive interaction of the li atom in the 3d( ) state with the droplet . in the li he ion signal , the blue shoulder of the 3d( ) peak extends to much higher wavenumbers than in the li he2 detected spectrum , and it is followed by a second peak at ( 31 536 10 ) cm , which we attribute either to a part of the 3d ( ) or ( ) state . to simulate the spectra , we use the orsay trento density functional as implemented in the code by f. dalfovo , with modifications by k. k. lehmann and r. schmied . the interaction between the helium droplet and the alkali atom is introduced by a pairwise additive ( pwa ) approach . for the excited states , the curves of pascale represent the set of li*he interactions of the electronic states relevant for this work . an effective coordinate zli is introduced as the distance between the center of mass of the droplet and the lithium atom . we accounted for the correct projection of the angular momentum on the helium lithium axis by means of wigner rotation matrices , following ref ( 13 ) and transforming the hamiltonian in a diagonal shape . in the frozen droplet picture , assuming a fast desorption of the lithium atom upon excitation , franck condon factors ( fcfs ) are calculated with the help of bcont 2.2 by leroy for bound - free transitions of the pseudodiatomic molecule . the majority of the li hen systems are in their vibronic ground state ( = 0 ) before the excitation . depending on the angular momentum the curve splits into ( = 0 ) states ( solid line ) , ( = 1 ) states ( dashed ) , and ( = 2 ) states ( dashed dotted ) . such large blue shifts on the order of several thousand wavenumbers have already been observed for calculations of excited states of na hen in ref ( 17 ) , where it was shown that the pwa approach works best at states with low principal quantum number n. in addition to the pwa approach , for na hen a perturbative configuration interaction ( pci ) approach was used , where the helium dopant interaction is introduced by a perturbation rather than a pair potential . although our potential energy curves ( see figure 2 ) for the 2s( ) , 2p(, ) , and 3s( ) reproduce the results of other calculations , the spectrum for the 3p and 3d states is far off the experimental one . however , the calculated theoretical spectrum for the transition from the ground state into the 3s( ) state has a maximum at 27 880 cm , which matches the experimental peak maximum ( 27 820 cm ) very well as can be seen from figure 4 in ref ( 24 ) . the difference between theory and experiment for the 3p and 3d spectra is expected to some extent due to several approximations in the pwa approach : ( i ) the ground state helium density is assumed to be maintained during the excitation ( frozen droplet approximation ) , ( ii ) interactions between 3p and 3d states are not included , but in the case of li the states lie relatively close , and ( iii ) the approach rests on the potentials calculated by pascale . time - of - flight mass spectrum as obtained while the laser is scanned across the two - photon transitions , which is shown in figure 1 . in addition to li dimers , which are formed on the droplets , also complexes of li , li2 , and residual water , which is present in the vacuum chamber , are observed in the mass spectrum . according to the pwa approach , the 3p( ) state lies energetically lower than the 3p( ) state , and the 3d( ) state is the energetically lowest among the three 3d molecular substates , which is in agreement with our assignment within the exciplex approach . as can be seen from the excitation spectrum in figure 1 , we do not observe a significant isotope effect within the accuracy of our data . figure 4 shows the time - of - flight mass spectrum as obtained , while the laser is scanned across the two - photon transitions ( figure 1 ) . because of the high photon density and the presence of the 308 nm light , li dimers ( li2 , lili , and li2 ) are also observed in the mass spectrum . we attribute the peaks at m / z = 24 and m / z = 31 to the products of an oxidative hydrolysis of lioh and li(lioh ) , respectively , which are produced upon a reaction with water in the helium droplet . because of the resonant excitation of an intermediate state , the reaction may be enhanced by the present laser photons . we find no evidence for the formation of li clusters ( i.e. the absence of these complexes is related to the high formation energies of lithium clusters , which are significantly higher than those of the heavier ak clusters and have been investigated in detail on / in helium nanodroplets . the excitation spectrum of li dimers in the range between 21 250 and 24 000 cm is shown in figure 5a . the ion yield for m / z = 12 ( li2 ) , 13 ( lili ) , and 14 ( li2 ) the composition of the molecules on the droplets is 85.71% li2 , 13.71% lili , and 0.55% li2 . a large fraction of the molecules fragments into li and li upon ionization , which is observed when monitoring li for the same excitation spectrum ( not shown ) . = 0 ) triplet transitions of li dimers on the surface of helium nanodroplets ( a ) . the only exception is the relatively low intensity of the = 0 state when compared to the fc factor , which is most probably caused by saturation effects . this broad wing is caused by the interaction of the excited electron of the molecule with the helium droplet , i.e. , the binding energies of li2 1g+ and li2 1u+ to a helium cluster of 30 atoms are of similar value around 50 cm , but the positions at the surface differ significantly : while the intermolecular axis of the li2 1g+ is oriented perpendicular to the surface , li2 1u+ lies flat on the surface . the recorded spectrum was fitted with a sum of asymmetric two - sigma functions1we identify the position of the recorded peaks at the wavelength where the second derivative of eq 1 has a maximum for each set of fit parameters . this allows a detailed comparison between the spectroscopy of free molecules and molecules on the droplet . the most accurate experimental data have been obtained by perturbation facilitated optical optical double resonance ( pfoodr ) spectroscopy , where for the 2g state all three isotopomers of the li dimer have been investigated . in order to use helium nanodroplets in future experiments as a matrix for the electronic spectroscopy of tailored molecules on their surface , we are interested in the accuracy of spectroscopic constants , which can be extracted from their spectra . hence , we calculate te from t in eq 2 by adding a constant offset of 8213.8 , 8215.2 , and 8216.4 cm for li2 , lili , and li2 , respectively , which corresponds to the absolute energy of the 1u+( = 0 ) state . the molecular parameters listed in table 1 demonstrate that parameters deduced from lithium molecules on the surface of helium nanodroplets agree well with free molecule parameters ( within the 2 uncertainty interval ) . we have shown that two - photon spectroscopy of li hen offers a possibility to access li hen rydberg states without uv lasers . we were able to assign the 3p 2s and 3d 2s transition of li atoms on helium nanodroplets by using two different methods . within the pseudodiatomic model , the states split into the 3p ( , ) and 3d ( ,, ) different molecular substates . the first approach ( exciplex approach ) used for the assignment exploits the fact that the exciplex formation process depends on the quantum number of the intermediate state . in the second part of the discussion of li hen hen potentials are generated by using a pairwise additive approach and assuming a frozen helium density that interacts with the excited dopant . condon factors of the bound free transitions overestimate the blue shift of the transitions and reveal the necessity of more sophisticated theoretical models for the accurate description of excited li hen states , which are capable of including quantum effects , which are non - negligible for lithium , as well as configuration interactions of the energetically close p and d states . as the lightest of the alkali metals , recording the blue or red shifts of rydberg states on droplets with respect to the free atom energies may provide further insight into the shielding effects of the helium droplet as obtained from our recent rydberg ritz analysis . in addition to the spectroscopy of li hen , we show for the first time a spectrum of a bound bound transition of li2 molecules on the surface of hen . the rempi - tof technique allows to study all three isotopomers ( li2 , lili , and li2 ) of the li dimer simultaneously . our investigation of li2 shows that despite the interaction between the molecule and the droplet , helium droplet isolation spectroscopy can serve as a tool to test ab initio molecular potentials of electronically excited states of species on the droplet surface .

in the beginning , medical science was capable of describing the features of inflammatory bowel disease ( ibd ) , and medical therapeutics were guided by trial and error , misguided hypotheses about disease pathogenesis , and some measure of serendipity . crohn s disease and ulcerative colitis the two major forms of idiopathic ibd have been recognized as distinct disease entities for over a century . as early as 1761 morgagni described intestinal inflammation that in modern times we would recognize as crohn s disease.1 after the identification of the causative agent of tuberculosis by koch in 1882 , it became clear that some individuals had a disease similar to intestinal tuberculosis but did not bear the tubercle bacillus . reports by fenwick in 1889 , dalziel in 1913 , weiner in 1914 , moschowitz and wilensky in 1923 and 1927 , and goldfarb and suissman in 1931 predated the landmark publication of crohn , ginzburg , and oppenheimer in 1932 describing terminal ileitis.1,2 later , lockhart - mummery and morson3 described granulomatous colitis , and the disease process was understood to potentially affect the large bowel as well . the historical origins of ulcerative colitis are less clear , with descriptions of bloody diarrhea and dysentery dating back to antiquity . generally , however , wilkes is credited with the first pathologic description of what was called simple ulcerative colitis in 1859.1 subsequently , in 1875 , wilkes and moxon described a syndrome of simple ulcerative colitis in greater detail.1 with the recognition of the nosologic distinctions of these diseases came attempts to treat them surgically and medically ( table 1 ) . depending upon the etiologic concept of the disease , for example , many conceived of the disease as being caused by an as yet unrecognized pathogen . accordingly , treatments have included potassium permanganate , dakin s solution , antidysentery serum , escherichia coli vaccine , antiamoebic drugs , and sulfonamides.1 these treatments did not prove to be durable or effective , and have all been abandoned over time . sulfasalazine , the first truly effective agent used in ibd , was discovered serendipitously when this antirheumatic agent was observed to produce resolution of both diarrhea and arthralgias in patients with ulcerative colitis being treated with this agent for their joint disease . table 1inflammatory bowel disease landmarks of the pastdistinction of crohn s disease and ulcerative colitis from infectious colitis made possible by the development of bacteriologic techniquesdistinction between crohn s disease and ulcerative colitis drawnthe inflammatory bowel diseases are understood to be immune - mediated diseasessulfasalazine and hydrocortisone used to treat ulcerative colitisthiopurine agents ( mercaptopurine and azathioprine ) used to treat ibdmethotrexate used to treat crohns disease and cyclosporine used to treat severe , steroid - refractory ulcerative colitisfamilial clustering implicates genetic factors in ibdbowel - sparing understood to be a key principle of surgical therapy , and the technique of stricturoplasty developedileal pouch - anal anastomosis developed as an alternative to ileostomy after total proctocolectomy for ulcerative colitisibd , inflammatory bowel disease inflammatory bowel disease landmarks of the past ibd , inflammatory bowel disease further experimentation revealed that the active moiety of sulfasalazine was 5-aminosalicylate ( 5-asa ) , whereas the sulfa moiety was responsible for many of the untoward effects of the drug.4 furthermore , delivery of the active 5-asa molecule was finally understood to rely upon the presence of the diazo bond between the sulfapyridine and 5-asa moieties for the delivery of the 5-asa to the distal bowel upon cleavage of the bond by the colonic flora.5 subsequently , a host of second - generation 5-asa agents were developed that utilized a variety other mechanisms to deliver the drug topically to the distal bowel . these have included time - released and ph - dependent agents , as well as non - sulfa - containing diazo - bonded agents such as olsalazine and balsalazide . the descriptive histology of ibd suggested that immune activation could also be targeted by therapeutic agents . the observation of acute and chronic inflammatory cells , and the common occurrence of extraintestinal immune - mediated manifestations eventually led to the use of adrenocorticotropic hormone6 and corticosteroids,7 which have proven to be highly effective in treating these diseases . the first randomized controlled trial of hydrocortisone in ulcerative colitis proved the agent to be remarkably effective,8 and finally produced effective therapy for severe ulcerative colitis , which previously had been associated with a considerably high case fatality rate . nevertheless , the corticosteroids have proven to be a double - edged sword in the treatment of ibd . evidence from population - based cohorts suggests that although less than half of patients with ibd require treatment with these agents , the need for corticosteroids is associated with a worse prognosis , including increased risk of surgery and risk of disability.9 in all , slightly more than half of individuals treated with glucocorticoids will be well and off steroids at 1 year after beginning these agents . mercaptopurine and azathioprine were initially disappointing in clinical trials in crohn s disease until appropriate dosing and duration of therapy were more fully explicated . with the landmark study of present et al.,10 mercaptopurine and azathioprine assumed their place in the ibd armamentarium as treatments primarily for patients who were steroid - refractory or steroiddependent . these agents are also thought to be effective in fistulizing crohn s disease.11 more recently , these agents have been more fully accepted as appropriate therapies for the treatment of steroid - dependent to - refractory ulcerative colitis , as well.12 a second - line agent shown to be effective in steroid - dependent crohn s disease is methotrexate . this agent is effective both in the short term , to induce steroid - free remission,13 and in the long term , to maintain remission.14 immunosuppressive therapy progressed further as increasingly potent agents became available . the introduction of cyclosporine for treatment of severe , steroid - refractory ulcerative colitis has provided an alternative to total proctocolectomy for some individuals.15 however , the narrow therapeutic margin of this agent has precluded widespread application , and in the united states its use remains confined largely to tertiary - care referral centers . cyclosporine appears to be most effective when used as a bridge to maintenance therapy with mercaptopurine or azathioprine.16 the continued role of this agent becomes less certain with the advent of potent biologic agents effective in severe ulcerative colitis . the earliest surgeries for crohn s disease often sought to divert the fecal stream by bypassing surgery , rather than resection . although effective , this technique has long been out of favor , with resection and primary anastomosis possible for the majority of patients with crohn s disease . more directly applicable to the optimum care of patients was the appreciation that wide margins of resection were not effective in preventing disease recurrence.17 the important principle of bowel preservation in crohn s disease became favored in an effort to prevent short - bowel syndrome from repeated small - bowel resections . this principle of sparing the small bowel was carried further with the development of stricturoplasty techniques.18 finally , the development in the 1980s of ileal pouch - anal anastomosis for patients with ulcerative colitis who required total proctocolectomy was a revolutionary surgical advance that has provided a much needed alternative to permanent endileostomy.19 at present , key concepts regarding the pathogenesis of inflammatory bowel disease are as follows ( table 2 ) . first , it is recognized that immune tolerance is the normal state of the intestinal immune system . second , it is apparent that a wide variety of cell types are orchestrated in a tightly regulated fashion to maintain immunologic tolerance . at the same time , the capacity to mount an immune and inflammatory response within the mucosa is maintained . third , the luminal flora is a key ingredient in the abnormal immune response of ibd . fourth , genetic factors predispose individuals to an abnormal immune response to the flora . finally , it is recognized that both the innate and adaptive immune responses play integrated roles in the homeostasis of the intestinal mucosal immune response . table 2landmarks of the presentcontrolled physiologic inflammation is understood to result from the normal state of immune tolerance in the intestinedevelopment of genetic animal models of colitis demonstrate diverse disturbances of immunity and intestinal barrier function capable of inducing a phenotype of gut inflammationanimal models demonstrate the central role of gut flora as a necessary factor in colitis , with the flora further implicated in serologic studies in humansnod2/card15 demonstrated as the first disease - associated gene in crohn s disease after ibd1 localized to chromosome 16 by genomewide scanningdefective innate immune response implicated in crohn s disease by nod2 physiologybiologic agents developed through monoclonal antibody technology and molecular biology techniquesinfliximab , the first of a new class of anti - tnf biologics , shown to be effective in crohn s disease and ulcerative colitisthe heterogeneity of crohn s disease and ulcerative colitis demonstrated in genetic and serologic studiestnf , tumor necrosis factor landmarks of the present tnf , tumor necrosis factor for many years it has been recognized that the intestinal mucosa contains a resident population of inflammatory cells . these cells are poised at the interface between the intestinal lumen and the systemic circulation in readiness for enteric infection and other insults to the mucosa . the key factor that differentiates individuals with ibd from normal individuals is the ability to downregulate that inflammatory state and return it to a condition of normal , controlled gut inflammation . by contrast , individuals susceptible to ibd will tend to enter a state of uncontrolled and chronic inflammation with failure to downregulate the inflammation caused by the insult . historically , it has been recognized that approximately one in five individuals newly diagnosed with ibd will report a family history . in addition , first - degree relatives , in particular siblings , are at increased risk of ibd , and this risk is somewhat higher in individuals of ashkenazi jewish background.20 third , there is a high concordance for crohn s disease among monozygotic twins , whereas a somewhat lesser concordance is observed in monozygotic twins with one affected individual with ulcerative colitis.21 these clues have long suggested that genetics play an important role in susceptibility to the inflammatory bowel diseases . environmental contributions to the pathogenesis of ibd have also long been suspected . for many years , predominant theories have surrounded the notion that the mucosal inflammation of ibd is an appropriate response to pathogenic microbiota.22 candidates for pathogenic agents have been diverse over the years , and have included diplostreptococcus , entamoeba histolytica , and , most persistently , mycobacterium paratuberculosis.23 more recently , lines of evidence suggest that normal , nonpathogenic flora ( at least in the conventional sense of pathogenicity ) are a necessary though not sufficient factor in the pathogenesis of ibd , whereas firm evidence of a pathogenic agent has been lacking . in addition to the host flora , other environmental factors have been noted to affect the expression of ibd . these factors may be considered to be disease modifiers , and most prominently include smoking,24 nonsteroidal anti - inflammatory drugs,25 and prior appendectomy.26 in particular , both smoking and appendectomy have been recognized to be distinct risk factors with dichotomous outcomes for crohn s disease and ulcerative colitis . smoking has been associated with increased risk of crohn s disease as well as worse outcomes over the course of the disease,27 whereas for ulcerative colitis , smoking has been recognized as having a protective effect.2830 the effects of tobacco smoke are complex and poorly understood . the role of smoking does not appear to be as simple as the presence or absence of nicotine , which may be an effective therapy for ulcerative colitis but is poorly tolerated.31 current investigation focuses on the role of carbon monoxide in smoke as a factor capable of modifying the expression of inflammation in the gut.32 circumstantial evidence has long pointed to the critical role of the luminal flora in ibd . the evidence is strongest in crohn s disease and relatively sparse in ulcerative colitis . in clinical practice antibiotics are widely used to treat perianal disease in crohn s disease.33 evidence from clinical trials suggests some effect of antibiotic therapy as a treatment for crohn s disease , primarily in disease with colonic localization.34 in addition , imidazole antibiotics such as metronidazole35 and ornidazole36 have been demonstrated reproducibly to have a prophylactic effect for disease recurrence after ileal resection . furthermore , as noted above , a time - honored , though outmoded technique to treat refractory crohn s disease has been to surgically divert the affected segment . experiments carried out in the 1980s demonstrated that the luminal contents contained factors , most likely bacterial factors , capable of reinducing inflammation in quiescent diverted bowel.37 in addition , elemental diet has been demonstrated to be an effective therapy for crohn s disease.38 effects of elemental diet may be complex , but are thought to include a favorable effect on the bowel flora composition that may minimize immunologic stimulation of the host immune response . another line of research being vigorously pursued is the potential role of probiotic therapies as treatments for ibd . thus far , clinical trials in humans have not demonstrated a robust effect of these agents in ibd . the clearest demonstration of benefit has been with a preparation known as vsl#3 in the treatment and prophylaxis of pouchitis occurring in ileal pouches created as curative therapy for ulcerative colitis.39,40 other studies have suggested modest benefit in ulcerative colitis and crohn s disease for a variety of probiotic agents , including nissle 1917 and vsl#3.41,42 finally , one intriguing study demonstrated excessive responsiveness of lamina propria mononuclear cells to autologous gut bacteria among patients with ibd.43 animal models of colitis have also provided essential clues to the role of the luminal flora in the pathogenesis of ibd . the list of genetic and acquired aberrations that culminate in phenotypic manifestations of intestinal inflammation is strikingly long and diverse.44 these models include animals with disturbed immune regulation , such as the interleukin ( il)-2 , t - cell receptor and il-10 knockouts , as well as models where disruption of the epithelial barrier function is paramount , such as the g2i knockout mouse . the sheer diversity of abnormalities that drive intestinal inflammation reminiscent of human ibd suggests that the gut has a limited phenotypic repertoire and is capable of expressing such disturbances in only a limited number of ways . the corollary is that human ibd may also be two phenotypic syndromes driven by diverse genetic abnormalities . this is clearly suggested by observations of genetic and serologic heterogeneity among individuals diagnosed with ulcerative colitis or crohn s disease.4548 in addition , the role of the intestinal flora in driving gut mucosal inflammation has been clearly demonstrated in these diverse models . in every model thus far explored , it is clear that removing intestinal flora from the equation by raising animal lines in gnotobiotic conditions greatly diminishes or prevents the onset of gut inflammation . with the introduction of nonpathogenic bacteria , it does not appear to be the case that pathogenic microbiota are necessary to drive the disease . furthermore , it has become clear that the characteristics of the intestinal flora also shape the phenotypic characteristics of the gut inflammation . for example in the hla b27 transgenic rat , monoassociation experiments with single , nonpathogenic bacterial species have been performed . the introduction of bacteroides vulgatus results in a moderately severe colitis , whereas the introduction of e. coli results in no colitis whatsoever . colonization with the entire mixture of the usual bacterial flora present in the cecum culminates in an aggressive colitis , whereas the introduction of cecal bacteria plus the probiotic species lactobacillus gg results in protection from colitis altogether.49 these experiments suggest that specific nonpathogenic bacteria may shape the expression of disease severity in profound ways . surprisingly , it also appears that specific nonpathogenic bacteria may also be a determinant of anatomic disease localization . il-10 knockout mice grown in germ - free conditions do not typically manifest colitis . with the introduction of commensal flora , the typical colonic inflammatory phenotype ensues . however , when monocolonization with enterococcus faecalis occurs , a left - sided colitis ensues . alternatively , upon colonization with e. coli , a distinctly right - sided colitis occurs.50 together , these findings suggest that the composition of the commensal flora may have a major impact on the phenotypic expression of ibd . it can not be assumed that the genetic aberrations in gene knockout or knock - in models of colitis are reflective of the actual genetic polymorphisms that cause human disease . however , great strides have been made in the identification of genetic defects associated with crohn s disease and ulcerative colitis . the greatest progress has been made to the application of genomewide scanning , which has been greatly facilitated by the development of microsatellite markers and more recently single - nucleotide polymorphism ( snp ) haplotype maps of the human genome . with the collaboration of numerous genetics research groups across the globe , specific genetic loci with strong associations with ibd have been discovered.51 this approach is distinct from the candidate gene approach , which explores genetic polymorphisms in a gene that is predicted to be of particular interest by virtue of its known function . rather , the greatest progress has been made by an unbiased appraisal of the human genome afforded by genomewide scanning . using these techniques , the first such ibd gene identified was the nod2/card15 gene on chromosome 16 , associated with the ibd1 locus found on genomewide scanning.5254 specific disease associated polymorphisms included the r702w , g908r , and 33 mutations associated with relative risks for crohn s disease of 14.3 , 34.1 , and 17.64 for homozygous recessive individuals over control populations.5254 explication of the cellular function of nod2 has shed light on the nature of the underlying immune defects of crohn s disease . nod2 ( nuclear type binding oligomerization domain 2 ) consists of two card domains , followed by a nod domain , and ending in a leucine - rich repeat . it is within the leucine - rich repeat domain that the disease - associated mutations have been noted . these are loss - of - function mutations , which appear to prevent the binding of muramyl dipeptide , which is ubiquitously present in virtually all bacterial cell walls.55 downstream events lead to the activation of nfb through an interaction with the card domains.56 thus , it appears that nod2 is intimately involved in intracellular bacterial sensing and generation of the innate immune response.57 less certain is how a loss - of - function mutation in the leucine - rich repeat causes the inflammation of crohn s disease . although many hypotheses have been generated regarding the mechanism of disease , none have been conclusively proven . one hypothesis is that with the loss of binding of muramyl dipeptide to nod2 , there is a compensatory response with failure to inhibit the activation of c - rel and p50 by binding of peptidoglycan to the toll - like receptor 2.58 this results in the downstream activation of il-12 and consequent inflammation . regardless of the precise mechanism through which nod2 polymorphisms generate a crohn s disease phenotype , this discovery has brought the role of the innate immune responses to the fore . the innate immune responses are the hardwired and immediate responses to pathogens that occur through binding of pathogen - associated molecular patterns to cell - bound receptors . the critical cellular elements in this response include macrophages , nk cells , and nonprofessional antigen - presenting cells . by contrast , much research of the last 20 years has focused on the adaptive immune system . the adaptive immune responses require priming and the generation of antigen - specific responses by b and t cells , and therefore may not be immediate . although cell - to - cell surface interactions may occur , soluble factors may also play a role in adaptive immune responses . the dendritic cells of the gut have become a focal point in our understanding of intestinal immune responses.59 elegant immunofluorescent microscopic techniques have demonstrated large populations of dendritic cells localized just beneath the intestinal epithelial cells.60 these arborized cells project long podocytes through the interstices of the intestinal epithelial cells and out to the intestinal lumen . here , extensive sampling of luminal antigens occurs . it is believed that the dendritic cells process these antigens , and then present them to the cells in the peyer s patches as well as in the mesenteric lymph nodes . depending upon the nature of the antigen and the activation state of the dendritic cell , the end result may be immune activation or tolerization . enterocytes may also present antigen , also likely leading to tolerization,61 whereas pathogenic microbes traversing m cells to the underlying peyer s patches are likely to trigger immune activation.62 in parallel to the growing importance of the innate immune system came the first studies of therapies intended to enhance the innate immune responses , rather than to suppress adaptive immune responses . korzenik and dieckgraefe63 reexamined data from the 1970s and 1980s that suggested that neutrophil responses were diminished in patients with crohn s disease . in addition , they noted that varied conditions characterized by neutrophil defects are also commonly associated with crohn s disease or a crohn s disease - like phenotype . they reasoned , therefore , that individuals with crohn s disease may also have neutrophil defects , and that stimulating neutrophils may be beneficial in the treatment of crohn s disease . this hypothesis led them to test granulocyte - colony stimulating factor ( g - csf ) and granulocyte - macrophage ( gm)-csf in open - label pilot trials in crohn s disease , with promising early results.64,65 a subsequent phase 2 study of sargramostim ( gm - csf ) in crohn s disease demonstrated evidence of activity of this agent in decreasing symptoms of active disease.66 unfortunately , a phase 3 study failed to demonstrate efficacy over placebo , although a separate study demonstrated efficacy of this agent in steroid sparing.67 more detailed descriptions of the immunologic pathways of the adaptive immune responses in crohn s disease and ulcerative colitis have also led to new and specific therapeutic agents . in the early 1990s , animal models of immunologic disease demonstrated that t helper ( th ) cell populations segregated according to distinct cytokine profiles.68 th1 cells are characterized by the expression of il-2 , interferon- , il-12 , and tumor necrosis factor ( tnf ) . th1 responses are most closely associated with the cell - mediated immune responses that characterize crohn s disease.69 by contrast , th2 responses are associated with humoral immunity and with a cytokine profile comprising il-4 , il-5 , il-6 , and il-13 . the cytokine profile expressed in ulcerative colitis tissues is most consistent with a variation on th2-like immune responses , lacking in the expression of il-4.69 understanding the role of cytokines has been a critical advance in ibd therapeutics , along with the advent of monoclonal antibody technology , which made possible the targeted inhibition of specific disease - related cytokines . clearly , the greatest advance in this area has been the advent of anti - tnf biologic therapies . early reports from the netherlands described two cases of crohn s disease treated with a chimeric monoclonal anti - tnf antibody called ca2.70 this was followed by a case series of ten patients with crohn s disease , who experienced a high rate of response and rapid mucosal healing on endoscopy.71 the first randomized controlled trial by targan et al.72 demonstrated that short - term induction occurred rapidly and in the majority of patients with active disease unresponsive to other therapies . the accent i73 and accent ii74 studies demonstrated the ability to maintain a durable response in a subset of patients with nonfistulizing and fistulizing crohn s disease , respectively , over the course of a year of follow - up . one of the drawbacks of chimeric monoclonal antibody therapy has been the generation of immune responses against the drug and loss of response . these include adalimumab , a fully human anti - tnf antibody,75 and certolizumab , a pegylated fab anti - tnf fragment.76 whether these agents are less immunogenic than infliximab is as yet uncertain . however , findings of long - term studies lasting from 6 to 12 months suggest that the efficacy of these agents is equivalent to that seen with infliximab.77,78 other issues with the anti - tnf biologics relate to weighing the risks and benefits of these agents for specific patients.79 described risks include rare occurrence of opportunistic infections , including intracellular infections such as tuberculosis and fungal and viral infections , demyelinating disorders , and rare reports of lymphoma.80 it will be critical to define populations for whom these risks are appropriate , or alternatively to identify patients at least risk for developing these complications . other cytokine - directed therapies are at earlier stages of development . randomized controlled trials with daily subcutaneous injections of il-10 provided disappointingly negative results.81 this result was unexpected after initially promising phase 2 studies , and especially given that the preclinical rationale of using this classic inhibitory cytokine was quite strong . in addition , studies using the humanized anti - il-2 receptor antibody daclizumab showed this agent to be ineffective as a treatment for ulcerative colitis.82 early studies with anti - il-12 antibody appear to demonstrate some activity in active crohn s disease.83 this agent , which binds to epitopes in the p40 subunit of il-12 , may also bind to the same subunit as a component of the heterodimeric proinflammatory cytokine il-23 . of note , the il-23 receptor gene was recently associated with crohn s disease ; namely , polymorphisms in this gene appear to confer protection against the disease.84 this would suggest that agents that inhibit il-23 may prove to be effective in crohn s disease . a second area where understanding of the pathogenesis has led to novel therapies is in the area of cellular adhesion and recruitment . although a normal state of physiologic inflammation exists in the mucosa of the bowel , it is only through the coordinated expression of specific adhesion molecules that cells are recruited from the peripheral circulation into the mucosa in response to inflammation . in the early 1990s , the details of cellular recruitment and adhesion were explicated in fine detail.85 when inflammation is present within a tissue , a series of events occur in rapid succession . first , the endothelium becomes activated , and the expression of e - and p - selectins on the endothelial surface expression occurs . l - selectin on the cell surface of leukocytes in circulation binds weakly to the selectins expressed on the endothelium . this , in turn , causes rolling of the leukocyte along the endothelial surface and activation of integrin expression on the leukocyte surface.85 the integrins then bind tightly to cellular adhesion molecules such as vcam , icam , and madcam86 on the endothelial surface . finally , the leukocytes diapedese into the mucosa . here , chemokines exist in a gradient within the mucosa , guiding the leukocytes further toward the mucosal surface . thus , adhesion and recruitment of leukocytes is a complex and critical process in inflammation that offers many potential targets for specific interventions against inflammation . the first agent to have targeted adhesion was the humanized anti-4 integrin antibody natalizumab . a large phase 2 study of natalizumab in crohn s disease appeared to demonstrate short - term activity but failed to meet its primary end point in a statistically significant way.87 a follow - up phase 3 study again demonstrated activity of this agent , but missed its primary end point , in large part owing to a high placebo response rate.88 however , a follow - on study demonstrated robust maintenance efficacy with natalizumab.88 an additional short - term study finally confirmed the efficacy of natalizumab among patients with elevated c - reactive protein.89 unexpectedly , however , three cases of progressive multifocal leukoencephalopathy ( pml ) , an otherwise extremely rare and usually fatal central nervous system disease caused by the jc virus , were found among 3000 patients treated for either multiple sclerosis or crohn s disease with this agent.90 a second selective adhesion molecule inhibitor , mln-02 , has been demonstrated in a phase 2 study to be efficacious in patients with active ulcerative colitis failing treatment with 5-aminosalicylates.91 a second pilot study was performed in crohn s disease with promising effect.92 at this time , it is unknown if the more specific adhesion of this antibody to 47 integrin , the ligand of madcam ( mucosal addressin cellular adhesion molecule , located only in gut endothelium ) , will improve the safety profile of this agent and avoid the occurrence of pml . a third area of interest for therapeutic intervention is the process of t - cell activation . the interactions between antigen - presenting cells and t cells may be conceived of as being analogous to a neurological synapse . as with the neuronal synapse , a variety of stimulatory and inhibitory signals are integrated when the antigen - presenting cell interacts with a t cell , yielding a net overall effect of activation or inhibition of the immune response.93,94 the basis of the antigen - specific response occurs with the presentation of an antigenic epitope in the context of major histocompatibility complex ( mhc ) class ii . mhc class ii / antigen binds to the t - cell receptor cd3 in an antigen - specific fashion . however , this primary signal is not sufficient in itself to activate the nave t cell . these include binding of tnf receptor to tnf on the cell surface , cd40 ligand to cd40 , and b7 to the costimulation receptor cd28.94 when such costimulatory signals are lacking , the end result is anergy or apoptosis of the t cell rather than activation . both the antigen - specific interaction of mhc class ii - cd3 and the costimulatory signals offer opportunities for therapeutic intervention . the humanized anti - cd3 antibody visilizumab has been explored as a treatment for refractory ulcerative colitis.95 open - label studies suggest that this agent may be efficacious even in situations where disease is severe and unresponsive to steroids . visilizumab is highly potent , with activity seen at doses as low as 5 g per kilogram . the long - term safety consequences of lysing activated t cells have yet to be determined , as studies have been relatively small and of short duration . other strategies have attempted to target the costimulatory signal rather than the t - cell receptor . abatacept is a fusion molecule of ctl4 and immunoglobulin , known as ctla - ig.96 this agent is approved in the united states for the treatment of rheumatoid arthritis and has been shown to be effective in patients who have not responded to anti - tnf biologic agents in that disease state.96 in principle , this agent would be of considerable interest as a potential therapy for ibd . as yet untapped are therapies that might exert therapeutic effect by enhancing regulatory t cells as opposed to inhibiting effector t cells . there is a growing appreciation for various populations of t cells that exert a downregulatory effect on immune responses and do so through a variety of mechanisms . distinct populations have been recognized as including tr1 cells , characterized by elaboration of il-10,97 th3 cells,98 characterized by membrane - bound transforming growth factor , and cd4 + cd25 + t regulatory cells , which inhibit through direct cell - to - cell contact.99 the earliest exploration of this therapeutic principle may involve the selection and engineering of t cells to deliver il-10.100 the clinical efficacy and safety of this approach is as yet unknown . in summary , in recent times we have learned that the intestinal flora is critical in generating the immune response of ibd . in crohn s disease an excess of effector t cells , characterized as either th1- or th2-like , is associated with the distinct clinical manifestations of crohn s disease and ulcerative colitis , respectively . these detailed explanations of the pathogenesis of ibd have led to more effective and focused treatments for these diseases . increasingly , we are entering a time when high - throughput technologies are revolutionizing the approach to medical discovery . high - throughput dna sequencers are capable of sequencing an individual genome in days rather than years . gene expression arrays simultaneously represent the expression of thousands of transcripts rather than a handful at a time . mass spectrometry combined with other techniques permits the simultaneous determination of thousands of proteins in a burgeoning area known as proteomics . nonclassical microbiologic techniques based upon molecular biology will facilitate an improved understanding of the role of the gut flora in ibd . the completion of the human genome project and snp haplotype mapping101 have made it possible to examine individual genotypes in relation to disease susceptibility as well as disease characteristics and prognosis . rather than starting from a prior hypothesis about a single gene , protein , or metabolite playing a role in the disease process , unbiased explorations of all possible biologic variations may be explored . advances in bioinformatics and the ability to integrate the volumes of data generated by such experiments will be critical in allowing medicine to progress to the point where personalized medicine may become fact rather than aspiration . when these new techniques have been more fully applied to the problems of patients with ibd , it is likely that we will improve both prediction of individual prognoses for the disease , as well as be able to target therapy to the mechanism of disease . we will no longer blithely talk about curing crohn s disease or ulcerative colitis ; rather , it is very likely that we will talk about very specific cures for each one of the many varied genetic or acquired aberrations that cause the crohn s disease or ulcerative colitis phenotype . we will also finally elucidate the environmental factors that have contributed to the rise of these diseases in westernized societies . in summary , a survey of the past and present advances in the inflammatory bowel diseases provides much cause for optimism . the accelerating pace of discovery in crohn s disease and ulcerative colitis provides hope that each year will bring a better life to the growing number of individuals who suffer from these curious diseases . dr . sands discloses that he has been a consultant and has received grant support and honoraria from centocor , abbott laboratories , elan pharmaceuticals , schering plough research institute , biogenidec , procter & gamble pharmaceuticals , salix , shire , prometheus laboratories , millennium pharmaceuticals , cerimon pharmaceuticals , genentech , wyeth , otsuka america pharmaceuticals inc .

crohn s disease and ulcerative colitis , collectively known as the inflammatory bowel diseases ( ibd ) , are largely diseases of the twentieth century , and are associated with the rise of modern , westernized industrial society . although the causes of these diseases remain incompletely understood , the prevailing model is that the intestinal flora drives an unmitigated intestinal immune response and inflammation in the genetically susceptible host . a review of the past and present of these diseases shows that detailed description preceded more fundamental elucidation of the disease processes . working out the details of disease pathogenesis , in turn , has yielded dividends in more focused and effective therapy for ibd . this article highlights the key descriptions of the past , and the pivotal findings of current studies in disease pathogenesis and its connection to medical therapy . future directions in the ibd will likely explicate the inhomogeneous causes of these diseases , with implications for individualized therapy .

Past: the era of description Present: the era of explanation Future: the era of prediction Disclosures

in the beginning , medical science was capable of describing the features of inflammatory bowel disease ( ibd ) , and medical therapeutics were guided by trial and error , misguided hypotheses about disease pathogenesis , and some measure of serendipity . crohn s disease and ulcerative colitis the two major forms of idiopathic ibd have been recognized as distinct disease entities for over a century . as early as 1761 morgagni described intestinal inflammation that in modern times we would recognize as crohn s disease.1 after the identification of the causative agent of tuberculosis by koch in 1882 , it became clear that some individuals had a disease similar to intestinal tuberculosis but did not bear the tubercle bacillus . reports by fenwick in 1889 , dalziel in 1913 , weiner in 1914 , moschowitz and wilensky in 1923 and 1927 , and goldfarb and suissman in 1931 predated the landmark publication of crohn , ginzburg , and oppenheimer in 1932 describing terminal ileitis.1,2 later , lockhart - mummery and morson3 described granulomatous colitis , and the disease process was understood to potentially affect the large bowel as well . generally , however , wilkes is credited with the first pathologic description of what was called simple ulcerative colitis in 1859.1 subsequently , in 1875 , wilkes and moxon described a syndrome of simple ulcerative colitis in greater detail.1 with the recognition of the nosologic distinctions of these diseases came attempts to treat them surgically and medically ( table 1 ) . table 1inflammatory bowel disease landmarks of the pastdistinction of crohn s disease and ulcerative colitis from infectious colitis made possible by the development of bacteriologic techniquesdistinction between crohn s disease and ulcerative colitis drawnthe inflammatory bowel diseases are understood to be immune - mediated diseasessulfasalazine and hydrocortisone used to treat ulcerative colitisthiopurine agents ( mercaptopurine and azathioprine ) used to treat ibdmethotrexate used to treat crohns disease and cyclosporine used to treat severe , steroid - refractory ulcerative colitisfamilial clustering implicates genetic factors in ibdbowel - sparing understood to be a key principle of surgical therapy , and the technique of stricturoplasty developedileal pouch - anal anastomosis developed as an alternative to ileostomy after total proctocolectomy for ulcerative colitisibd , inflammatory bowel disease inflammatory bowel disease landmarks of the past ibd , inflammatory bowel disease further experimentation revealed that the active moiety of sulfasalazine was 5-aminosalicylate ( 5-asa ) , whereas the sulfa moiety was responsible for many of the untoward effects of the drug.4 furthermore , delivery of the active 5-asa molecule was finally understood to rely upon the presence of the diazo bond between the sulfapyridine and 5-asa moieties for the delivery of the 5-asa to the distal bowel upon cleavage of the bond by the colonic flora.5 subsequently , a host of second - generation 5-asa agents were developed that utilized a variety other mechanisms to deliver the drug topically to the distal bowel . the first randomized controlled trial of hydrocortisone in ulcerative colitis proved the agent to be remarkably effective,8 and finally produced effective therapy for severe ulcerative colitis , which previously had been associated with a considerably high case fatality rate . with the landmark study of present et al.,10 mercaptopurine and azathioprine assumed their place in the ibd armamentarium as treatments primarily for patients who were steroid - refractory or steroiddependent . these agents are also thought to be effective in fistulizing crohn s disease.11 more recently , these agents have been more fully accepted as appropriate therapies for the treatment of steroid - dependent to - refractory ulcerative colitis , as well.12 a second - line agent shown to be effective in steroid - dependent crohn s disease is methotrexate . the introduction of cyclosporine for treatment of severe , steroid - refractory ulcerative colitis has provided an alternative to total proctocolectomy for some individuals.15 however , the narrow therapeutic margin of this agent has precluded widespread application , and in the united states its use remains confined largely to tertiary - care referral centers . this principle of sparing the small bowel was carried further with the development of stricturoplasty techniques.18 finally , the development in the 1980s of ileal pouch - anal anastomosis for patients with ulcerative colitis who required total proctocolectomy was a revolutionary surgical advance that has provided a much needed alternative to permanent endileostomy.19 at present , key concepts regarding the pathogenesis of inflammatory bowel disease are as follows ( table 2 ) . first , it is recognized that immune tolerance is the normal state of the intestinal immune system . third , the luminal flora is a key ingredient in the abnormal immune response of ibd . finally , it is recognized that both the innate and adaptive immune responses play integrated roles in the homeostasis of the intestinal mucosal immune response . table 2landmarks of the presentcontrolled physiologic inflammation is understood to result from the normal state of immune tolerance in the intestinedevelopment of genetic animal models of colitis demonstrate diverse disturbances of immunity and intestinal barrier function capable of inducing a phenotype of gut inflammationanimal models demonstrate the central role of gut flora as a necessary factor in colitis , with the flora further implicated in serologic studies in humansnod2/card15 demonstrated as the first disease - associated gene in crohn s disease after ibd1 localized to chromosome 16 by genomewide scanningdefective innate immune response implicated in crohn s disease by nod2 physiologybiologic agents developed through monoclonal antibody technology and molecular biology techniquesinfliximab , the first of a new class of anti - tnf biologics , shown to be effective in crohn s disease and ulcerative colitisthe heterogeneity of crohn s disease and ulcerative colitis demonstrated in genetic and serologic studiestnf , tumor necrosis factor landmarks of the present tnf , tumor necrosis factor for many years it has been recognized that the intestinal mucosa contains a resident population of inflammatory cells . in addition , first - degree relatives , in particular siblings , are at increased risk of ibd , and this risk is somewhat higher in individuals of ashkenazi jewish background.20 third , there is a high concordance for crohn s disease among monozygotic twins , whereas a somewhat lesser concordance is observed in monozygotic twins with one affected individual with ulcerative colitis.21 these clues have long suggested that genetics play an important role in susceptibility to the inflammatory bowel diseases . for many years , predominant theories have surrounded the notion that the mucosal inflammation of ibd is an appropriate response to pathogenic microbiota.22 candidates for pathogenic agents have been diverse over the years , and have included diplostreptococcus , entamoeba histolytica , and , most persistently , mycobacterium paratuberculosis.23 more recently , lines of evidence suggest that normal , nonpathogenic flora ( at least in the conventional sense of pathogenicity ) are a necessary though not sufficient factor in the pathogenesis of ibd , whereas firm evidence of a pathogenic agent has been lacking . these factors may be considered to be disease modifiers , and most prominently include smoking,24 nonsteroidal anti - inflammatory drugs,25 and prior appendectomy.26 in particular , both smoking and appendectomy have been recognized to be distinct risk factors with dichotomous outcomes for crohn s disease and ulcerative colitis . smoking has been associated with increased risk of crohn s disease as well as worse outcomes over the course of the disease,27 whereas for ulcerative colitis , smoking has been recognized as having a protective effect.2830 the effects of tobacco smoke are complex and poorly understood . the role of smoking does not appear to be as simple as the presence or absence of nicotine , which may be an effective therapy for ulcerative colitis but is poorly tolerated.31 current investigation focuses on the role of carbon monoxide in smoke as a factor capable of modifying the expression of inflammation in the gut.32 circumstantial evidence has long pointed to the critical role of the luminal flora in ibd . the evidence is strongest in crohn s disease and relatively sparse in ulcerative colitis . in clinical practice antibiotics are widely used to treat perianal disease in crohn s disease.33 evidence from clinical trials suggests some effect of antibiotic therapy as a treatment for crohn s disease , primarily in disease with colonic localization.34 in addition , imidazole antibiotics such as metronidazole35 and ornidazole36 have been demonstrated reproducibly to have a prophylactic effect for disease recurrence after ileal resection . experiments carried out in the 1980s demonstrated that the luminal contents contained factors , most likely bacterial factors , capable of reinducing inflammation in quiescent diverted bowel.37 in addition , elemental diet has been demonstrated to be an effective therapy for crohn s disease.38 effects of elemental diet may be complex , but are thought to include a favorable effect on the bowel flora composition that may minimize immunologic stimulation of the host immune response . the clearest demonstration of benefit has been with a preparation known as vsl#3 in the treatment and prophylaxis of pouchitis occurring in ileal pouches created as curative therapy for ulcerative colitis.39,40 other studies have suggested modest benefit in ulcerative colitis and crohn s disease for a variety of probiotic agents , including nissle 1917 and vsl#3.41,42 finally , one intriguing study demonstrated excessive responsiveness of lamina propria mononuclear cells to autologous gut bacteria among patients with ibd.43 animal models of colitis have also provided essential clues to the role of the luminal flora in the pathogenesis of ibd . this is clearly suggested by observations of genetic and serologic heterogeneity among individuals diagnosed with ulcerative colitis or crohn s disease.4548 in addition , the role of the intestinal flora in driving gut mucosal inflammation has been clearly demonstrated in these diverse models . furthermore , it has become clear that the characteristics of the intestinal flora also shape the phenotypic characteristics of the gut inflammation . colonization with the entire mixture of the usual bacterial flora present in the cecum culminates in an aggressive colitis , whereas the introduction of cecal bacteria plus the probiotic species lactobacillus gg results in protection from colitis altogether.49 these experiments suggest that specific nonpathogenic bacteria may shape the expression of disease severity in profound ways . however , great strides have been made in the identification of genetic defects associated with crohn s disease and ulcerative colitis . using these techniques , the first such ibd gene identified was the nod2/card15 gene on chromosome 16 , associated with the ibd1 locus found on genomewide scanning.5254 specific disease associated polymorphisms included the r702w , g908r , and 33 mutations associated with relative risks for crohn s disease of 14.3 , 34.1 , and 17.64 for homozygous recessive individuals over control populations.5254 explication of the cellular function of nod2 has shed light on the nature of the underlying immune defects of crohn s disease . these are loss - of - function mutations , which appear to prevent the binding of muramyl dipeptide , which is ubiquitously present in virtually all bacterial cell walls.55 downstream events lead to the activation of nfb through an interaction with the card domains.56 thus , it appears that nod2 is intimately involved in intracellular bacterial sensing and generation of the innate immune response.57 less certain is how a loss - of - function mutation in the leucine - rich repeat causes the inflammation of crohn s disease . the dendritic cells of the gut have become a focal point in our understanding of intestinal immune responses.59 elegant immunofluorescent microscopic techniques have demonstrated large populations of dendritic cells localized just beneath the intestinal epithelial cells.60 these arborized cells project long podocytes through the interstices of the intestinal epithelial cells and out to the intestinal lumen . it is believed that the dendritic cells process these antigens , and then present them to the cells in the peyer s patches as well as in the mesenteric lymph nodes . depending upon the nature of the antigen and the activation state of the dendritic cell , the end result may be immune activation or tolerization . in addition , they noted that varied conditions characterized by neutrophil defects are also commonly associated with crohn s disease or a crohn s disease - like phenotype . they reasoned , therefore , that individuals with crohn s disease may also have neutrophil defects , and that stimulating neutrophils may be beneficial in the treatment of crohn s disease . this hypothesis led them to test granulocyte - colony stimulating factor ( g - csf ) and granulocyte - macrophage ( gm)-csf in open - label pilot trials in crohn s disease , with promising early results.64,65 a subsequent phase 2 study of sargramostim ( gm - csf ) in crohn s disease demonstrated evidence of activity of this agent in decreasing symptoms of active disease.66 unfortunately , a phase 3 study failed to demonstrate efficacy over placebo , although a separate study demonstrated efficacy of this agent in steroid sparing.67 more detailed descriptions of the immunologic pathways of the adaptive immune responses in crohn s disease and ulcerative colitis have also led to new and specific therapeutic agents . th1 responses are most closely associated with the cell - mediated immune responses that characterize crohn s disease.69 by contrast , th2 responses are associated with humoral immunity and with a cytokine profile comprising il-4 , il-5 , il-6 , and il-13 . early reports from the netherlands described two cases of crohn s disease treated with a chimeric monoclonal anti - tnf antibody called ca2.70 this was followed by a case series of ten patients with crohn s disease , who experienced a high rate of response and rapid mucosal healing on endoscopy.71 the first randomized controlled trial by targan et al.72 demonstrated that short - term induction occurred rapidly and in the majority of patients with active disease unresponsive to other therapies . however , findings of long - term studies lasting from 6 to 12 months suggest that the efficacy of these agents is equivalent to that seen with infliximab.77,78 other issues with the anti - tnf biologics relate to weighing the risks and benefits of these agents for specific patients.79 described risks include rare occurrence of opportunistic infections , including intracellular infections such as tuberculosis and fungal and viral infections , demyelinating disorders , and rare reports of lymphoma.80 it will be critical to define populations for whom these risks are appropriate , or alternatively to identify patients at least risk for developing these complications . in addition , studies using the humanized anti - il-2 receptor antibody daclizumab showed this agent to be ineffective as a treatment for ulcerative colitis.82 early studies with anti - il-12 antibody appear to demonstrate some activity in active crohn s disease.83 this agent , which binds to epitopes in the p40 subunit of il-12 , may also bind to the same subunit as a component of the heterodimeric proinflammatory cytokine il-23 . of note , the il-23 receptor gene was recently associated with crohn s disease ; namely , polymorphisms in this gene appear to confer protection against the disease.84 this would suggest that agents that inhibit il-23 may prove to be effective in crohn s disease . in the early 1990s , the details of cellular recruitment and adhesion were explicated in fine detail.85 when inflammation is present within a tissue , a series of events occur in rapid succession . first , the endothelium becomes activated , and the expression of e - and p - selectins on the endothelial surface expression occurs . this , in turn , causes rolling of the leukocyte along the endothelial surface and activation of integrin expression on the leukocyte surface.85 the integrins then bind tightly to cellular adhesion molecules such as vcam , icam , and madcam86 on the endothelial surface . a large phase 2 study of natalizumab in crohn s disease appeared to demonstrate short - term activity but failed to meet its primary end point in a statistically significant way.87 a follow - up phase 3 study again demonstrated activity of this agent , but missed its primary end point , in large part owing to a high placebo response rate.88 however , a follow - on study demonstrated robust maintenance efficacy with natalizumab.88 an additional short - term study finally confirmed the efficacy of natalizumab among patients with elevated c - reactive protein.89 unexpectedly , however , three cases of progressive multifocal leukoencephalopathy ( pml ) , an otherwise extremely rare and usually fatal central nervous system disease caused by the jc virus , were found among 3000 patients treated for either multiple sclerosis or crohn s disease with this agent.90 a second selective adhesion molecule inhibitor , mln-02 , has been demonstrated in a phase 2 study to be efficacious in patients with active ulcerative colitis failing treatment with 5-aminosalicylates.91 a second pilot study was performed in crohn s disease with promising effect.92 at this time , it is unknown if the more specific adhesion of this antibody to 47 integrin , the ligand of madcam ( mucosal addressin cellular adhesion molecule , located only in gut endothelium ) , will improve the safety profile of this agent and avoid the occurrence of pml . as with the neuronal synapse , a variety of stimulatory and inhibitory signals are integrated when the antigen - presenting cell interacts with a t cell , yielding a net overall effect of activation or inhibition of the immune response.93,94 the basis of the antigen - specific response occurs with the presentation of an antigenic epitope in the context of major histocompatibility complex ( mhc ) class ii . abatacept is a fusion molecule of ctl4 and immunoglobulin , known as ctla - ig.96 this agent is approved in the united states for the treatment of rheumatoid arthritis and has been shown to be effective in patients who have not responded to anti - tnf biologic agents in that disease state.96 in principle , this agent would be of considerable interest as a potential therapy for ibd . in summary , in recent times we have learned that the intestinal flora is critical in generating the immune response of ibd . in crohn s disease an excess of effector t cells , characterized as either th1- or th2-like , is associated with the distinct clinical manifestations of crohn s disease and ulcerative colitis , respectively . we will no longer blithely talk about curing crohn s disease or ulcerative colitis ; rather , it is very likely that we will talk about very specific cures for each one of the many varied genetic or acquired aberrations that cause the crohn s disease or ulcerative colitis phenotype . we will also finally elucidate the environmental factors that have contributed to the rise of these diseases in westernized societies . in summary , a survey of the past and present advances in the inflammatory bowel diseases provides much cause for optimism . the accelerating pace of discovery in crohn s disease and ulcerative colitis provides hope that each year will bring a better life to the growing number of individuals who suffer from these curious diseases .

however , progression of the scoliotic deformity after posterior spinal fusion has been described [ 14 ] . various etiologies for loss of correction have been proposed , including pseudarthrosis , implant failure , incorrect selection of fusion levels , adding - on , biologic plasticity of the fusion mass [ 6 , 7 ] , and the crankshaft phenomenon ( continued anterior growth of the spine ) . recent studies have proposed that pedicle screws are better able to control the three columns of the spine and may decrease the incidence of deformity progression after posterior spinal instrumentation and fusion [ 9 , 10 ] . in our anecdotal experience , this has not been the case . therefore , the purpose of this study was two - fold : ( 1 ) to assess the incidence of spinal deformity progression after posterior segmental instrumentation and fusion in the treatment of idiopathic scoliosis at a single institution , and ( 2 ) to analyze the variables associated with deformity progression in this patient population . after obtaining institutional review board approval , a retrospective review of a consecutive series of idiopathic scoliosis patients treated at a single institution was performed . all patients with major thoracic curves ( lenke types 14 ) treated with posterior segmental instrumentation were included if they had minimum 2-year clinical and radiographic follow - up . exclusion criteria included anterior spinal release or instrumentation and fusion , and incomplete radiographic or clinical data . data were collected from the pre - operative , immediate post - operative ( 46 weeks post - op ) , 1-year post - operative , and 2-year post - operative visits . a total of 402 idiopathic scoliosis patients were surgically treated at our institution between november 2003 and august 2008 . of these , 285 gave their consent to enter the prospective patient database that was used to analyze the current cohort , and 230 of the 285 patients ( 80 % ) had at least 2-year post - operative follow - up . eighty - nine patients were included in this analysis based on our inclusion criteria of main thoracic scoliosis ( lenke types 14 ) and posterior - only segmental instrumentation . segmental instrumentation was defined as more than 80 % of fixation points were instrumented . thirty - three percent of patients had all pedicle screw constructs , and 67 % of patients had hybrid constructs with hooks , sublamina wires , and pedicle screws . although the instrumentation type differed , similar techniques were used to perform the deformity correction and to achieve a solid arthrodesis . deformity correction was achieved using compression techniques on the convexity and distraction on the concavity , as well as in situ bending . thorough decortication of the posterior elements was performed at the end of the procedure and local autograft as well as cancellous allograft bone chips were used in the fusion mass . clinical measures included height , weight , and inclinometer measurements ( proximal thoracic , thoracic , and thoracolumbar ) . height measurements were performed at each clinic visit using an ayrton stadiometer model s100 ( prior lake , mn ) , with a reported precision of 0.15 cm . radiographic parameters included proximal thoracic , main thoracic , and thoracolumbar coronal cobb angles , thoracic and thoracolumbar sagittal cobb angles , apical vertebral translation ( avt ) , coronal and sagittal balance , t1 tilt , risser grade , and state of the triradiate cartilage ( trc ) . we also determined the implant density for each patient , as well as the percent of fixation sites instrumented with pedicle screws . subjective scores were collected at each clinic visit using the scoliosis research society questionnaire ( srs-22 ) . based on previous studies , deformity progression was defined as a 10 increase in the major coronal cobb angle between the first post - operative and 2-year post - operative radiographs [ 1113 ] . the stable vertebra ( sv ) ( most distal vertebra in the major curve that is bisected by the center sacral vertical line ) , neutral vertebra ( nv ) ( most distal vertebra in the major curve that is neutrally rotated based on the symmetric appearance of the pedicles ) , touch vertebra ( tv ) ( last vertebra with the pedicle touched by the center sacral vertical line ) , and the last instrumented vertebra ( liv ) were recorded . clinical and radiographic data between the two groups [ deformity progression versus no progression after instrumented fusion ( stable ) ] were analyzed to determine the variables associated with deformity progression . patient and curve characteristics were summarized pre - operatively , at first erect , at 1-year follow - up , and at 2-year follow - up for all subjects and between deformity progression and control subjects . continuous characteristics were compared using univariate logistic regression and ordinal characteristics were compared using the cochran armitage test for trend or a mann whitney u - test . change in measurement over time was analyzed between deformity groups using mixed model analysis with a compound symmetry correlation structure . univariable and multivariable logistic regression was used to assess pre - operative and post - operative risk factors of deformity progression . all tests were two - sided and p - values less than 0.05 were considered significant . clinical and radiographic data between the two groups [ deformity progression versus no progression after instrumented fusion ( stable ) ] were analyzed to determine the variables associated with deformity progression . patient and curve characteristics were summarized pre - operatively , at first erect , at 1-year follow - up , and at 2-year follow - up for all subjects and between deformity progression and control subjects . continuous characteristics were compared using univariate logistic regression and ordinal characteristics were compared using the cochran armitage test for trend or a mann whitney u - test . change in measurement over time was analyzed between deformity groups using mixed model analysis with a compound symmetry correlation structure . univariable and multivariable logistic regression was used to assess pre - operative and post - operative risk factors of deformity progression . all tests were two - sided and p - values less than 0.05 were considered significant . seventy - six patients ( 86 % ) were in the stable control group and 13 patients ( 14 % ) were found to experience deformity progression of more than 10 in the coronal plane within 2 years of surgery ( fig . 1 ) . the stable group comprised 14 boys and 62 girls , and the deformity progression group comprised three boys and ten girls . the average follow - up for this cohort was 4.2 years ( range 2.08.3 years).fig . 1a 12 years and 8 months old , risser grade 0 , pre - menarchal female with lenke type 1b idiopathic scoliosis . pre - operative posterior - anterior ( pa ) ( a ) , lateral ( b ) , left bend ( c ) , and right bend ( d ) radiographs demonstrate a 57 main thoracic curve . first - erect post - operative pa ( e ) and lateral ( f ) , and 5-year post - operative pa ( g ) and lateral ( h ) radiographs demonstrate correction of the coronal plane deformity down to 15 with a t5t12 posterior instrumented spinal fusion . the deformity then progressed to 41 at final follow - up . at the most recent follow - up , the patient has developed a 16 thoracic rib prominence with worsening trunk shift to the right and waist asymmetry a 12 years and 8 months old , risser grade 0 , pre - menarchal female with lenke type 1b idiopathic scoliosis . pre - operative posterior - anterior ( pa ) ( a ) , lateral ( b ) , left bend ( c ) , and right bend ( d ) radiographs demonstrate a 57 main thoracic curve . first - erect post - operative pa ( e ) and lateral ( f ) , and 5-year post - operative pa ( g ) and lateral ( h ) radiographs demonstrate correction of the coronal plane deformity down to 15 with a t5t12 posterior instrumented spinal fusion . the deformity then progressed to 41 at final follow - up . at the most recent follow - up , the patient has developed a 16 thoracic rib prominence with worsening trunk shift to the right and waist asymmetry patients in the deformity progression group tended to be younger based on chronologic age . the average age in the deformity progression group was 13.7 years and in the stable group it was 14.7 years ( p = 0.14 ) . the 14 boys in the stable group had a median age of 15.1 years and the 62 girls in the stable group had a median age of 14.2 years . the three boys in the deformity progression group had a median age of 13.7 years and the ten girls in the deformity progression group had a median age of 12.6 years . there was a significant difference in the change in height from 1-year follow - up to 2-year follow - up with respect to deformity progression . the average change in height for the deformity progression group was 2.6 cm , compared to 0.8 cm for the stable group ( p = 0.01 ) . there was also a significant difference in the change in height from pre - operative to 2-year follow - up with respect to deformity progression . the average change in height for the deformity progression group was 6.4 cm , compared to 3.5 cm for the stable group ( p = 0.003 ) . the implants were removed at 2.5 and 3 years post - op , respectively . they were followed for at least 1 year after the revision surgery , with no significant increase in the spinal deformity at the final follow - up . one patient underwent removal of implants at 3 years post - op for late infection after her deformity had already progressed . at the final follow - up a second patient required an irrigation and debridement on post - operative day 10 for persistent wound drainage . additionally , one patient in the deformity progression group was found to have a broken screw at 1.5 years post - op . further evaluation with a computed tomography scan and nuclear bone scan over the next year did not demonstrate pseudarthrosis or additional implant failure . tables 1 , 2 , 3 , and 4 present radiographic data from the pre - operative , first - erect post - operative , 1-year post - operative , and 2-year post - operative visits , respectively . there was no significant relationship between menarchal status , trc , lenke classification , t1 tilt , or coronal balance and deformity progression . while more than 50 % of the patients in the deformity progression group were risser grade 0 or 1 , the distribution of the risser grades between the two groups was not statistically significant ( table 5 ) . the mean change in the major coronal cobb angle was 1.7 in the stable group ( pre - op : 58.5 ; post - op : 22.1 ; 2-year post - op : 23.8 ) versus 14.7 in the deformity progression group ( pre - op : 52.9 ; post - op : 17.5 ; 2-year post - op : 32.2).table 1pre - operative clinical and radiographic datavariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdage ( years)14.52.1913.71.8714.72.220.14height ( cm)160.18.84161.311.86159.98.290.59weight ( cm)54.711.4552.112.0655.211.360.37proximal inclinometer [ median ( iqr)]4(26)5(03)4(26)0.16thoracic inclinometer [ median ( iqr)]15(1215)12.5(1214)15(1215)0.49thoracolumbar inclinometer [ median ( iqr)]6(47)7(27)6(47)0.95touch vertebrae [ median ( iqr)]22(2022)22(2223)22(2021)0.11cobb proximal thoracic27.69.7126.79.1727.89.850.70cobb main thoracic57.711.8152.97.7858.512.220.12cobb thoracolumbar36.111.7233.212.4036.611.610.32avt proximal4.15.3454.923.95.430.39avt main48.415.1843.210.1449.215.770.19avt lumbar16.712.7115.412.2616.912.850.53coronal balance15.710.7720.813.3014.810.120.12t1 tilt3.84.4343.673.84.570.28sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translationtable 2first - erect clinical and radiographic datavariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdtime from surgery to first erect ( days)29.713.7727.111.7630.114.100.45percent correction57.311.8452.67.7658.212.260.12cobb proximal thoracic12.18.4210.812.7612.37.530.54cobb main thoracic21.48.4417.56.6922.18.560.08cobb thoracolumbar19.610.0818.48.0219.810.420.64avt proximal4.14.926.35.543.84.740.19avt main13.28.539.55.5613.88.820.20avt lumbar17.313.0115.514.6817.612.780.27coronal balance16.211.6917.415.791610.960.21t1 tilt4.64.415.55.974.44.120.49uiv tilt11.71.323.68.03.16.50.70liv tilt21.22.034.58.56.17.40.52sd standard deviation ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebratable 3one - year post - operative clinical and radiographic datavariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdtime from surgery to 1-year follow - up ( days)369.659.22349.279.65373.254.720.18height ( cm)164.28.2316511.711647.540.69weight ( cm)59.413.4555.411.0360.113.790.24proximal inclinometer [ median ( iqr)]2(03)2.5(04)2(02)0.18thoracic inclinometer [ median ( iqr)]7(57)7(47)6.5(57)0.69thoracolumbar inclinometer [ median ( iqr)]0(02)0(02)1(02)0.84cobb proximal thoracic13.99.2415.59.8913.79.170.52cobb main thoracic24.98.5526.66.6324.68.850.43cobb thoracolumbar19.410.1019.19.3319.410.300.91avt proximal4.35.264.23.724.35.510.91avt main16.811.5716.58.5716.812.080.93avt lumbar15.310.7212.29.0515.810.960.16coronal balance13.79.4515.212.8613.58.790.39t1 tilt4.84.795.34.974.74.790.58uiv tilt7.45.359.54.0375.490.09liv tilt8.86.3611.26.078.46.360.21sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebratable 4two - year post - operative clinical and radiographic datavariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdtime from surgery to 2-year follow - up ( days)780.4134.64798.7123.31777.4136.940.61height ( cm)164.410.78167.612.98163.910.360.24weight ( cm)60.614.3458.110.1961.114.950.49proximal inclinometer [ median ( iqr)]1(02)0(02)2(02)0.54thoracic inclinometer [ median ( iqr)]7(47)6.5(58)7(47)0.55thoracolumbar inclinometer [ median ( iqr)]0(02)0(02)0(02)0.65cobb proximal thoracic13.68.2915.59.4513.38.100.39cobb main thoracic259.3032.27.3223.89.090.006cobb thoracolumbar18.210.1521.99.8517.510.130.15avt proximal4.75.636.86.354.45.460.21avt main19.211.8222.212.9518.711.630.28avt lumbar14.310.512.89.9914.510.630.44coronal balance11.99.5112.88.8611.89.660.04t1 tilt4.64.756.95.574.24.520.20uiv tilt6.96.0111.65.246.15.790.02liv tilt8.85.90134.678.15.820.04sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebratable 5pre - operative and 2-year post - operative risser gradesvariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuepre - operative risser grade0.32 014 ( 16 % ) 4 ( 31 % ) 10 ( 13 % ) 117 ( 19 % ) 3 ( 23 % ) 14 ( 18 % ) 211 ( 12 % ) 1 ( 8 % ) 10 ( 13 % ) 315 ( 17 % ) 2 ( 15 % ) 13 ( 17 % ) 413 ( 15 % ) 2 ( 15 % ) 11 ( 15 % ) 519 ( 21 % ) 1 ( 8 % ) 18 ( 24 % ) two - year post - operative risser grade0.46 00 ( 0 % ) 0 ( 0 % ) 0 ( 0 % ) 15 ( 6 % ) 0 ( 0 % ) 5 ( 7 % ) 28 ( 9 % ) 2 ( 15 % ) 6 ( 8 % ) 36 ( 7 % ) 1 ( 8 % ) 5 ( 7 % ) 426 ( 29 % ) 4 ( 31 % ) 22 ( 28 % ) 544 ( 49 % ) 6 ( 46 % ) 38 ( 50 % ) pre - operative clinical and radiographic data sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation first - erect clinical and radiographic data sd standard deviation ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebra one - year post - operative clinical and radiographic data sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebra two - year post - operative clinical and radiographic data sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebra pre - operative and 2-year post - operative risser grades the mean absolute liv angulation for the deformity progression group was 4.5 8.5 at first erect and for the stable group , it was 6.1 7.4 ( p = 0.52 ) . at 2-year follow - up , however , the mean absolute liv angulation for the deformity progression group was 13.0 4.7 and for the stable group , it was 8.1 5.8 ( p = 0.04 ) . the mean change in liv angulation from first erect to 2-year follow - up for the deformity progression group was 5.5 4.4 , compared to 0.5 4.7 for the stable group ( p = 0.005 ) . the mean absolute upper instrumented vertebra ( uiv ) angulation for the deformity progression group was 3.6 8.0 at first erect and for the stable group , it was 3.1 6.5 ( p = 0.70 ) . at 2-year follow - up , however , the mean absolute uiv angulation for the deformity progression group was 11.6 5.2 and for the stable group , it was 6.1 5.8 ( p = 0.02 ) . the median change in uiv angulation from first erect to 2-year follow - up for the deformity progression group was 4.3 5.8 , compared to 0.8 5.3 for the stable group ( p = 0.03 ) . the mean absolute main thoracic avt for the deformity progression group was 9.5 5.6 mm at first erect and for the stable group , it was 13.8 8.8 mm ( p = 0.20 ) . at 2-year follow - up the mean absolute avt for the deformity progression group was 22.2 12.9 mm and for the stable group , it was 18.7 11.6 mm ( p = 0.28 ) . there was , however , a significant median change in avt in the deformity progression group from first erect to 2-year follow - up of 12.6 11.3 mm , compared to 4.8 9.3 mm for the stable group ( p = 0.02 ) . both the percent of fixation sites instrumented with pedicle screws and implant density were significantly different between the two groups . the percent of fixation sites instrumented with pedicle screws ( 71 % ) was significantly greater in the deformity progression group compared to the stable group ( 47 % ) ( p = 0.004 ) . additionally , the mean implant density in the deformity progression group ( 1.79 ) was also significantly greater than the median implant density in the stable group ( 1.63 ) ( p = 0.029 ) . selection of fusion levels was also assessed in these patients with main thoracic curves ( table 6 ) . patients in the deformity progression group had a significantly greater difference between the lowest instrumented vertebra and stable vertebra compared to the patients in the stable group ( p = 0.001 ) . this was also true for the difference between the lowest instrumented vertebra and the touch vertebra between the two groups ( p = 0.04 ) . the difference between the liv and the neutral vertebra however was not significantly different between the two groups ( p = 0.43).table 6selection of fusion levelsvariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdliv sv0.31.91.80.9901.90.001liv liv lower instrumented vertebra , sv stable vertebra , nv neutral vertebra , tv vertebra last touched by the center sacral vertical line selection of fusion levels sd standard deviation ; liv lower instrumented vertebra , sv stable vertebra , nv neutral vertebra , tv vertebra last touched by the center sacral vertical line table 7 presents the srs-22 scores from the pre - operative and 2-year post - operative visits . subjects in the deformity progression group showed no significant change in the appearance domain score from pre - operative to 2-year follow - up ( p = 0.221 ) . however , subjects in the stable group showed a significant increase ( improvement ) in the appearance domain score , with a median pre - operative score of 3.4 0.6 and a 2-year follow - up score of 4.4 0.53 ( p < 0.001 ) . subjects in the deformity progression group showed a significantly lower srs-30 appearance domain score at 2-year follow - up than the stable group . the mean score at 2-year follow - up for the deformity progression group was 4.0 0.66 and for the stable group , it was 4.4 0.53 ( p = 0.044).table 7pre - operative and 2-year post - operative scoliosis research society questionnaire ( srs-22 ) scoresvariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdpre - operative srs-22 scores pain4.10.634.20.664.10.630.88 appearance3.40.643.50.703.40.630.86 activity4.20.454.30.564.20.430.80 mental3.90.673.80.823.90.650.43 satisfaction3.21.523.11.713.31.490.67 subtotal3.90.433.90.573.90.410.91 total score3.90.423.90.603.90.390.87two - year post - operative srs-22 scores pain4.40.604.20.654.40.590.21 appearance4.30.5640.664.40.530.02 activity4.40.354.30.374.40.350.21 mental4.20.7340.514.20.760.56 satisfaction4.40.794.30.944.40.770.61 subtotal4.30.434.10.384.30.430.09 total score4.30.434.10.414.40.420.10 pre - operative and 2-year post - operative scoliosis research society questionnaire ( srs-22 ) scores the average age in the deformity progression group was 13.7 years and in the stable group it was 14.7 years ( p = 0.14 ) . the 14 boys in the stable group had a median age of 15.1 years and the 62 girls in the stable group had a median age of 14.2 years . the three boys in the deformity progression group had a median age of 13.7 years and the ten girls in the deformity progression group had a median age of 12.6 years . there was a significant difference in the change in height from 1-year follow - up to 2-year follow - up with respect to deformity progression . the average change in height for the deformity progression group was 2.6 cm , compared to 0.8 cm for the stable group ( p = 0.01 ) . there was also a significant difference in the change in height from pre - operative to 2-year follow - up with respect to deformity progression . the average change in height for the deformity progression group was 6.4 cm , compared to 3.5 cm for the stable group ( p = 0.003 ) . the implants were removed at 2.5 and 3 years post - op , respectively . they were followed for at least 1 year after the revision surgery , with no significant increase in the spinal deformity at the final follow - up . one patient underwent removal of implants at 3 years post - op for late infection after her deformity had already progressed . at the final follow - up a second patient required an irrigation and debridement on post - operative day 10 for persistent wound drainage . additionally , one patient in the deformity progression group was found to have a broken screw at 1.5 years post - op . . further evaluation with a computed tomography scan and nuclear bone scan over the next year did not demonstrate pseudarthrosis or additional implant failure . tables 1 , 2 , 3 , and 4 present radiographic data from the pre - operative , first - erect post - operative , 1-year post - operative , and 2-year post - operative visits , respectively . there was no significant relationship between menarchal status , trc , lenke classification , t1 tilt , or coronal balance and deformity progression . while more than 50 % of the patients in the deformity progression group were risser grade 0 or 1 , the distribution of the risser grades between the two groups was not statistically significant ( table 5 ) . the mean change in the major coronal cobb angle was 1.7 in the stable group ( pre - op : 58.5 ; post - op : 22.1 ; 2-year post - op : 23.8 ) versus 14.7 in the deformity progression group ( pre - op : 52.9 ; post - op : 17.5 ; 2-year post - op : 32.2).table 1pre - operative clinical and radiographic datavariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdage ( years)14.52.1913.71.8714.72.220.14height ( cm)160.18.84161.311.86159.98.290.59weight ( cm)54.711.4552.112.0655.211.360.37proximal inclinometer [ median ( iqr)]4(26)5(03)4(26)0.16thoracic inclinometer [ median ( iqr)]15(1215)12.5(1214)15(1215)0.49thoracolumbar inclinometer [ median ( iqr)]6(47)7(27)6(47)0.95touch vertebrae [ median ( iqr)]22(2022)22(2223)22(2021)0.11cobb proximal thoracic27.69.7126.79.1727.89.850.70cobb main thoracic57.711.8152.97.7858.512.220.12cobb thoracolumbar36.111.7233.212.4036.611.610.32avt proximal4.15.3454.923.95.430.39avt main48.415.1843.210.1449.215.770.19avt lumbar16.712.7115.412.2616.912.850.53coronal balance15.710.7720.813.3014.810.120.12t1 tilt3.84.4343.673.84.570.28sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translationtable 2first - erect clinical and radiographic datavariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdtime from surgery to first erect ( days)29.713.7727.111.7630.114.100.45percent correction57.311.8452.67.7658.212.260.12cobb proximal thoracic12.18.4210.812.7612.37.530.54cobb main thoracic21.48.4417.56.6922.18.560.08cobb thoracolumbar19.610.0818.48.0219.810.420.64avt proximal4.14.926.35.543.84.740.19avt main13.28.539.55.5613.88.820.20avt lumbar17.313.0115.514.6817.612.780.27coronal balance16.211.6917.415.791610.960.21t1 tilt4.64.415.55.974.44.120.49uiv tilt11.71.323.68.03.16.50.70liv tilt21.22.034.58.56.17.40.52sd standard deviation ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebratable 3one - year post - operative clinical and radiographic datavariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdtime from surgery to 1-year follow - up ( days)369.659.22349.279.65373.254.720.18height ( cm)164.28.2316511.711647.540.69weight ( cm)59.413.4555.411.0360.113.790.24proximal inclinometer [ median ( iqr)]2(03)2.5(04)2(02)0.18thoracic inclinometer [ median ( iqr)]7(57)7(47)6.5(57)0.69thoracolumbar inclinometer [ median ( iqr)]0(02)0(02)1(02)0.84cobb proximal thoracic13.99.2415.59.8913.79.170.52cobb main thoracic24.98.5526.66.6324.68.850.43cobb thoracolumbar19.410.1019.19.3319.410.300.91avt proximal4.35.264.23.724.35.510.91avt main16.811.5716.58.5716.812.080.93avt lumbar15.310.7212.29.0515.810.960.16coronal balance13.79.4515.212.8613.58.790.39t1 tilt4.84.795.34.974.74.790.58uiv tilt7.45.359.54.0375.490.09liv tilt8.86.3611.26.078.46.360.21sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebratable 4two - year post - operative clinical and radiographic datavariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdtime from surgery to 2-year follow - up ( days)780.4134.64798.7123.31777.4136.940.61height ( cm)164.410.78167.612.98163.910.360.24weight ( cm)60.614.3458.110.1961.114.950.49proximal inclinometer [ median ( iqr)]1(02)0(02)2(02)0.54thoracic inclinometer [ median ( iqr)]7(47)6.5(58)7(47)0.55thoracolumbar inclinometer [ median ( iqr)]0(02)0(02)0(02)0.65cobb proximal thoracic13.68.2915.59.4513.38.100.39cobb main thoracic259.3032.27.3223.89.090.006cobb thoracolumbar18.210.1521.99.8517.510.130.15avt proximal4.75.636.86.354.45.460.21avt main19.211.8222.212.9518.711.630.28avt lumbar14.310.512.89.9914.510.630.44coronal balance11.99.5112.88.8611.89.660.04t1 tilt4.64.756.95.574.24.520.20uiv tilt6.96.0111.65.246.15.790.02liv tilt8.85.90134.678.15.820.04sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebratable 5pre - operative and 2-year post - operative risser gradesvariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuepre - operative risser grade0.32 014 ( 16 % ) 4 ( 31 % ) 10 ( 13 % ) 117 ( 19 % ) 3 ( 23 % ) 14 ( 18 % ) 211 ( 12 % ) 1 ( 8 % ) 10 ( 13 % ) 315 ( 17 % ) 2 ( 15 % ) 13 ( 17 % ) 413 ( 15 % ) 2 ( 15 % ) 11 ( 15 % ) 519 ( 21 % ) 1 ( 8 % ) 18 ( 24 % ) two - year post - operative risser grade0.46 00 ( 0 % ) 0 ( 0 % ) 0 ( 0 % ) 15 ( 6 % ) 0 ( 0 % ) 5 ( 7 % ) 28 ( 9 % ) 2 ( 15 % ) 6 ( 8 % ) 36 ( 7 % ) 1 ( 8 % ) 5 ( 7 % ) 426 ( 29 % ) 4 ( 31 % ) 22 ( 28 % ) 544 ( 49 % ) 6 ( 46 % ) 38 ( 50 % ) pre - operative clinical and radiographic data sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation first - erect clinical and radiographic data sd standard deviation ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebra one - year post - operative clinical and radiographic data sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebra two - year post - operative clinical and radiographic data sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebra pre - operative and 2-year post - operative risser grades the mean absolute liv angulation for the deformity progression group was 4.5 8.5 at first erect and for the stable group , it was 6.1 7.4 ( p = 0.52 ) . at 2-year follow - up , however , the mean absolute liv angulation for the deformity progression group was 13.0 4.7 and for the stable group , it was 8.1 5.8 ( p = 0.04 ) . the mean change in liv angulation from first erect to 2-year follow - up for the deformity progression group was 5.5 4.4 , compared to 0.5 4.7 for the stable group ( p = 0.005 ) . the mean absolute upper instrumented vertebra ( uiv ) angulation for the deformity progression group was 3.6 8.0 at first erect and for the stable group , it was 3.1 6.5 ( p = 0.70 ) . at 2-year follow - up , however , the mean absolute uiv angulation for the deformity progression group was 11.6 5.2 and for the stable group , it was 6.1 5.8 ( p = 0.02 ) . the median change in uiv angulation from first erect to 2-year follow - up for the deformity progression group was 4.3 5.8 , compared to 0.8 5.3 for the stable group ( p = 0.03 ) . the mean absolute main thoracic avt for the deformity progression group was 9.5 5.6 mm at first erect and for the stable group , it was 13.8 8.8 mm ( p = 0.20 ) . at 2-year follow - up the mean absolute avt for the deformity progression group was 22.2 12.9 mm and for the stable group , it was 18.7 11.6 mm ( p = 0.28 ) . there was , however , a significant median change in avt in the deformity progression group from first erect to 2-year follow - up of 12.6 11.3 mm , compared to 4.8 9.3 mm for the stable group ( p = 0.02 ) . both the percent of fixation sites instrumented with pedicle screws and implant density were significantly different between the two groups . the percent of fixation sites instrumented with pedicle screws ( 71 % ) was significantly greater in the deformity progression group compared to the stable group ( 47 % ) ( p = 0.004 ) . additionally , the mean implant density in the deformity progression group ( 1.79 ) was also significantly greater than the median implant density in the stable group ( 1.63 ) ( p = 0.029 ) . selection of fusion levels was also assessed in these patients with main thoracic curves ( table 6 ) . patients in the deformity progression group had a significantly greater difference between the lowest instrumented vertebra and stable vertebra compared to the patients in the stable group ( p = 0.001 ) . this was also true for the difference between the lowest instrumented vertebra and the touch vertebra between the two groups ( p = 0.04 ) . the difference between the liv and the neutral vertebra however was not significantly different between the two groups ( p = 0.43).table 6selection of fusion levelsvariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdliv sv0.31.91.80.9901.90.001liv nv2.42.221.92.292.42.210.43liv tv0.42.331.71.930.22.330.04sd standard deviation ; liv lower instrumented vertebra , sv stable vertebra , nv neutral vertebra , tv vertebra last touched by the center sacral vertical line selection of fusion levels sd standard deviation ; liv lower instrumented vertebra , sv stable vertebra , nv neutral vertebra , tv vertebra last touched by the center sacral vertical line table 7 presents the srs-22 scores from the pre - operative and 2-year post - operative visits . subjects in the deformity progression group showed no significant change in the appearance domain score from pre - operative to 2-year follow - up ( p = 0.221 ) . however , subjects in the stable group showed a significant increase ( improvement ) in the appearance domain score , with a median pre - operative score of 3.4 0.6 and a 2-year follow - up score of 4.4 0.53 ( p < 0.001 ) . subjects in the deformity progression group showed a significantly lower srs-30 appearance domain score at 2-year follow - up than the stable group . the mean score at 2-year follow - up for the deformity progression group was 4.0 0.66 and for the stable group , it was 4.4 0.53 ( p = 0.044).table 7pre - operative and 2-year post - operative scoliosis research society questionnaire ( srs-22 ) scoresvariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdpre - operative srs-22 scores pain4.10.634.20.664.10.630.88 appearance3.40.643.50.703.40.630.86 activity4.20.454.30.564.20.430.80 mental3.90.673.80.823.90.650.43 satisfaction3.21.523.11.713.31.490.67 subtotal3.90.433.90.573.90.410.91 total score3.90.423.90.603.90.390.87two - year post - operative srs-22 scores pain4.40.604.20.654.40.590.21 appearance4.30.5640.664.40.530.02 activity4.40.354.30.374.40.350.21 mental4.20.7340.514.20.760.56 satisfaction4.40.794.30.944.40.770.61 subtotal4.30.434.10.384.30.430.09 total score4.30.434.10.414.40.420.10 pre - operative and 2-year post - operative scoliosis research society questionnaire ( srs-22 ) scores in 1973 , dubousset first reported deformity progression after posterior spinal fusion in young patients with paralytic scoliosis . this was thought to occur as a result of continued anterior spinal growth and was termed the crankshaft phenomenon . then , in 1989 , dubousset et al . reviewed all idiopathic and paralytic cases fused before the age of 11 years at texas scottish rite hospital for children and miami children s hospital between 1966 and 1984 , and concluded that deformity progression was inevitable in patients with considerable remaining growth and recommended anterior fusion to achieve stable correction . since that time , several authors have attempted to determine the variables associated with deformity progression . lee and nachemson evaluated this phenomenon in 63 consecutive patients with idiopathic scoliosis who were all risser grade 0 at the time of surgery . they found that patients treated with hybrid constructs with chronologic age of 11 years or younger , especially with a skeletal age of 10 years or younger , were more likely to experience deformity progression . in this cohort , the average deformity progression was 9 in the coronal plane and 7 in the axial plane , as determined by the perdriolle method . . performed a similar analysis in 43 patients and found that an open trc and a younger chronologic age at the time of surgery were significantly predictive of the amount of deformity progression the patient experienced . hamill et al . also evaluated the state of the trc and found that an open trc alone did not indicate a high likelihood of deformity progression . a few years later , sanders et al . repeated the analysis in a different cohort of 43 patients and found that posterior spinal fusion performed before or during the peak height velocity was a strong predictor of the crankshaft phenomenon . our results confirmed the importance of chronologic age , as the patients in the deformity progression group tended to be younger ( mean 13.7 vs. 14.7 years old ) . between 1 and 2 years post - op , the mean change in height for the deformity progression group was 2.6 4.2 cm , compared to 0.8 2.8 cm for the stable group ( p = 0.01 ) , indicating that they were closer to their peak height velocity at the time of surgery . as found in previous studies [ 9 , 12 ] , risser grade and the state of the trc were not significant variables in our analysis . in terms of the amount of deformity progression after posterior fusion , several radiographic variables were significantly different between the two groups in this cohort . as expected , based on our definition of deformity progression , the residual scoliosis at 2 years post - op was statistically significant in the coronal plane , as measured by the main thoracic cobb angle ( mean 32.2 vs. 23.8 ) . additionally , the change in uiv angulation , liv angulation , and main thoracic avt were all significantly greater in the deformity progression group . pedicle screws interfered with our ability to use the nash moe and perdriolle methods , and we had insufficient data on implant dimensions to use the position of the pedicle screws to determine vertebral rotation . recent studies have concluded that posterior segmental constructs are better able to control the crankshaft phenomenon and may mitigate the need for a combined anterior fusion . burton et al . evaluated 18 risser grade 0 patients with idiopathic scoliosis and found no evidence of crankshaft phenomenon in patients treated with segmental hook and screw constructs . compared hybrid versus all pedicle screw constructs in 67 idiopathic scoliosis patients and found that radiographic measures of deformity progression were significantly better in the all pedicle screw group . similarly , sarlak et al . reported on seven juvenile idiopathic patients treated with segmental pedicle screw constructs . with a minimum of 5 years follow - up , they reported some deformity progression in three patients ; however , they felt that it was not enough to recommend combined anterior spinal fusion in this skeletally immature patient population . additionally , a recent study by hwang et al . identified a 12 % loss of coronal correction at 2-year follow - up that was not associated with infection , adding - on , or pseudarthrosis . they found that loss of correction was statistically associated with a larger cobb magnitude , apical translation , and t1 tilt angle . the selection of fusion levels was not analyzed ; however , patients with all pedicle screw constructs were found to have a lower rate of loss of correction compared to patients with hybrid fixation ( 10 vs. 20 % ) . in our study , a majority of the patients ( 67 % ) were treated with a hybrid segmental construct with pedicle screws at the base and around the apex of the deformity , occasional sublamina wire fixation at the apex , and hooks at the top of the construct . implant density was actually found to be significantly greater in the deformity progression group as compared to the stable group ( mean 1.79 vs. 1.63 ) . this may represent anticipation of post - operative deformity progression , and , therefore , surgeon bias to use a greater number of implants . thirty - three percent of the patients were treated with segmental pedicle screw constructs with one or two hooks at the top . the percent of fixation sites instrumented with pedicle screws was also significantly greater in the deformity progression group . the selection of fusion levels appeared to play a more important role in these patients . in general , main thoracic curves can be treated with selective or non - selective fusion based on the magnitude and flexibility of the compensatory curves . the lowest instrumented vertebra is typically selected by assessing the bend films , the position of the center sacral vertical line on the standing radiograph , as well as the end and neutral vertebrae of the main thoracic curve . patients in this cohort who experienced deformity progression had a lowest instrumented vertebra which was nearly two motion segments cephalad to the stable vertebra ( table 6 ) . in comparison , patients in the stable group were , on average , fused to the stable vertebra . patients in both groups were fused about two vertebrae distal to the neutral vertebra . while deformity progression was clearly evident in our patient cohort , it is difficult to differentiate the concepts of crankshaft phenomenon and adding - on below the liv . burton et al . defined crankshaft as 10 of curve progression in the coronal cobb angle or 10 of change in rib vertebral angle difference , and they defined adding - on as an increase in the scoliotic deformity outside the fused levels of 5 or more . cho et al . defined adding - on as an increase in cobb angle of at least 5 and distalization of the end vertebra , or a change in disc angulation of 5 or greater below the liv . ultimately , a three - dimensional analysis of the fused segment will be required to clearly identify where the deformity progression occurred , either in the fused segment or over the adjacent levels . only the appearance domain score was statistically different between the two groups at 2-years post - op ( 4.0 in the deformity progression group versus 4.4 in the stable group ) . recently published the minimum clinically important difference ( mcid ) for each domain of the srs questionnaire and found the mcid of the appearance domain to be 0.98 . additionally , none of the 16 patients in this cohort required a revision surgery to correct the deformity that had worsened over the post - operative period . this finding likely represents a surgeon bias to avoid a complex revision procedure ; however , none of the patients developed more than a 45 main thoracic coronal plane deformity . primarily , it was performed retrospectively and included only 24 % ( 98 out of 402 ) of the idiopathic scoliosis patients surgically treated at our institution during the determined time frame . additionally , the sample size of the two groups was substantially different ; however , we used appropriate statistical analyses to account for this difference . additionally , a number of variables , including rod type and diameter , as well as surgical technique including derotation maneuvers , could not be controlled for in this analysis and could have biased our observation . it is clear from the current study that deformity progression can occur in growing children who undergo posterior spinal fusion for idiopathic scoliosis . the peak height velocity ( phv ) has been identified as a risk factor ; however , the phv occurs prior to traditional determinants of growth remaining , such as risser sign and bone age . additionally , segmental pedicle screw constructs were not able to control deformity progression in this cohort , and the selection of fusion levels seemed to play a more important role , as patients in the deformity progression group were fused almost two levels cephalad to the stable vertebra .

study designretrospective chart and radiographic review.purposeto assess the incidence of and variables associated with spinal deformity progression after posterior segmental instrumentation and fusion at a single institution . progression of the scoliotic deformity after posterior instrumented spinal fusion has been described . recent studies have concluded that segmental pedicle screw constructs are better able to control deformity progression.methodsretrospective review of a consecutive series of idiopathic scoliosis patients ( n = 89 ) with major thoracic curves ( lenke types 14 ) treated with posterior segmental instrumentation and fusion . deformity progression was defined as a 10 increase in cobb angle between the first - erect and 2-year post - operative radiographs . clinical and radiographic data between the two cohorts ( deformity progression versus stable ) were analyzed to determine the variables associated with deformity progression.resultspatients in the deformity progression group ( n = 13 ) tended to be younger ( median 13.7 vs. 14.7 years ) and experienced a significant change in height ( p = 0.01 ) during the post - operative period compared to the stable group ( n = 76 ) . at 2-years post - op , the patients in the deformity progression group had experienced a significantly greater change in upper instrumented vertebra ( uiv ) angulation , lower instrumented vertebra ( liv ) angulation , and apical vertebral translation ( avt ) . two - year post - op scoliosis research society questionnaire ( srs-22 ) scores in the appearance domain were also significantly worse in the deformity progression group . patients in the deformity progression group had a significantly greater difference between the lowest instrumented vertebra and stable vertebra compared to patients in the stable group ( p = 0.001).conclusionsdeformity progression after posterior spinal fusion does occur after modern segmental instrumentation . segmental pedicle screw constructs do not prevent deformity progression . skeletally immature patients with a significant growth potential are at the highest risk for deformity progression . in immature patients , extending the fusion distally to the stable vertebra may minimize deformity progression.level of evidencelevel iii .

Level of evidence Introduction Materials and methods Statistical analysis Results Clinical measures Radiographic measures Subjective scores Discussion Conclusion

the mean change in the major coronal cobb angle was 1.7 in the stable group ( pre - op : 58.5 ; post - op : 22.1 ; 2-year post - op : 23.8 ) versus 14.7 in the deformity progression group ( pre - op : 52.9 ; post - op : 17.5 ; 2-year post - op : 32.2).table 1pre - operative clinical and radiographic datavariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdage ( years)14.52.1913.71.8714.72.220.14height ( cm)160.18.84161.311.86159.98.290.59weight ( cm)54.711.4552.112.0655.211.360.37proximal inclinometer [ median ( iqr)]4(26)5(03)4(26)0.16thoracic inclinometer [ median ( iqr)]15(1215)12.5(1214)15(1215)0.49thoracolumbar inclinometer [ median ( iqr)]6(47)7(27)6(47)0.95touch vertebrae [ median ( iqr)]22(2022)22(2223)22(2021)0.11cobb proximal thoracic27.69.7126.79.1727.89.850.70cobb main thoracic57.711.8152.97.7858.512.220.12cobb thoracolumbar36.111.7233.212.4036.611.610.32avt proximal4.15.3454.923.95.430.39avt main48.415.1843.210.1449.215.770.19avt lumbar16.712.7115.412.2616.912.850.53coronal balance15.710.7720.813.3014.810.120.12t1 tilt3.84.4343.673.84.570.28sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translationtable 2first - erect clinical and radiographic datavariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdtime from surgery to first erect ( days)29.713.7727.111.7630.114.100.45percent correction57.311.8452.67.7658.212.260.12cobb proximal thoracic12.18.4210.812.7612.37.530.54cobb main thoracic21.48.4417.56.6922.18.560.08cobb thoracolumbar19.610.0818.48.0219.810.420.64avt proximal4.14.926.35.543.84.740.19avt main13.28.539.55.5613.88.820.20avt lumbar17.313.0115.514.6817.612.780.27coronal balance16.211.6917.415.791610.960.21t1 tilt4.64.415.55.974.44.120.49uiv tilt11.71.323.68.03.16.50.70liv tilt21.22.034.58.56.17.40.52sd standard deviation ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebratable 3one - year post - operative clinical and radiographic datavariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdtime from surgery to 1-year follow - up ( days)369.659.22349.279.65373.254.720.18height ( cm)164.28.2316511.711647.540.69weight ( cm)59.413.4555.411.0360.113.790.24proximal inclinometer [ median ( iqr)]2(03)2.5(04)2(02)0.18thoracic inclinometer [ median ( iqr)]7(57)7(47)6.5(57)0.69thoracolumbar inclinometer [ median ( iqr)]0(02)0(02)1(02)0.84cobb proximal thoracic13.99.2415.59.8913.79.170.52cobb main thoracic24.98.5526.66.6324.68.850.43cobb thoracolumbar19.410.1019.19.3319.410.300.91avt proximal4.35.264.23.724.35.510.91avt main16.811.5716.58.5716.812.080.93avt lumbar15.310.7212.29.0515.810.960.16coronal balance13.79.4515.212.8613.58.790.39t1 tilt4.84.795.34.974.74.790.58uiv tilt7.45.359.54.0375.490.09liv tilt8.86.3611.26.078.46.360.21sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebratable 4two - year post - operative clinical and radiographic datavariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdtime from surgery to 2-year follow - up ( days)780.4134.64798.7123.31777.4136.940.61height ( cm)164.410.78167.612.98163.910.360.24weight ( cm)60.614.3458.110.1961.114.950.49proximal inclinometer [ median ( iqr)]1(02)0(02)2(02)0.54thoracic inclinometer [ median ( iqr)]7(47)6.5(58)7(47)0.55thoracolumbar inclinometer [ median ( iqr)]0(02)0(02)0(02)0.65cobb proximal thoracic13.68.2915.59.4513.38.100.39cobb main thoracic259.3032.27.3223.89.090.006cobb thoracolumbar18.210.1521.99.8517.510.130.15avt proximal4.75.636.86.354.45.460.21avt main19.211.8222.212.9518.711.630.28avt lumbar14.310.512.89.9914.510.630.44coronal balance11.99.5112.88.8611.89.660.04t1 tilt4.64.756.95.574.24.520.20uiv tilt6.96.0111.65.246.15.790.02liv tilt8.85.90134.678.15.820.04sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebratable 5pre - operative and 2-year post - operative risser gradesvariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuepre - operative risser grade0.32 014 ( 16 % ) 4 ( 31 % ) 10 ( 13 % ) 117 ( 19 % ) 3 ( 23 % ) 14 ( 18 % ) 211 ( 12 % ) 1 ( 8 % ) 10 ( 13 % ) 315 ( 17 % ) 2 ( 15 % ) 13 ( 17 % ) 413 ( 15 % ) 2 ( 15 % ) 11 ( 15 % ) 519 ( 21 % ) 1 ( 8 % ) 18 ( 24 % ) two - year post - operative risser grade0.46 00 ( 0 % ) 0 ( 0 % ) 0 ( 0 % ) 15 ( 6 % ) 0 ( 0 % ) 5 ( 7 % ) 28 ( 9 % ) 2 ( 15 % ) 6 ( 8 % ) 36 ( 7 % ) 1 ( 8 % ) 5 ( 7 % ) 426 ( 29 % ) 4 ( 31 % ) 22 ( 28 % ) 544 ( 49 % ) 6 ( 46 % ) 38 ( 50 % ) pre - operative clinical and radiographic data sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation first - erect clinical and radiographic data sd standard deviation ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebra one - year post - operative clinical and radiographic data sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebra two - year post - operative clinical and radiographic data sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebra pre - operative and 2-year post - operative risser grades the mean absolute liv angulation for the deformity progression group was 4.5 8.5 at first erect and for the stable group , it was 6.1 7.4 ( p = 0.52 ) . the mean change in the major coronal cobb angle was 1.7 in the stable group ( pre - op : 58.5 ; post - op : 22.1 ; 2-year post - op : 23.8 ) versus 14.7 in the deformity progression group ( pre - op : 52.9 ; post - op : 17.5 ; 2-year post - op : 32.2).table 1pre - operative clinical and radiographic datavariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdage ( years)14.52.1913.71.8714.72.220.14height ( cm)160.18.84161.311.86159.98.290.59weight ( cm)54.711.4552.112.0655.211.360.37proximal inclinometer [ median ( iqr)]4(26)5(03)4(26)0.16thoracic inclinometer [ median ( iqr)]15(1215)12.5(1214)15(1215)0.49thoracolumbar inclinometer [ median ( iqr)]6(47)7(27)6(47)0.95touch vertebrae [ median ( iqr)]22(2022)22(2223)22(2021)0.11cobb proximal thoracic27.69.7126.79.1727.89.850.70cobb main thoracic57.711.8152.97.7858.512.220.12cobb thoracolumbar36.111.7233.212.4036.611.610.32avt proximal4.15.3454.923.95.430.39avt main48.415.1843.210.1449.215.770.19avt lumbar16.712.7115.412.2616.912.850.53coronal balance15.710.7720.813.3014.810.120.12t1 tilt3.84.4343.673.84.570.28sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translationtable 2first - erect clinical and radiographic datavariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdtime from surgery to first erect ( days)29.713.7727.111.7630.114.100.45percent correction57.311.8452.67.7658.212.260.12cobb proximal thoracic12.18.4210.812.7612.37.530.54cobb main thoracic21.48.4417.56.6922.18.560.08cobb thoracolumbar19.610.0818.48.0219.810.420.64avt proximal4.14.926.35.543.84.740.19avt main13.28.539.55.5613.88.820.20avt lumbar17.313.0115.514.6817.612.780.27coronal balance16.211.6917.415.791610.960.21t1 tilt4.64.415.55.974.44.120.49uiv tilt11.71.323.68.03.16.50.70liv tilt21.22.034.58.56.17.40.52sd standard deviation ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebratable 3one - year post - operative clinical and radiographic datavariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdtime from surgery to 1-year follow - up ( days)369.659.22349.279.65373.254.720.18height ( cm)164.28.2316511.711647.540.69weight ( cm)59.413.4555.411.0360.113.790.24proximal inclinometer [ median ( iqr)]2(03)2.5(04)2(02)0.18thoracic inclinometer [ median ( iqr)]7(57)7(47)6.5(57)0.69thoracolumbar inclinometer [ median ( iqr)]0(02)0(02)1(02)0.84cobb proximal thoracic13.99.2415.59.8913.79.170.52cobb main thoracic24.98.5526.66.6324.68.850.43cobb thoracolumbar19.410.1019.19.3319.410.300.91avt proximal4.35.264.23.724.35.510.91avt main16.811.5716.58.5716.812.080.93avt lumbar15.310.7212.29.0515.810.960.16coronal balance13.79.4515.212.8613.58.790.39t1 tilt4.84.795.34.974.74.790.58uiv tilt7.45.359.54.0375.490.09liv tilt8.86.3611.26.078.46.360.21sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebratable 4two - year post - operative clinical and radiographic datavariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuemeansdmeansdmeansdtime from surgery to 2-year follow - up ( days)780.4134.64798.7123.31777.4136.940.61height ( cm)164.410.78167.612.98163.910.360.24weight ( cm)60.614.3458.110.1961.114.950.49proximal inclinometer [ median ( iqr)]1(02)0(02)2(02)0.54thoracic inclinometer [ median ( iqr)]7(47)6.5(58)7(47)0.55thoracolumbar inclinometer [ median ( iqr)]0(02)0(02)0(02)0.65cobb proximal thoracic13.68.2915.59.4513.38.100.39cobb main thoracic259.3032.27.3223.89.090.006cobb thoracolumbar18.210.1521.99.8517.510.130.15avt proximal4.75.636.86.354.45.460.21avt main19.211.8222.212.9518.711.630.28avt lumbar14.310.512.89.9914.510.630.44coronal balance11.99.5112.88.8611.89.660.04t1 tilt4.64.756.95.574.24.520.20uiv tilt6.96.0111.65.246.15.790.02liv tilt8.85.90134.678.15.820.04sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebratable 5pre - operative and 2-year post - operative risser gradesvariableall subjects ( n = 89)deformity progression ( n = 13)control ( n = 76)p - valuepre - operative risser grade0.32 014 ( 16 % ) 4 ( 31 % ) 10 ( 13 % ) 117 ( 19 % ) 3 ( 23 % ) 14 ( 18 % ) 211 ( 12 % ) 1 ( 8 % ) 10 ( 13 % ) 315 ( 17 % ) 2 ( 15 % ) 13 ( 17 % ) 413 ( 15 % ) 2 ( 15 % ) 11 ( 15 % ) 519 ( 21 % ) 1 ( 8 % ) 18 ( 24 % ) two - year post - operative risser grade0.46 00 ( 0 % ) 0 ( 0 % ) 0 ( 0 % ) 15 ( 6 % ) 0 ( 0 % ) 5 ( 7 % ) 28 ( 9 % ) 2 ( 15 % ) 6 ( 8 % ) 36 ( 7 % ) 1 ( 8 % ) 5 ( 7 % ) 426 ( 29 % ) 4 ( 31 % ) 22 ( 28 % ) 544 ( 49 % ) 6 ( 46 % ) 38 ( 50 % ) pre - operative clinical and radiographic data sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation first - erect clinical and radiographic data sd standard deviation ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebra one - year post - operative clinical and radiographic data sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebra two - year post - operative clinical and radiographic data sd standard deviation ; iqr interquartile range ( 25th percentile75th percentile ) ; avt apical vertebral translation ; uiv upper instrumented vertebra ; liv lower instrumented vertebra pre - operative and 2-year post - operative risser grades the mean absolute liv angulation for the deformity progression group was 4.5 8.5 at first erect and for the stable group , it was 6.1 7.4 ( p = 0.52 ) .

there is extensive current interest in the high - content , high - throughput screening of live cell populations , and the experimental techniques being developed for this purpose are leading to important biological insights with applications to new clinical therapies [ 14 ] . due to the large amount of data generated by these experiments , automated data processing systems for segmentation and tracking of live cells in sequences of microscope images are a virtual necessity . several comprehensive systems for segmentation and tracking have recently been described in the literature ( see , e.g. , [ 5 , 6 ] and references therein ) . in most existing approaches , cells are segmented using advanced level set , active contour or watershed methods , or ingenious combinations of them [ 57 ] . in this paper , we investigate the use of a multilevel aggregation algorithm as an alternative method for segmenting live cell bright field microscope images . saliency measure for this algorithm to decide when aggregates of pixels constitute salient segments that should not be grouped further . we investigate the performance of the multilevel aggregation algorithm for bright field cell segmentation in images and sequences of images , and discuss its advantages and drawbacks compared to other methods as a possible building block in comprehensive automatic cell segmentation and tracking systems . figure 1 shows a microscope image with approximately two dozen mouse c2c12 myoblast cells on a dark grey background . c2c12 myoblasts ( atcc crl-1772 ) are muscle cell precursors , which have varied shape and size as well as good motility in vitro . in tissue culture , they will grow to a fully confluent monolayer with well - separated cell nuclei and a small amount of overlapping cytoplasm between adjacent cells . our goal is to obtain separate segments for each cell in images like figure 1 . two major challenges are , first , that the cells appear in two distinct basic shapes ( cells that are close to division show as bright circular shapes , while regular cells have more stretched shapes in which the cytoplasm and nucleus can be discerned ) , and , second , that cells may be touching or overlapping . while our ultimate goal is to automatically segment sequences of complex images like figure 1 , we start out in this paper with segmentation of simplified cases ( isolated cells and small groups of cells ) . we use a variant of the segmentation by weighted aggregation technique [ 8 , 9 ] to segment the microscope images , which itself is based on algebraic multigrid ( amg ) methods for solving linear systems of equations [ 10 , 11 ] . the swa method attempts to segment the image into salient ( or prominent ) groups of pixels by using a multilevel aggregation procedure that groups blocks of pixels at various scales , based on multiscale feature vectors for the pixel blocks . the swa method has more recently also been used as the basis of a more general multilevel clustering algorithm [ 12 , 13 ] . the variant of the method that we use is described in detail below , and it is explained how it is tailored to the application at hand . our algorithmic contribution is a new scale invariant saliency measure for deciding when aggregates of pixels constitute salient segments that should not be grouped further . our new saliency measure is a variant of the saliency measure employed in [ 8 , 9 ] that takes scaling into account . furthermore , we use the so - called first pass of the standard amg graph coarsening algorithm [ 10 , 11 ] to coarsen the pixel graph , rather than the direct aggregation methods that are used in swa algorithms described in the literature [ 8 , 9 , 12 , 14 ] . we test our approach on parts of real microscope images , and show how segmentation based on multilevel intensity similarity alone does not lead to satisfactory results . however , the addition of multilevel variance of mean intensities ( as a measure of texture ) to the feature vector of each aggregate , as in [ 9 , 14 ] , leads to correct cell segmentation . in the second part of the paper , preliminary results are presented for applying the multilevel aggregation algorithm in space time to temporal sequences of microscope images , with the goal of obtaining space time segments ( object tunnels ) that track individual cells . this parallels previous research results on space time segmentation using level set methods [ 15 , 16 ] and on comprehensive segmentation and tracking systems for sequences of cell images [ 5 , 6 ] , but space time segmentation using swa has , to our knowledge , not been investigated in the literature yet . the ultimate goal of this space time segmentation is to determine a space time segment ( object tunnel ) for each individual cell , taking into account cell divisions . ideally , when a cell divides , the segment of the mother cell should terminate and cell segments should start for the two daughter cells . in this sense , the desired outcome of our space time segmentation is more complicated than for related problems such as cerebral vasculature segmentation in ct scans , for which three - dimensional level set algorithms have been developed . also , the full space time segmentation problem is complicated since cells change shape when they divide and cells may touch and overlap . in this paper , we present preliminary results on our experience with extending the spatial swa segmentation algorithm to space time segmentation of sequences of images , for simplified cases of isolated cells and pairs of cells . we identify problems that remain and suggest possible extensions of the algorithm that may handle them , and we comment on the potential of the swa segmentation method as a building block in comprehensive segmentation and tracking systems along the lines of [ 5 , 6 ] and references therein . while the general ideas and concepts of the swa framework are described in several places [ 8 , 9 , 12 , 14 ] , most of the description in the literature is formulated in general terms , and not all algorithmic details are spelled out . in this paper , we want to present methods and results that are fully reproducible by the reader , and for this reason we provide a complete description of the version of the swa algorithm we settled on . one property of the swa algorithm is that it has a significant number of parameters that have to be chosen judiciously to obtain the desired segmentation . while this may be perceived as a potential drawback of the approach , it also opens opportunities to finetune the algorithm for the application class at hand . indeed , the problem of segmenting an image without further specifications is often ill - posed . for example , a satellite image with roads , buildings , cars and people can be segmented at the level of the roads and buildings , or at the more detailed level of individual cars and people . finetuning the swa parameters the question about how to choose the swa parameters is not discussed much in the literature [ 8 , 9 , 12 , 14 ] , and in most cases specific parameters are not given for segmentation results that are presented as examples . again in the interest of reproducibility , we make sure in this paper to include full details about the parameters we choose for each image or image sequence for which we give segmentation results , and we also formulate some general guidelines for choosing parameters . automatic segmentation and tracking of live cells in bright field microscopy images is a difficult task [ 5 , 18 ] . for this reason , fluorescence microscopy is often used in place of bright field microscopy to image live cells . in fluorescence microscopy , cells are made fluorescent by applying fluorescent dyes , or by making the cells artificially express fluorescent proteins . the strong fluorescence signals facilitate tracking , but may also have significant drawbacks . in many cases , the fluorescence introduced is toxic for the cells , and it may change cell behaviour . on the contrary , bright field microscopy is well suited for live cell studies as the imaging conditions can usually be chosen to minimize phototoxicity . for example , the experiments reported in show how the shape of a cell can be linked to genetic factors , in this case signaling proteins . in this study , the cells were fixed and permeabilized prior to phalloidin staining and , since the cells are no longer alive , it is impossible to examine the same cells at a later time . however , the image segments we obtain from our analysis can be used to categorize cell shapes while keeping the cells in culture . this ensures minimal interference with normal cell function through toxicity from photochemical effects and the high intensity illumination that is required to excite fluorescent probes . in other types of experiments , fluorescent markers are used to measure concentrations of certain specific proteins that are under study . a limited number of fluorescent marker channels with nonoverlapping spectra ( typically up to three ) are used in combination . if one or two of these channels are used solely for tracking purposes , the number of channels available for measuring protein concentrations is reduced , which may be a significant limitation . for these reasons , it can be an advantage if methods can be derived that manage to track cells based on bright field images . in addition , bright field images typically contain many features of interest , and accurate segmentation methods for bright field images are intrinsically useful since they allow the study of cell morphology and internal cell structure , and their dynamics . for example , the morphology of a cell can be indicative of cell health or can indicate various stages of pathology . also , accurate bright field segmentation of cells and cell organelles may allow for more precise integration of fluorescence signals over relevant parts of precisely identified cells , enhancing the accuracy of protein expression level measurements . finally , it should also be noted that the contrast of bright field images is often enhanced by the use of phase contrast microscopy . this allows us to see detailed structure within cells on figure 1 ( including cell nuclei and nucleoli ) , but it also generates bright halos around cells which may complicate segmentation since they do not uniformly enclose cells . the microscope images used in this paper ( including figure 1 ) were obtained as follows . cells were plated at a low density in glass chamber slides and imaged at 20x using a phase contrast objective ( nikon teu 2000 with cfi plan fluor elwd 20x ) and a cooled ccd camera ( hamamatsu c9100 - 12 ) . each image contains 512 by 512 pixels , with a resolution of 0.8 m / pixel and 14 effective bits per pixel . unless otherwise noted , all cell culture reagents were purchased from invitrogen . c2c12 myoblasts were cultivated in growth medium ( dulbecco 's modified eagle medium supplemented with 20% fetal bovine serum ( sigma ) , 2 mm glutamine , 50 units penicillin and 50 g / ml streptomycin in 10 cm cell culture plates ( corning ) . cells were treated with 1.5 ml of 0.05% trypsin - edta for 5 minutes , manually dispersed by pipetting and diluted in growth medium to 50,000 cells / ml . subsequently , 3 ml of this suspension was added to one lab - tek chambered coverglass well ( nunc ) and cells were allowed to settle undisturbed at 37c and 10% co2 for 16 hours . the slide was then placed on the motorized stage of a nikon teu 2000 equipped with a solent environmental chamber where the cells were kept at the same co2 and temperature conditions as before . , we give a brief description of the swa algorithm and introduce a new scale invariant saliency measure . in section 3 we investigate how the algorithm performs for segmenting bright field cell images . in section 4 we explain how the approach is extended to segmentation in space time in a straightforward way , and investigate the performance of space time segmentation for simple sequences of real cell images . section 6 discusses conclusions and future work . finally , appendix a gives a pseudocode - style step - by - step description of our algorithmic implementation . in this section , we describe the multilevel segmentation algorithm that we employ in this paper . we start with an overview of the basic swa algorithm from [ 8 , 9 , 1214 , 21 ] , followed by a subsection on some specific more detailed aspects of the algorithm . in the third subsection , we propose and discuss a new scale invariant saliency measure for the swa algorithm . for a complete description of the algorithm we use , we refer the reader to appendix a , which provides an actual pseudocode - style step - by - step description of our implementation . figure 2 shows a schematic representation of the swa algorithm . a high - level description of the swa algorithm proceeds as follows . in the first , top - down phase of the algorithm , pixels are recursively grouped into increasingly large overlapping blocks . at any level in the process , the top - down phase ends when all remaining blocks have become salient . in the second , bottom - up phase of the algorithm , overlapping blocks that were identified as salient are recursively sharpened , until all fine - level pixels are assigned to a unique segment at the top level . the problem of image segmentation can be viewed in terms of segmentation of a weighted undirected graph , with each node of the graph representing a pixel , and each edge corresponding to a link between neighbouring nodes , weighted by the similarity in intensity between the two neighbouring pixels . the swa algorithm starts with this weighted graph on the finest level ( which we call level 1 ) and forms a weighted graph of reduced size on level 2 , with the nodes of the level-2 graph representing overlapping blocks of level-1 nodes . each level-2 block is formed around a level-1 seed point , which is called a c - point ( short for coarse point ) on level 1 . nodes on level 1 that are not chosen as c - points are called f - points ( short for fine points ) . the overlapping blocks are chosen such that they group neighbouring nodes that have similar intensities . this graph coarsening can be done in a variety of ways , and the particular approach we employ is explained in the next subsection and in appendix a.3 . this graph coarsening process is then repeated on level r , r = 2,3 , 4 , , to obtain the coarse - level graph on level r + 1 . the left branch of the diagram in figure 2 shows for an example image how the nodes ( shown as red dots ) are coarsened during the first , top - down phase of the algorithm . ( for each coarse - level overlapping block , one finest - level node corresponding to the c - point of the block on the previous level is shown as a representative . ) in more specific terms , we start from the pixel graph of the original image , with the intensities of the pixels stored in level-1 intensity vector i. ( we scale each input image such that the maximum intensity value equals 1 . ) pixels that are neighbours in the horizontal or vertical directions are connected by weighted edges in the graph , with edge weights aij defined using an exponential function of the intensity difference between the pixels they connect : ( 1)aij={e|iiij|if i , j are neighbours,0,otherwise , with 0 a user - defined parameter . c - points are then chosen for the level-1 graph and their indices are stored in vector c. an interpolation matrix from level 2 to level 1 is defined as ( 2)pij={1if ic , i = cj,0if ic , icj , aicjkcaikif ic . here we use the shorthand notation i c to mean that i = cj for some j. each column j of p represents a level-2 node , and element pij indicates which fraction of level-1 node i belongs to level-2 block j. ( the rows of p sum to one . ) the edge weights between nodes of the level-2 graph are calculated by an averaging process known as galerkin coarsening : ( 3)a = ptap . in a similar way , interpolation operators p are derived between the other recursive levels , and coarse - level graph weights are calculated by ( 4)a[r+1]=p[r , r+1]ta[r]p[r , r+1 ] . on each level , the blocks are tested for saliency : a salient ( or prominent ) block is a block that is sufficiently different from all the blocks it is connected to , as determined by a saliency measure . once a block is designated salient , it is not allowed to merge with other blocks on coarser levels . the coarsening process terminates at the level where all blocks have become salient , at which point we have found all segments in the image . in figure 2 , the bottommost image is at the coarsest level where coarsening stops because each of the two nodes represents a salient segment ( the white and the black segment ) . the right branch of the diagram in figure 2 represents the second , bottom - up phase of the algorithm . recall that the segments obtained on the coarsest level correspond to overlapping blocks on the finest level . it is important to retain overlapping blocks in the first phase of the algorithm , since a pixel that may appear to belong primarily to a certain block according to fine - level information , may be reclassified as belonging to a different block later when coarser - level information is taken into account . the goal of the second phase is to uniquely assign every finest - level pixel to one of the segments . the nodes on the second - to - coarsest level are first assigned to the segments obtained on the coarsest level according to the weights in the interpolation matrix , and this is repeated recursively for nodes on increasingly fine levels . in this way , the representation of the so - far overlapping segments is obtained on each recursive level . on each level , the overlap between segments is reduced by a sharpening procedure , with a sharpening threshold d1 , which we normally take equal to 15% . in this sharpening procedure , nodes that belong for more than 85% to a segment are assigned for 100% to that segment , while nodes that belong for less than 15% to a segment are fully decoupled from that segment . finally , on the finest level the swa algorithm is in many ways similar to amg methods for solving linear systems of equations [ 10 , 11 ] , and the v - shaped process of figure 2 is called a v - cycle in multigrid terminology . contrary to the amg v - cycle , where cycles are repeated to iteratively produce better approximations to linear equations , we perform the image segmentation v - cycle only once , giving the final segmentation . the recursive part of the algorithm is preceded and followed by a nonrecursive part on the finest level . in the recursive part we consider two adjacent levels : level r ( the current fine level ) and level r + 1 ( the current coarse level ) . on level r , we find a suitable subset of fine - level nodes ( the c - points ) that will form the seed points for the coarse - level blocks . then , as long as we are not yet on the coarsest level ( i.e. , not all nodes are salient ) , we calculate the coarse - level graph weights from the fine - level weights , and coarsen again recursively . once on the coarsest level , the recursive function returns the segments found , and performs a bottom - up process that ultimately leads to each pixel in the ( finest - level ) image being assigned to exactly one of the ( initially overlapping ) segments found on the coarsest level . the nonrecursive and recursive parts of the algorithm are described in detail in appendices a.1 and a.2 , respectively , and the coarsening algorithm is given in appendix a.3 . the following are further details and enhancements of the swa algorithm . in order to coarsen the graph at the current level , we use the so - called first pass of the standard amg coarsening algorithm [ 10 , 11 ] . this algorithm coarsens the connectivity graph with weights aij by first dividing the connections in sets of weak and strong connections based on their weights , and then approximately determining a subset of the nodes of the graph that is a maximal independent set in the subgraph formed by only retaining the strongly connected edges . strength parameter ( see ( a.22 ) ) is used as a threshold to determine strong connections in the coarsening algorithm . the nodes in the maximal independent set , which are selected such that they are not linked by strong connections , become the c - points on the current level . nodes that have strong connections to many other nodes are more likely to be chosen as c - points . note also that , on coarse level r , nodes that have already been designated salient on previous levels ( see section 2.3 ) , automatically become c - points at the beginning of the coarsening on level r. note that we use standard amg coarsening [ 10 , 11 ] to coarsen the graph , rather than the direct aggregation methods that are used in swa algorithms described in the literature [ 8 , 9 , 12 , 14 ] . we find the standard amg coarsening a simple and effective graph coarsening algorithm that leads to good results , as confirmed by extensive experiments . calculating a by ( 5)a[r+1]=p[r , r+1]ta[r]p[r , r+1 ] gives the coupling weight between two blocks based on finer - level coupling weights across their shared boundary . however , it is beneficial to also consider the direct similarity between the blocks in terms of their average intensity . the average intensity of each block on level r + 1 can easily be calculated from the average intensity on level r by multiplication with a scaled interpolation matrix , see ( a.11 ) and ( a.10 ) . the coupling weights can then be rescaled as follows to incorporate similarity in average intensity : ( 6)aij[r+1]aij[r+1]e|ii[r+1]ij[r+1]| . here , ii is the average intensity of block i on level r + 1 , and 0 is a user - defined parameter . note that , while the diagonal terms of a do not affect the selection of c - points on level r , they do affect the off - diagonal terms of a that subsequently influence the selection of c - points on level r + 1 . diagonal terms of a represent the internal similarity of blocks , and should be taken into account when calculating similarity between coarse - level blocks . another blockwise quantity we use to better connect similar blocks is multilevel variance in intensity ( as a measure of texture ) . every node on level r + 1 corresponds to an overlapping group of nodes on level r , every node on level r corresponds to an overlapping group of nodes on level r 1 , and so forth . the variance in intensity of the level - r nodes that correspond to a level - r + 1 node is easily calculated using the standard expression var(x ) = e((x e(x ) ) ) = e(x ) e(x ) , see ( a.12 ) . these variances are calculated between all consecutive levels , and are assigned to coarse - level nodes by recursive averaging ( see ( a.13 ) ) . an r - component multilevel feature vector with intensity variances can thus be associated with every node on level r + 1 , see ( a.14 ) . in each such feature vector , the last component gives the variance between the average intensities of the level - r nodes that correspond to the level - r + 1 node , the one - before - last component gives the average over those level - r nodes of the variance between the average intensities of the level - r 1 nodes that correspond to each of those level - r nodes , and so forth . the averages in the feature vectors can be calculated efficiently via recursive averaging , see ( a.13 ) . the coupling weights between blocks can then be rescaled according to the similarity in these variance vectors , with a scaling parameter , see ( a.17 ) . in practice , it turns out that variance for small blocks is less relevant and may cause incorrect segmentation . we therefore only use variance rescaling on levels larger than level , with a user - defined parameter . in order to avoid small segments , we only allow detection of salient segments on levels larger than level , with a user - defined parameter . in summary , we list the free parameters in our algorithm , to be chosen such that correct segmentation is obtained for the application at hand : top - level intensity scaling factor , coarse - level intensity rescaling factor , coarse - level variance rescaling factor , coarsening strength threshold , saliency threshold ( see the next section ) , sharpening threshold d1 , segment detection threshold level , and variance rescaling threshold level . we propose a saliency measure that is a modified version of the saliency measures used in [ 8 , 9 , 1214 , 21 ] . the saliency measures used in [ 8 , 9 , 1214 , 21 ] can be derived and motivated as follows . on each level r , define the energy functional ( 7)[r](u[r])=i > jaij[r](ui[r]uj[r])2i > jaij[r]ui[r]uj[r]=u[r]tl[r]u[r](1/2)u[r]tw[r]u[r ] . here , a is the coupling matrix on level r , the matrices l and w are given by ( 8)lij[r]={aij[r]if ij,kiaik[r]if i = j , w[r]=a[r ] , and u is a boolean state vector for a particular block on level r such that uj = 1 if node j belongs to the block and uj = 0 otherwise . l is called the laplacian of the ( weighted ) graph . note that w = a on all levels so there is no real need to introduce the variables w , but below we choose to use the w in expressions for the saliency measure . the swa algorithm seeks segments ( boolean vectors u ) that yield low values of the energy functional . on the finest level , this formulation is equivalent to a normalized cut formulation for the segmentation problem [ 14 , 22 ] . in the normalized cut approach , a generalized eigenvalue problem is formulated on the finest level that is related to functional ( 7 ) . the few eigenvectors with lowest eigenvalues are computed and are used to segment the image using a clustering method . note that amg solvers can also be developed that directly calculate the eigenvectors of this fine - level eigenvalue problem in an efficient manner . in the swa approach , however , we do not directly calculate the fine - level eigenvectors , but , instead , consider increasingly coarse versions of the coupling matrix and detect salient segments based on connection strength in those coarse - level coupling matrices , guided by functional ( 7 ) . this approach allows us to take into account coarse - level features ( like multilevel variance ) for segment detection . at level r , the swa algorithm checks for each node i whether it is salient . ( recall that node i is interpreted as an overlapping block of finest - level pixels . ) to this end , define the boolean vector for the single - node segment with node i on level r by uj = ij ( i.e. , ui = 1 , and uj = 0 for j i ) . the saliency measure i of node i is then given by ( 9)i[r]=(u[r],i)=lii[r](1/2)wii[r ] , and node i is designated a salient segment if its saliency measure is smaller than a user - defined constant : ( 10)i[r]<. ( note that saliency measure ( 9 ) can not be used on the finest level , since wii = 0 for all finest - level nodes i. in practice , this is normally resolved by not allowing salient nodes on the finest level . ) it can be understood easily that saliency measure ( 9 ) is sensitive to the scales of segments , and , depending on the application , this may lead to difficulties . in what follows , we illustrate the scale sensitivity of saliency measure ( 9 ) by a simple example , and propose a new variant of saliency measure ( 9 ) that takes into account scaling . saliency measure ( 9 ) for node i on level r can be related to the block of pixels on the finest levels that corresponds to node i ( which we can call block i ) . indeed , the saliency measure can be interpreted as the sum of the coupling coefficients along the border of block i divided by the sum of the coupling coefficients along connections internal to block i . it can easily be seen that this interpretation is exact for any nonoverlapping block , as is illustrated by the following simple example . consider figure 4 which represents an example image with a square block of white pixels in the centre , and assume that this block corresponds to node i on level 2 in the aggregation process ( we thus call it block i ) . the ith column of the interpolation matrix between levels 2 and 1 , p , contains 1 s in the rows corresponding to the pixels in block i , and 0 s in the other rows . it is convenient to represent the ith column of p in so - called stencil form , as ( 11)pi=. let us define two more matrices , the adjacency matrix v and the unweighted graph laplacian g , which are boolean versions of w and l , not weighted by the coupling strengths in a : ( 12)vij={0if aij=0,1if aij0,gij={vijif ij,kivikif i = j . the stencils of the operators v and g are given by ( 13)v=,g=[11411 ] . ( note that these stencils represent the nonzero elements in each of the columns ( or rows ) of v and g. since each column has the same pattern of nonzeros , we don not give the stencils of v and g a subscript index . note that columns corresponding to pixels close to the boundaries of the image would have a different nonzero pattern , but we assume that the white block in figure 4 is embedded in a larger black image and is located far from the image boundary , such that we don not have to worry about boundaries . ) let us now calculate the ith diagonal elements of the level-2 versions of v and g , namely , v = pvp and g = pgp . first , we want to show that diagonal element gii = pigpi equals the boundary length of block i , which in this example is 3 4 = 12 . applying matrix g to column pi gives , in stencil notation , ( 14)gpi=[0111012221110111212101110 ] . multiplying this with row pi results in a scalar value of ( 15)pitgpi=12 , which is the boundary length of block i , as desired . next we want to show that ( 1/2)vii = ( 1/2 ) pivpi equals the number of internal connections within block i. first apply matrix v to column pi to get , in stencil notation , ( 16)vpi=. then multiply this with row pi to obtain ( 17)pitvpi=24 , which equals twice the number of internal connections , or approximately twice the area of block i. due to this interpretation of the diagonal elements of matrices g and v in terms of block boundary length and block area , we will in what follows refer to the matrices g as boundary length matrices , and to v as area matrices . it is now easy to see that the ith diagonal element of l corresponds to the sum of the coupling coefficients along the boundary of block i , or , in other words , to the boundary length of block i weighted by the similarity with neighbouring blocks along the block boundary . similarly , the ith diagonal element of w = a corresponds to twice the sum of the coupling coefficients along connections internal to block i , or , in other words , to twice the area of block i , weighted by the mutual similarity of neighbouring finer - level blocks in the interior of block i. we therefore call the matrices w weighted area matrices . note that this interpretation also follows directly from plugging u = pi into ( 7 ) ( middle expression ) . with these interpretations of the diagonal elements of l , w , g and v in hand , we can now analyze the scale behaviour of saliency measure ( 9 ) , and propose a new scale invariant version of it . figure 5 shows two examples of images with a white shape on a black background . the original saliency measure ( 18)i[r]=lii[r](1/2)wii[r ] , is much smaller for shape ( a ) than for shape ( b ) : shape ( a ) has a shorter boundary and a larger area than shape ( b ) , and the similarity - weighted boundary length lii is proportional to the block boundary length , while the similarity - weighted area ( 1/2)wii is proportional to the area . for the example considered , this is undesirable , since both shapes should be deemed equally salient : their saliency measure should be of similar value for the swa algorithm to segment them correctly if they were to occur together in a larger image . it is also clear that saliency measure ( 18 ) tends to assume geometrically smaller values as levels get coarser , since , in two dimensions for example , shape areas normally grow by a factor of four when boundary lengths double . consider two white blobs with the shape of figure 5(a ) , a large one and a small one . the large blob would reduce to a single node at a coarser level than the small blob , and the salience measure ( 18 ) for the large blob would be smaller than for the small blob , since the ratio of boundary length to area is smaller for the large blob than for the small blob . we can thus say that saliency measure ( 18 ) is not scale invariant ( and also not shape - invariant , according to the example of figure 5 ) . potential scaling difficulties with saliency measure ( 18 ) have been discussed in the literature and some ad hoc fixes have been proposed , but a generally applicable modification that addresses these scaling issues has not been proposed yet . in order to remedy the potential scaling difficulties of saliency measure ( 18 ) , we propose the following new saliency measure : ( 19)i[r]=lii[r]/gii[r]wii[r]/vii[r ] . the motivation behind this new saliency measure is simple : we normalize the similarity - weighted boundary length by dividing it by the unweighted boundary length , and we normalize the similarity - weighted area by dividing it by the unweighted area . as a consequence , the quantities lii / gii and wii / vii become scale invariant ( they lie between 0 and 1 , since all coupling weights also lie between 0 and 1 ) . the new saliency measure can then simply be interpreted as ( 20)i[r]=average similarity along boundary of block iaverage similarity in interior of block i. this interpretation also provides an easy intuitive understanding of the saliency measure : a block is salient if it has low average similarity to its neighbouring blocks , but this has to be evaluated relative to how similar its own finer - level blocks are to each other . it is also clear that the new saliency measure may make it easier for many applications to choose a saliency threshold below which nodes are to be considered salient : due to the scale invariance ( and shape - invariance ) of the new measure the saliency threshold can remain constant on all levels , and segments with different shapes can be detected more consistently . we have experimented extensively comparing the scale invariant saliency measure ( 19 ) and the original saliency measure ( 18 ) . even though the scale invariant saliency measure is somewhat more expensive to compute ( a second three - way sparse matrix product v = pvp has to be evaluated on each level ) , we have found that it is much easier for our application to find a suitable value of the saliency threshold that works well on all recursive levels and for the different shapes the cells in our images assume . for our application , the extra work leads to a significantly more robust , less parameter - dependent algorithm and is thus worthwhile . it has to be noted , though , that for some applications the original saliency measure ( 18 ) may give good results ( with less work ) , or may be preferable for other reasons . the original saliency measure is expected to work well for segments of similar size and shape . also , the original saliency measure favours large segments ( since the saliency measure tends to assume smaller values on coarser levels ) , and this may be beneficial in applications in which only large segments are of interest . ( e.g. , one could consider an image with many white blobs of different sizes on a black background , and if only the large blobs are important , the original saliency measure can be used to select them . ) to finalize this section on a scale invariant saliency measure , we want to make three remarks . first , the example in figure 4 that led to the interpretations of lii , gii , wii and vii in terms of block boundary lengths and areas , was set in the context of nonoverlapping blocks . in the first stage of the swa v - cycle , the interpretation in terms of block boundaries and areas becomes less straightforward in this case and the heuristics become approximate , but the formulas remain well posed and extensive testing indicates that the measure performs as expected for the case of overlapping blocks as well . second , saliency measure ( 19 ) is not defined on the finest level since wii = 0 and vii = 0 for all finest - level nodes i. in many applications , salient segments on the finest level are not of interest , and can be disallowed . if they are to be allowed , the areas of finest - level pixels can be set to one , and scale invariant saliency measure i = lii / gii can be used . third , scale invariant saliency measure ( 19 ) only uses the diagonal elements of the boundary length and area matrices l , g , w and v , and the off - diagonal information in these matrices remains unused . the off - diagonal elements of these matrices can be used to refine ( 19 ) . for example , if block i shares a very short boundary with block j to which it is very similar , but is very different from all other neighbouring blocks , then its average similarity along the boundary , lii / gii , may be very small , even though block i is very similar to one of its neighbours . in this case , it may be desirable not to designate block i as salient , and to aggregate it with block j. such a situation can be detected by comparing the sizes of lij / gij for all neighbours j of i ( these ratios of off - diagonal elements can be interpreted as average similarities between blocks along their mutual boundary ) , and block i may only be considered salient if all of these are small ( relative to block i 's internal similarity ) . improvements of this kind may be important for certain types of applications , and remain a topic of further research . in this section we evaluate the performance of the multilevel segmentation algorithm by applying it to bright field phase contrast cell microscopy images . first we demonstrate the advantage of segmenting taking the multilevel intensity variance into account , versus not taking it into account . the image of figure 6(a ) has two regions that differ in texture but are identical in average intensity . using only intensity , only one segment is found ( figure 6(b ) ) because the algorithm can not distinguish the patterned shape from the uniform background . however , when intensity variance is incorporated , the two segments are identified separately ( figure 6(c ) ) . ( all cell images in this and the next section are obtained by cutting out a square n n region from larger 512 by 512 images as in figure 1 . ) however , correct segmentation is obtained : the variance of the background is low , but cells have high internal variance . by preferentially grouping blocks with high variance and blocks with low variance , cell parts are forced to merge together rather than merge with the background . figures 8 , 9 and 10 show further examples of successful segmentation results for increasingly difficult cases of cells with elongated expansions , touching cells , and cells that are close to division ( bright and circular ) . note that the segmentation parameters employed differ somewhat between the examples ; parameter selection is discussed at the end of this section . also , if the background is not contiguous , the swa algorithm finds a segment for each of the separate background regions . these background segments can easily be grouped together since they have very similar feature vectors at the level where they are detected as salient segments . finally , figure 11 shows segmentation results for a more difficult example involving four whole cells at once , one of which is close to division . with one choice of parameters , we get figure 11(b ) , which correctly identifies the 2 cells at the bottom with sharper boundaries but does not segment the top 2 cells correctly . with another choice of parameters , we get figure 11(c ) , where all four cells are found . however , part of the top - right cell is segmented with the background , and the nucleus of the bottom cell has its own segment . the results of figures 710 demonstrate that the swa algorithm is capable of correctly segmenting bright field cell images of moderate complexity . first , it is undesirable that parameters have to be changed depending on the image , since we are pursuing an automatic method . ( the next subsection explains how we have chosen the parameters for our algorithm to obtain the results shown above . ) however , the goal of the swa approach is to include enough multilevel features in the feature vectors of the overlapping blocks on the various levels to allow the algorithm to find correct segments for all images in a certain class , for example , the bright field cell images obtained by our experiment , with a single set of parameters . if this is achieved , the free parameters of the method are not a drawback , but are actually used to finetune the algorithm for the application class at hand , steering the segmentation in the desired direction . in our current implementation we have included multilevel intensity and multilevel intensity variance , and have shown that this allows the algorithm to correctly segment relatively complex bright field cell images . since these images are difficult to segment , this is a significant achievement , even if somewhat different parameters are required for different images . note also that the multilevel information about the segments gained by the swa algorithm leads to several other advantages over commonly used segmentation methods , as discussed in section 6 . however , it is clear that further research is needed before a fully automatic reliable swa segmentation method is obtained for the bright field images under consideration . a first type of improvement can be offered by including more features in the feature vector . for example , geometrical shape moments can be calculated for overlapping blocks at all levels , giving information about block shape and orientation that can be used to preferentially group together blocks that have similar shape or orientation [ 9 , 14 ] . we expect that this can be used to deal better with the difference between cells that are close to division ( they appear bright and circular ) and regular cells . the algorithm performs well on bright field images containing few cells , but the quality of the segmentation deteriorates when more cells are present . this is mainly due to the low - contrast boundaries and the broken halos that surround the cells . one way to overcome this problem may be to detect and promote boundary integrity across neighbouring aggregates as in . a second type of improvement , however , may be achieved by simply considering the extra information that is present in temporal sequences of images . the swa algorithm can take immediate advantage of this information by applying it directly to these image sequences in space time . however , before exploring swa segmentation in space time in section 4 , we first discuss how we chose the parameters for the segmentation results of figures 711 . to apply the algorithm to an image , we need to choose values for segmentation parameters , , , , , d1 , and . in order to find suitable parameters , we normally start with an initial parameter choice , and then finetune individual parameters according to the following guidelines . a suitable initial parameter set for our types of images to increase the contrast level of the image , increase top - level intensity scaling factor , which amplifies the intensity difference between pixel pairs . for images containing broad bright or dark boundaries of regions ( such as the halos in the bright field cell images ) , we do not want these boundaries to become salient segments themselves . since a large coarse - level intensity rescaling factor causes blocks with high average intensity to become more disconnected from their neighbours , a bright block can very easily become a salient segment . to avoid this , decrease to a small value , say in the range . in order to separate desired segments that differ more in average intensity and less in intensity variance , choose a larger intensity rescaling factor and a smaller variance rescaling factor . do the opposite if the desired segments differ more in variance . if the algorithm finds too many ( small ) segments , try decreasing ( more strong connections ) , decreasing ( stricter saliency threshold ) , increasing ( segments allowed only on coarser levels ) . decreasing ( more strong connections ) , decreasing ( stricter saliency threshold ) , increasing ( segments allowed only on coarser levels ) . on the contrary , if too few ( large ) segments are found , then shift the parameter values in the opposite direction the process for segmenting image sequences in space time ( see next section ) is similar but we usually start with a smaller , such as 0.03 . this is so because a node in an image sequence typically has more neighbours than a node in a single image . if we used the same , there would be fewer strong connections since our strength criterion defines strength relative to row sums of the coupling matrix ( see ( a.22 ) ) . in this section we describe results obtained when applying the multilevel swa algorithm to sequences of images . in our experimental technique cell trajectories in space time thus form object tunnels that are found efficiently by the swa algorithm . the extra temporal information makes it easier to resolve difficult cases such as touching cells , dividing cells , and cells that temporarily overlap . the resulting space time segments can also be used for tracking cell motion . by stacking up the images , the problem of segmenting multiple images it is not difficult to modify the swa algorithm to suit a 3d problem because the algorithm is already designed to coarsen arbitrary graphs , which can represent geometric grids of any dimension . additionally , swa can easily be applied to datasets with three spatial dimensions and one temporal dimension with little or no modicfication . figure 12 shows a cell with an irregular shape moving from the top - left corner of the window to the bottom - right corner , while slightly changing its shape , from image t = 1 to image t = 9 . applying the swa algorithm with appropriately chosen parameters to this stack of images , we find the two segments shown in figure 13 . note that the cell boundary is quite accurately captured in every frame , even though the boundary of the cell is irregular . figure 14 shows a plot of this result in 3d with the red tube representing the cell wall . in figure 14(a ) , the images are stacked with the t = 1 slice on top . in figure 14(b ) , the stack is reversed to show the t = 9 slice on top . the next set of images ( figure 15 ) shows a slow - moving triangular cell , and figure 16 shows cell contours annotated by a human expert . figure 17 shows the swa segmentation results , and figure 18 gives a 3d representation of them . in comparison with the human expert , the swa algorithm segments 71634 out of the 121 121 5 = 73205 pixels correctly , yielding a 97.85% accuracy . figure 19 shows a sequence of 11 cell images which represent a cell dividing into two cells . in figure 20 , this may be useful if one desires one large connected segment for all cells in a tree - like structure . figure 22 shows that segmentation parameters can be modified to obtain two segments , one for each of the daughter cells . even though the first few images contain only one cell , the information from later times causes the algorithm to interpret the mother cell as two cells that are about to separate . having two segments instead of one large segment for all three cells may be useful if one desires separate segments for each unique cell . in fact , if earlier images were included , one would want a third segment for the mother cell in this case . these two results illustrate how a judicious choice of segmentation parameters allow the user of the swa algorithm to steer the segmentation process towards the outcome that is desirable for the application at hand . figures 21 and 23 show 3d trajectories for the two segmentations . while more research is clearly needed to handle these rather complex cell divisions properly , the preliminary results shown point to interesting possibilities to use the space time swa algorithm as a building block of comprehensive tracking systems . multilevel algorithms often enjoy the desirable properties of fast execution time and low memory cost that are linearly proportional to the number of data elements [ 911 ] . in this section , we demonstrate how we achieved linear runtime as a function of the number of pixels in the input image for our implementation of the swa algorithm . we show that this , as expected , also holds for the space time version of the algorithm . this makes the space time swa algorithm a highly attractive building block for segmentation and tracking systems applied to long sequences of high - resolution images . also , if applications were to arise in which image sequences with very large numbers of pixels have to be segmented in short time , efficient parallel versions of the swa algorithm could be developed , along the lines of successful parallel implementations of amg , which have been shown to scale well on large parallel computers ( see , e.g. , , and references therein ) . we test runtime scaling by fixing a set of segmentation parameters and running the algorithm on images of different resolution , from 512 512 to 10 10 . ( we do this using an original 512 512 image that is downsampled to increasingly lower resolution . ) for each resolution , we determine the typical runtime by calculating the average runtime over three trials . figure 24 shows that the runtime scales almost linearly as a function of the number of pixels . our implementation is a mixed matlab - c research code , taking advantage of matlab 's sparse matrix data structures and operations , and employing c for the compute - intensive parts of the code that can not easily be done efficiently in matlab ( in particular , the amg coarsening step ) . this implementation could obviously be accelerated significantly for a production environment by migrating the whole implementation to c. however , even in our mixed matlab - c implementation , we obtain almost linear scaling , and a quite reasonable runtime of approximately 14 seconds for segmenting a 512 512 image . figure 25 shows that we also obtain linear runtime for a 3d 512 512 5 image downsampled to increasingly lower resolution . in this paper , we have investigated the use of a multilevel aggregation algorithm as a method for segmenting live cell bright field microscope images . we use a variant of the segmentation by weighted aggregation ( swa ) technique [ 8 , 9 ] and incorporate an improved scale invariant saliency measure , which can identify salient segments more accurately . we have shown that bright field cell images of moderate complexity are segmented correctly by incorporating multilevel intensity and intensity variance . we have shown how the swa algorithm can be applied without significant modification to temporal sequences of images , producing segments that track cell contours in space and time . correct segmentation still depends on a judicious choice of segmentation parameters , and further research is needed before a fully automatic reliable swa segmentation method is obtained for the bright field images under consideration . in future work , we plan to investigate improving segmentation results by including more features in the feature vector . for example , we are considering to include shape moments , anisotropic texture , cross - correlation between features , and boundary detection [ 9 , 14 , 21 ] . we also plan to apply the multilevel aggregation algorithm to segmentation of cell parts ( cytoplasm , nucleus and nucleoli ) . while the robust application of multilevel aggregation to bright field cell images requires further research , it is already clear that the swa segmentation approach may have several advantages over more commonly used segmentation techniques , which include level set , active contour , and watershed methods . first of all , multilevel aggregation is fast and linearly scalable in the number of pixels to be segmented . the algorithm is conceptually simple since there is no need to define initial level set seeds , or extract markers as in watershed . the space time segmentation approach uses extra temporal information that makes it easier to resolve difficult cases like touching cells , dividing cells and cells that temporarily overlap . nevertheless , it seems that extensive further research is required to handle these difficulties properly . the feature vector for each resulting segment contains multilevel information about the segment that can be exploited in various ways . it can be used to classify segments as background , dividing cell , or regular cell , and it can potentially be used ( in 3d ) to separate tracks of dividing cells from tracks of regular cells , to aid the proper handling of cell division events . the multilevel segmentation approach provides a single , unified technique that produces accurate cell trajectory segments ( for moderately complex cases so far ) and gives a lot of additional useful information . it is clear that it will have to be combined with other advanced methods of geometric , modeling and statistical nature in order to obtain a complete and robust segmentation and tracking system , along the lines of the sophisticated and comprehensive segmentation and tracking methods that recently have been described ( see [ 5 , 6 ] and references therein ) . however , due to its conceptual simplicity , linear efficiency , and tunability , and due to the useful additional multilevel information it provides , multilevel aggregation may simplify the development of such comprehensive systems , and it thus promises to be an attractive alternative to level set , active contour and watershed approaches as a basic building block for robust segmentation and tracking systems . output : an n m boolean segmentation matrix u describing m segments ( uij = 1 means that pixel i belongs to the jth segment ) . define global segmentation parameters : ( top - level intensity scaling factor ) , ( coarse - level intensity rescaling factor ) , ( coarse - level variance rescaling factor ) , ( coarsening strength threshold ) , ( saliency threshold ) , d1 ( sharpening threshold ) , ( segment detection threshold level ) , ( variance rescaling threshold level ) . ( top - level intensity scaling factor ) , ( coarse - level intensity rescaling factor ) , ( coarse - level variance rescaling factor ) , ( coarsening strength threshold ) , ( saliency threshold ) , d1 ( sharpening threshold ) , ( segment detection threshold level ) , ( variance rescaling threshold level ) . initialize level 1 variables , m , i , a , s , l , g , v and : current level = 1 . , m}. let i be an m 1 vector with ij as the intensity of pixel j , where the intensity values may range from 0 to 1 . ( each input image is scaled such that the maximum intensity value equals 1 . ) let coupling matrix a be an m m matrix with ( a.1)aij={e|iiij|if i , j are horizontal orvertical neighbours,0,otherwise.let variance matrix s be an m m zero matrix . let weighted boundary length matrix l be an m m matrix with ( a.2)lij={aijif ij,kiaikif i = j.let area matrix v be an m m matrix with ( a.3)vij={0if aij=0,1if aij0.let boundary length matrix g , be an m m matrix with ( a.4)gij={vijif ij,kivikif i = j.let saliency vector be an m 1 vector with ( a.5)i = liigii . , m}. let i be an m 1 vector with ij as the intensity of pixel j , where the intensity values may range from 0 to 1 . ( each input image is scaled such that the maximum intensity value equals 1 . ) let coupling matrix a be an m m matrix with ( a.1)aij={e|iiij|if i , j are horizontal orvertical neighbours,0,otherwise . let weighted boundary length matrix l be an m m matrix with ( a.2)lij={aijif ij,kiaikif i = j . let area matrix v be an m m matrix with ( a.3)vij={0if aij=0,1if aij0 . let boundary length matrix g , be an m m matrix with ( a.4)gij={vijif ij,kivikif i = j . let saliency vector be an m 1 vector with ( a.5)i = liigii . determine overlapping segments by calling the recursive graph segmentation function : ( a.6)u = imagevcycle(,m , i , a , s , v, ) . assign pixels uniquely to segments : set ( a.7)uij{1if j = min { j : max k uik = uij},0,otherwise . input : , m , i , a , s , v , . output : u , in which uij indicates the fraction of node i on level r belonging to segment j. if , set all i = . ( no salient segments allowed on current level . ) coarsen the current graph : set ( a.8)c[r]=coarsenamg(a[r],[r],, ) , where coarsenamg is a function described in appendix a.3 . let m be the length of c - point vector c. if m = m ( no further coarsening obtained ) , output u as an m m identity matrix ( every current node is a segment ) let interpolation matrix p be an m m matrix with ( a.9)pij[r , r+1]={1if ic[r ] , i = cj[r],0if ic[r ] , icj[r],aicj[r]kc[r]aikif ic[r ] . let column - scaled interpolation matrix p[r , r+1 ] be an m m matrix with ( a.10)pij[r , r+1]=pij[r , r+1]kpkj[r , r+1 ] . let coarse - level intensity vector i be an m 1 vector with ( a.11)i[r+1]=(pij[r , r+1])ti[r ] . for each block on the current coarse level , r + 1 , compute a new intensity variance measure relative to level r : ( a.12)scoarse[r+1]=(p[r , r+1])t(i[r])2(i[r+1])2 , and average the previously calculated variance measures for levels finer than level r + 1 : ( a.13)sfine[r+1]=(p[r , r+1])ts[r ] . here , ( i ) and ( i ) are the vectors i and i squared componentwise . then coarse - level intensity variance matrix s is an m ( r + 1 ) matrix given by ( a.14)s[r+1]=[sfine[r+1 ] scoarse[r+1 ] ] . define coarse - level coupling matrix a , an m m matrix , in three steps . let ( a.15)a[r+1]=p[r , r+1]ta[r]p[r , r+1].rescale using coarse - level intensity : ( a.16)aij[r+1]aij[r+1]e|ii[r+1]ij[r+1]| i , j.if , rescale using multilevel variance : set , for all i , j , ( a.17)aij[r+1]aij[r+1]e||si[r+1]sj[r+1]||2 , where si is the ith row of coarse - level intensity variance matrix s. let ( a.15)a[r+1]=p[r , r+1]ta[r]p[r , r+1 ] . if , rescale using multilevel variance : set , for all i , j , ( a.17)aij[r+1]aij[r+1]e||si[r+1]sj[r+1]||2 , where si is the ith row of coarse - level intensity variance matrix s. let coarse - level weighted area matrix w = a. let coarse - level area matrix v = pvp . define coarse - level weighted boundary length matrix l , an m m matrix , in two steps : let l = a . l ii kilik for all i. l ii kilik for all i. define coarse - level boundary length matrix g , an m m matrix , in two steps : let g = v . g ii kigik for all i. g ii kigik for all i. let coarse - level saliency vector be an m 1 vector , determined as follows . for each c - point i , if it was salient on level r , keep it salient on level r + 1 . if not , determine its saliency using the saliency measure . if ci = 0 , then i = 0 , otherwise ( a.18)i[r+1]={lii[r+1]/gii[r+1]wii[r+1]/vii[r+1]if lii[r+1 ] /gii[r+1]wii[r+1]/vii[r+1]>0otherwise . recursively segment the coarse graph : let ( a.19)u[r+1]=imagevcycle([r+1],m[r+1],i[r+1],a[r+1 ] , s[r+1],v[r+1],[r+1 ] ) . find the current segmentation matrix from the coarse - level segmentation matrix : let ( a.20)u[r]=p[r , r+1]u[r+1 ] . sharpen overlapping segments : for all i , j , set ( a.21)uij[r]{0if uij[r]<d1,1if uij[r]>1d1,uij[r]otherwise return current segmentation matrix u. input : a , , , . output : c , indices of c - points chosen on level r. let m be the number of rows ( or columns ) in a. let a be an m m matrix containing only strong connections . ( one may also use the criterion aij max kiaik for strong connections , which is more standard in the amg context . in our experience , either criterion may be used , but may have to be chosen differently . ) let be an m 1 vector in which i denotes the number of nonzero entries in column i of a[r ] . let t be an m 1 zero vector that keeps track of the set to which each node is assigned . for node i , ti = 0 means it is unassigned , ti = 1 means it is a c - point , and ti = 2 means it is an f - point . for i = 1,2 , , m , if i < , then set ti 1 and i 0 . ( so that salient nodes , or segments , are designated as c - points . ) while not all ti > 0 , do the following . let j be the smallest value satisfying tj = 0 and j = max kk . ( nodes that strongly influence many other nodes are likely to be chosen as c - points . ) set tj 1 and j 0 . let k={k : akj[r]>0,tk=0}. ( k is the set of unassigned nodes that are strongly influenced by node j. ) for all k k , do the following let h={h : akh[r]>0,th=0}. ( h is the set of nodes that strongly influence k. ) for all h h , set h = h + 1 . ( nodes in h are made more likely to become c - points . ) let j be the smallest value satisfying tj = 0 and j = max kk . ( nodes that strongly influence many other nodes are likely to be chosen as c - points . ) set tj 1 and j 0 . let k={k : akj[r]>0,tk=0}. ( k is the set of unassigned nodes that are strongly influenced by node j. ) for all k k , do the following . set tk 2 and k 0 . let h={h : akh[r]>0,th=0}. ( h is the set of nodes that strongly influence k. ) for all h h , set h = h + 1 . ( nodes in h are made more likely to become c - points . ) let h={h : akh[r]>0,th=0}. ( h is the set of nodes that strongly influence k. ) for all h h , set h = h + 1 . ( nodes in h are made more likely to become c - points . ) let the vector of c - points be c = { i : ti = 1}. let k be the number of consecutive images we wish to segment in space time . the following modifications need to be made to the nonrecursive part of the algorithm ( appendix a.1 ) . to highlight the changes , we do not rewrite any steps that do not change . read in the k greyscale images as n n intensity matrices , i1 , i2 , , ik . let n = n. ( the global segmentation parameters are defined as before . ) initialize variables , m , i , a , s , l , g , w , v and : set the current number of nodes m = nk . obtain the m 1 intensity vector i by reshaping the n nk intensity matrix given by ( a.23)[i1i2ik1ik ] . a is an m m matrix with ( a.24)aij={e|iiij|if i , j are horizontal , verticalor time - wise neighbours,0,otherwise.the other variables are defined as before . set the current number of nodes m = nk . obtain the m 1 intensity vector i by reshaping the n nk intensity matrix given by ( a.23)[i1i2ik1ik ] . a is an m m matrix with ( a.24)aij={e|iiij|if i , j are horizontal , verticalor time - wise neighbours,0,otherwise . everything else in the algorithm , including the recursive part and the coarsening , remains unchanged .

a multilevel aggregation method is applied to the problem of segmenting live cell bright field microscope images . the method employed is a variant of the so - called segmentation by weighted aggregation technique , which itself is based on algebraic multigrid methods . the variant of the method used is described in detail , and it is explained how it is tailored to the application at hand . in particular , a new scale - invariant saliency measure is proposed for deciding when aggregates of pixels constitute salient segments that should not be grouped further . it is shown how segmentation based on multilevel intensity similarity alone does not lead to satisfactory results for bright field cells . however , the addition of multilevel intensity variance ( as a measure of texture ) to the feature vector of each aggregate leads to correct cell segmentation . preliminary results are presented for applying the multilevel aggregation algorithm in space time to temporal sequences of microscope images , with the goal of obtaining space - time segments ( object tunnels ) that track individual cells . the advantages and drawbacks of the space - time aggregation approach for segmentation and tracking of live cells in sequences of bright field microscope images are presented , along with a discussion on how this approach may be used in the future work as a building block in a complete and robust segmentation and tracking system .

1. Introduction 2. Algorithm Description 3. Segmentation Results 4. Space Time Segmentation and Tracking 5. Scalability 6. Conclusions and Future Work A. Detailed Description of Multilevel Segmentation Algorithm

there is extensive current interest in the high - content , high - throughput screening of live cell populations , and the experimental techniques being developed for this purpose are leading to important biological insights with applications to new clinical therapies [ 14 ] . due to the large amount of data generated by these experiments , automated data processing systems for segmentation and tracking of live cells in sequences of microscope images are a virtual necessity . several comprehensive systems for segmentation and tracking have recently been described in the literature ( see , e.g. in this paper , we investigate the use of a multilevel aggregation algorithm as an alternative method for segmenting live cell bright field microscope images . saliency measure for this algorithm to decide when aggregates of pixels constitute salient segments that should not be grouped further . we investigate the performance of the multilevel aggregation algorithm for bright field cell segmentation in images and sequences of images , and discuss its advantages and drawbacks compared to other methods as a possible building block in comprehensive automatic cell segmentation and tracking systems . we use a variant of the segmentation by weighted aggregation technique [ 8 , 9 ] to segment the microscope images , which itself is based on algebraic multigrid ( amg ) methods for solving linear systems of equations [ 10 , 11 ] . the swa method attempts to segment the image into salient ( or prominent ) groups of pixels by using a multilevel aggregation procedure that groups blocks of pixels at various scales , based on multiscale feature vectors for the pixel blocks . the variant of the method that we use is described in detail below , and it is explained how it is tailored to the application at hand . our algorithmic contribution is a new scale invariant saliency measure for deciding when aggregates of pixels constitute salient segments that should not be grouped further . our new saliency measure is a variant of the saliency measure employed in [ 8 , 9 ] that takes scaling into account . furthermore , we use the so - called first pass of the standard amg graph coarsening algorithm [ 10 , 11 ] to coarsen the pixel graph , rather than the direct aggregation methods that are used in swa algorithms described in the literature [ 8 , 9 , 12 , 14 ] . we test our approach on parts of real microscope images , and show how segmentation based on multilevel intensity similarity alone does not lead to satisfactory results . however , the addition of multilevel variance of mean intensities ( as a measure of texture ) to the feature vector of each aggregate , as in [ 9 , 14 ] , leads to correct cell segmentation . in the second part of the paper , preliminary results are presented for applying the multilevel aggregation algorithm in space time to temporal sequences of microscope images , with the goal of obtaining space time segments ( object tunnels ) that track individual cells . this parallels previous research results on space time segmentation using level set methods [ 15 , 16 ] and on comprehensive segmentation and tracking systems for sequences of cell images [ 5 , 6 ] , but space time segmentation using swa has , to our knowledge , not been investigated in the literature yet . in this paper , we present preliminary results on our experience with extending the spatial swa segmentation algorithm to space time segmentation of sequences of images , for simplified cases of isolated cells and pairs of cells . we identify problems that remain and suggest possible extensions of the algorithm that may handle them , and we comment on the potential of the swa segmentation method as a building block in comprehensive segmentation and tracking systems along the lines of [ 5 , 6 ] and references therein . while the general ideas and concepts of the swa framework are described in several places [ 8 , 9 , 12 , 14 ] , most of the description in the literature is formulated in general terms , and not all algorithmic details are spelled out . while this may be perceived as a potential drawback of the approach , it also opens opportunities to finetune the algorithm for the application class at hand . indeed , the problem of segmenting an image without further specifications is often ill - posed . finetuning the swa parameters the question about how to choose the swa parameters is not discussed much in the literature [ 8 , 9 , 12 , 14 ] , and in most cases specific parameters are not given for segmentation results that are presented as examples . automatic segmentation and tracking of live cells in bright field microscopy images is a difficult task [ 5 , 18 ] . for this reason , fluorescence microscopy is often used in place of bright field microscopy to image live cells . however , the image segments we obtain from our analysis can be used to categorize cell shapes while keeping the cells in culture . if one or two of these channels are used solely for tracking purposes , the number of channels available for measuring protein concentrations is reduced , which may be a significant limitation . in addition , bright field images typically contain many features of interest , and accurate segmentation methods for bright field images are intrinsically useful since they allow the study of cell morphology and internal cell structure , and their dynamics . , we give a brief description of the swa algorithm and introduce a new scale invariant saliency measure . in section 4 we explain how the approach is extended to segmentation in space time in a straightforward way , and investigate the performance of space time segmentation for simple sequences of real cell images . in the third subsection , we propose and discuss a new scale invariant saliency measure for the swa algorithm . the problem of image segmentation can be viewed in terms of segmentation of a weighted undirected graph , with each node of the graph representing a pixel , and each edge corresponding to a link between neighbouring nodes , weighted by the similarity in intensity between the two neighbouring pixels . this graph coarsening can be done in a variety of ways , and the particular approach we employ is explained in the next subsection and in appendix a.3 . ( for each coarse - level overlapping block , one finest - level node corresponding to the c - point of the block on the previous level is shown as a representative . ) pixels that are neighbours in the horizontal or vertical directions are connected by weighted edges in the graph , with edge weights aij defined using an exponential function of the intensity difference between the pixels they connect : ( 1)aij={e|iiij|if i , j are neighbours,0,otherwise , with 0 a user - defined parameter . on each level , the blocks are tested for saliency : a salient ( or prominent ) block is a block that is sufficiently different from all the blocks it is connected to , as determined by a saliency measure . it is important to retain overlapping blocks in the first phase of the algorithm , since a pixel that may appear to belong primarily to a certain block according to fine - level information , may be reclassified as belonging to a different block later when coarser - level information is taken into account . in this way , the representation of the so - far overlapping segments is obtained on each recursive level . on each level , the overlap between segments is reduced by a sharpening procedure , with a sharpening threshold d1 , which we normally take equal to 15% . once on the coarsest level , the recursive function returns the segments found , and performs a bottom - up process that ultimately leads to each pixel in the ( finest - level ) image being assigned to exactly one of the ( initially overlapping ) segments found on the coarsest level . the nonrecursive and recursive parts of the algorithm are described in detail in appendices a.1 and a.2 , respectively , and the coarsening algorithm is given in appendix a.3 . in order to coarsen the graph at the current level , we use the so - called first pass of the standard amg coarsening algorithm [ 10 , 11 ] . this algorithm coarsens the connectivity graph with weights aij by first dividing the connections in sets of weak and strong connections based on their weights , and then approximately determining a subset of the nodes of the graph that is a maximal independent set in the subgraph formed by only retaining the strongly connected edges . note also that , on coarse level r , nodes that have already been designated salient on previous levels ( see section 2.3 ) , automatically become c - points at the beginning of the coarsening on level r. note that we use standard amg coarsening [ 10 , 11 ] to coarsen the graph , rather than the direct aggregation methods that are used in swa algorithms described in the literature [ 8 , 9 , 12 , 14 ] . another blockwise quantity we use to better connect similar blocks is multilevel variance in intensity ( as a measure of texture ) . in each such feature vector , the last component gives the variance between the average intensities of the level - r nodes that correspond to the level - r + 1 node , the one - before - last component gives the average over those level - r nodes of the variance between the average intensities of the level - r 1 nodes that correspond to each of those level - r nodes , and so forth . in summary , we list the free parameters in our algorithm , to be chosen such that correct segmentation is obtained for the application at hand : top - level intensity scaling factor , coarse - level intensity rescaling factor , coarse - level variance rescaling factor , coarsening strength threshold , saliency threshold ( see the next section ) , sharpening threshold d1 , segment detection threshold level , and variance rescaling threshold level . we propose a saliency measure that is a modified version of the saliency measures used in [ 8 , 9 , 1214 , 21 ] . here , a is the coupling matrix on level r , the matrices l and w are given by ( 8)lij[r]={aij[r]if ij,kiaik[r]if i = j , w[r]=a[r ] , and u is a boolean state vector for a particular block on level r such that uj = 1 if node j belongs to the block and uj = 0 otherwise . in the swa approach , however , we do not directly calculate the fine - level eigenvectors , but , instead , consider increasingly coarse versions of the coupling matrix and detect salient segments based on connection strength in those coarse - level coupling matrices , guided by functional ( 7 ) . it can be understood easily that saliency measure ( 9 ) is sensitive to the scales of segments , and , depending on the application , this may lead to difficulties . in what follows , we illustrate the scale sensitivity of saliency measure ( 9 ) by a simple example , and propose a new variant of saliency measure ( 9 ) that takes into account scaling . the ith column of the interpolation matrix between levels 2 and 1 , p , contains 1 s in the rows corresponding to the pixels in block i , and 0 s in the other rows . note that columns corresponding to pixels close to the boundaries of the image would have a different nonzero pattern , but we assume that the white block in figure 4 is embedded in a larger black image and is located far from the image boundary , such that we don not have to worry about boundaries . ) then multiply this with row pi to obtain ( 17)pitvpi=24 , which equals twice the number of internal connections , or approximately twice the area of block i. due to this interpretation of the diagonal elements of matrices g and v in terms of block boundary length and block area , we will in what follows refer to the matrices g as boundary length matrices , and to v as area matrices . with these interpretations of the diagonal elements of l , w , g and v in hand , we can now analyze the scale behaviour of saliency measure ( 9 ) , and propose a new scale invariant version of it . it is also clear that the new saliency measure may make it easier for many applications to choose a saliency threshold below which nodes are to be considered salient : due to the scale invariance ( and shape - invariance ) of the new measure the saliency threshold can remain constant on all levels , and segments with different shapes can be detected more consistently . even though the scale invariant saliency measure is somewhat more expensive to compute ( a second three - way sparse matrix product v = pvp has to be evaluated on each level ) , we have found that it is much easier for our application to find a suitable value of the saliency threshold that works well on all recursive levels and for the different shapes the cells in our images assume . also , the original saliency measure favours large segments ( since the saliency measure tends to assume smaller values on coarser levels ) , and this may be beneficial in applications in which only large segments are of interest . , one could consider an image with many white blobs of different sizes on a black background , and if only the large blobs are important , the original saliency measure can be used to select them . ) in the first stage of the swa v - cycle , the interpretation in terms of block boundaries and areas becomes less straightforward in this case and the heuristics become approximate , but the formulas remain well posed and extensive testing indicates that the measure performs as expected for the case of overlapping blocks as well . second , saliency measure ( 19 ) is not defined on the finest level since wii = 0 and vii = 0 for all finest - level nodes i. in many applications , salient segments on the finest level are not of interest , and can be disallowed . if they are to be allowed , the areas of finest - level pixels can be set to one , and scale invariant saliency measure i = lii / gii can be used . third , scale invariant saliency measure ( 19 ) only uses the diagonal elements of the boundary length and area matrices l , g , w and v , and the off - diagonal information in these matrices remains unused . first we demonstrate the advantage of segmenting taking the multilevel intensity variance into account , versus not taking it into account . however , part of the top - right cell is segmented with the background , and the nucleus of the bottom cell has its own segment . however , the goal of the swa approach is to include enough multilevel features in the feature vectors of the overlapping blocks on the various levels to allow the algorithm to find correct segments for all images in a certain class , for example , the bright field cell images obtained by our experiment , with a single set of parameters . if this is achieved , the free parameters of the method are not a drawback , but are actually used to finetune the algorithm for the application class at hand , steering the segmentation in the desired direction . in our current implementation we have included multilevel intensity and multilevel intensity variance , and have shown that this allows the algorithm to correctly segment relatively complex bright field cell images . however , it is clear that further research is needed before a fully automatic reliable swa segmentation method is obtained for the bright field images under consideration . a second type of improvement , however , may be achieved by simply considering the extra information that is present in temporal sequences of images . on the contrary , if too few ( large ) segments are found , then shift the parameter values in the opposite direction the process for segmenting image sequences in space time ( see next section ) is similar but we usually start with a smaller , such as 0.03 . by stacking up the images , the problem of segmenting multiple images it is not difficult to modify the swa algorithm to suit a 3d problem because the algorithm is already designed to coarsen arbitrary graphs , which can represent geometric grids of any dimension . in figure 20 , this may be useful if one desires one large connected segment for all cells in a tree - like structure . these two results illustrate how a judicious choice of segmentation parameters allow the user of the swa algorithm to steer the segmentation process towards the outcome that is desirable for the application at hand . while more research is clearly needed to handle these rather complex cell divisions properly , the preliminary results shown point to interesting possibilities to use the space time swa algorithm as a building block of comprehensive tracking systems . in this section , we demonstrate how we achieved linear runtime as a function of the number of pixels in the input image for our implementation of the swa algorithm . this makes the space time swa algorithm a highly attractive building block for segmentation and tracking systems applied to long sequences of high - resolution images . also , if applications were to arise in which image sequences with very large numbers of pixels have to be segmented in short time , efficient parallel versions of the swa algorithm could be developed , along the lines of successful parallel implementations of amg , which have been shown to scale well on large parallel computers ( see , e.g. our implementation is a mixed matlab - c research code , taking advantage of matlab 's sparse matrix data structures and operations , and employing c for the compute - intensive parts of the code that can not easily be done efficiently in matlab ( in particular , the amg coarsening step ) . in this paper , we have investigated the use of a multilevel aggregation algorithm as a method for segmenting live cell bright field microscope images . we use a variant of the segmentation by weighted aggregation ( swa ) technique [ 8 , 9 ] and incorporate an improved scale invariant saliency measure , which can identify salient segments more accurately . we have shown that bright field cell images of moderate complexity are segmented correctly by incorporating multilevel intensity and intensity variance . we have shown how the swa algorithm can be applied without significant modification to temporal sequences of images , producing segments that track cell contours in space and time . in future work , we plan to investigate improving segmentation results by including more features in the feature vector . while the robust application of multilevel aggregation to bright field cell images requires further research , it is already clear that the swa segmentation approach may have several advantages over more commonly used segmentation techniques , which include level set , active contour , and watershed methods . it can be used to classify segments as background , dividing cell , or regular cell , and it can potentially be used ( in 3d ) to separate tracks of dividing cells from tracks of regular cells , to aid the proper handling of cell division events . it is clear that it will have to be combined with other advanced methods of geometric , modeling and statistical nature in order to obtain a complete and robust segmentation and tracking system , along the lines of the sophisticated and comprehensive segmentation and tracking methods that recently have been described ( see [ 5 , 6 ] and references therein ) . however , due to its conceptual simplicity , linear efficiency , and tunability , and due to the useful additional multilevel information it provides , multilevel aggregation may simplify the development of such comprehensive systems , and it thus promises to be an attractive alternative to level set , active contour and watershed approaches as a basic building block for robust segmentation and tracking systems . for each block on the current coarse level , r + 1 , compute a new intensity variance measure relative to level r : ( a.12)scoarse[r+1]=(p[r , r+1])t(i[r])2(i[r+1])2 , and average the previously calculated variance measures for levels finer than level r + 1 : ( a.13)sfine[r+1]=(p[r , r+1])ts[r ] .

the structure and dynamics of chromatin clearly differentiate the eukaryotes from the other superkingdoms of life . eukaryotic chromatin undergoes a variety of structural and compositional changes that accompany the progression of the cell cycle and changes in gene expression . these dynamics are regulated both by a variety of protein - protein and protein - dna interactions and by catalytic modification and reorganization of proteins and nucleic acids that comprise the chromosomes . in recent years it has become clear that covalent modification of histones , transcription factors and other chromosomal proteins plays a major part in the dynamics of chromatin . the covalent modifications include hydroxylation of proline and lysine , methylation of lysine and arginine , phosphorylation of serine and threonine , and acetylation and ubiquitination of lysine . other enzymes , such as deacetylases and phosphatases , remove covalent modifications from proteins and thus reverse their effects . the covalent modifications may change the local or global charge properties of chromosomal proteins and regulate their interactions with dna . additionally , the modifications also seem to form a ' code ' that is recognized by specific groups of regulatory proteins . besides covalent modifications , atp - dependent enzymes such as the swi2/snf2 atpases and other chaperone - like proteins remodel the chromatin by rearranging the binding pattern of histones and other chromosomal proteins . these covalent and non - covalent catalytic actions on the chromatin can result in condensation or decondensation of chromatin , either locally or on the chromosomal scale , and thereby regulate access of transcription factors and other proteins to the chromatin . many of these chromatin - modifying activities are organized into large multisubunit complexes , with the core enzyme accompanied by several noncatalytic subunits . the subunits of these complexes are characterized by a number of conserved domains that interact with other proteins or dna . most of these domains are evolutionarily mobile modules and combine with each other in a very wide range of domain architectures . well studied examples of these are the bromodomain that interacts with acetylated peptides , chromodomains that mediate specific interactions with proteins and rna , the phd finger mediating protein - protein interactions , the myb and sant domains that interact with both dna and proteins , and the sap and at - hook domains that interact with dna . these domains often occur together in large proteins linked to catalytic domains and may serve to tether these proteins to different components of chromatin and deliver catalytic activities to specific locations . the computational analysis of chromatin proteins has helped in the identification of a large number of conserved modules in the chromosomal proteins . these studies , followed by biochemical and structural characterization of these modules has thrown considerable light on the biology of chromatin dynamics and the roles these domains have played in course of evolution . using computational analysis , we also show that this domain is found linked to catalytic domains such as oxidoreductase and jun activating binding protein 1 ( jab ) domains and these proteins may define a novel class of chromatin - modifying enzymes . in the course of our systematic survey of eukaryotic chromosomal proteins , we observed a conserved globular module shared by the swi3p and rsc8p proteins from the yeast saccharomyces cerevisiae that did not map to any previously characterized domain . these homologous proteins are parts of the multisubunit swi / snf and rsc complexes that drive chromatin remodeling through the snf2/swi2-like atpase subunit . the region of similarity encompassing this segment has been termed ' conserved region 1 ' in . both these proteins additionally contain a myb - related helix - turn - helix domain termed the sant domain at their carboxyl terminus and a further carboxy - terminal conserved -helical extension restricted to orthologs of rsc8p and swi3p . given that these subunits are key components of the respective chromatin - remodeling complexes , and mediate multiple interactions , we sought to further investigate the provenance of their conserved amino - terminal module using computational methods . a psi - blast search initiated with this region from rsc8p ( gi : 14318562 , region 80 - 177 ) not only recovered its paralogs and orthologs from other organisms such as moira from drosophila and baf155 and baf170 from vertebrates , but also several other uncharacterized proteins from diverse eukaryotes with statistically significant expected values ( e - values ) at the point of detection . for example , the search recovered the nuclear protein spac23e2.02 from schizosaccharomyces pombe in iteration 4 ( e = 2 10 ) , the human proteins kiaa1915 ( e = 10 ) and kiaa0601 ( e = 10 ) in iteration 2 and the arabidopsis thaliana protein at2g47620 in iteration 3 ( e = 10 ) . we prepared a multiple alignment of these regions from all the proteins detected in these searches ( figure 1 ) and used it to construct a psi - blast position - specific score matrix ( pssm ) and a hidden markov model for further searches against the complete or nearly complete proteome databases of individual eukaryotes . these searches allowed us to detect additional copies of this region in the carboxyl termini of the asynaptic 1 protein ( a hop1p homolog ) from plants . additionally these searches also recovered a conserved globular region in the extreme carboxyl terminus of the ada2-like proteins from eukaryotes , with moderate significance . while these newly detected proteins have a somewhat distinct conservation pattern from the originally detected set ( figure 1 ) , their secondary structure predicted using the jpred program closely matches that predicted for the originally detected set . this observation , together with the information that the ada2-like proteins are chromatinic proteins , just like the members of the original set , suggests that these domains are probably distant relatives of each other ( figure 1 ) . the conserved region described above occurred in various distinct domain - architecture contexts , suggesting that it is an evolutionarily mobile domain ( figure 2 ) . we named this domain the swirm domain after the proteins swi3p , rsc8p and moira in which it was first recognized . secondary - structure predictions , based on the multiple alignment , indicate the presence of four distinct a helices . hence , it is predicted that the swirm domain is likely to form a globular structure in the form of a tetrahelical bundle ( figure 1 ) . however , a direct comparison showed no specific relationship between the sequence conservation pattern of the swirm domain and those of other -helical bundles that are commonly encountered in chromosomal proteins such as the bromodomain , the myb or sant domains and other helical dna - binding domains . the differences in conservation pattern between the distantly related ada2p carboxy - terminal domains and the canonical swirm domains suggest that the former are likely to interact with a very distinct set of partners . rsc8p ( swh3p ) has been shown to mediate multiple interactions in the rsc complex : it undergoes dimerization via the carboxy - terminal coiled - coil segment , associates with the swi2/snf2 atpase sth1p by forming two distinct contacts , and it forms a complex with rsc6p subunit . deletion analyses have shown that the region of the protein that includes the swirm domain is probably required for at least one of the contacts with sth1p and perhaps even those with rsc6p . this suggests that the swirm domain is most likely to mediate protein - protein interactions . the rsc8p- and swi3p - related proteins have fairly complex architectures , implying that the different modules may mediate distinct interactions . rsc8p has an additional zz domain between the swirm and myb domains , whereas the animal and dictyostelium versions , like moira , have an additional amino - terminal chromodomain ( figure 2 ) . however , the common denominator in all these proteins are the swirm and myb domains , suggesting that they mediate the evolutionarily conserved interactions of these proteins , such as those with the swi2/snf2 atpase . the presence of a divergent swirm domain , linked to a horma domain , in asy1 , the plant ortholog of the yeast hop1p , is consistent with the role of these proteins in mediating protein - protein interactions in the process of chromosome synapsis . the ada2p - like proteins ( figure 2 ) are chromatin - associated proteins and are known to mediate protein - protein interactions . they contain a distinct carboxy - terminal globular domain that is distantly related to the swirm domain , suggesting that this domain could mediate a subset of their interactions with the transcriptional machinery . a striking group of proteins with the swirm domain is the one typified by spac23e2.02 and spbc146.09c from s. pombe . between one and four orthologs of this protein are encoded by different crown - group eukaryotes ( figures 1,3 ) and contain the swirm domain linked to a predicted fad - dependent oxidoreductase domain . a similar observation regarding the presence of a fad - binding domain has been reported for kiaa0601 , a vertebrate protein of this family . kiaa0601 , along with the co - repressor corest , is stably associated with the histone deacetylase complex . furthermore , spac23e2.02 has been shown to be a nuclear protein and along with its paralog spbc146.09c , contains at its extreme carboxyl terminus an hmg1 domain , which is a common dna - binding module found in diverse chromosomal proteins ( figures 2,3 ) . the closest relatives of the predicted oxidoreductase domains linked to the swirm domain are the polyamine oxidases and the monoamine oxidases ( figure 3 ) . these enzymes are involved in the oxidation of amino groups of polyamines such as spermine and spermidine and monoamines such as dopamine and serotonin . the availability of the crystal structure for the polyamine oxidase from maize allowed us to evaluate the sequence conservation of the oxidoreductase domain fused to the swirm domains . the alignment of this domain from the spac23e2.02-like proteins spans the entire length of the amino oxidase and contains the hallmark residues required to bind the fad cofactor ( figure 3 ) . these include the glycine - rich loop bounded by a -strand and an a helix that is typical of rossmann fold nucleotide - binding proteins , a conserved glutamate specific to the amino oxidase family , that interacts with ribose in fad and other residues of the substrate and cofactor - binding site . thus , these oxidoreductase domains are likely to function as amino oxidases ; this , together with their linkage within the same polypeptide to domains typical of chromatin proteins , and evidence for nuclear localization , suggests that they are novel chromatin - modifying enzymes . consistent with this , the complex containing kiaa0601 has been shown to contain fad and was suggested to be a chromatin - modifying enzyme . building on these observations , we suggest two functional possibilities for these proteins : first , they could act as novel protein - modifying enzymes that oxidize the amino groups of lysines or arginines present on histones or transcription factors ; alternatively they could affect chromatin structure by oxidizing basic polyamines present in the chromatin and thereby reducing the charge balance . it is also conceivable that the fad cofactor of these enzymes functions analogously to nad , in a deacetylation reaction of acetyllysines similar to that carried out by sir2 enzymes . whereas both these enzymes are derived versions of the rossmann fold and bind a dinucleotide cofactor , the swirm amino oxidase proteins do not possess equivalents of the unique inserts with residues that allow the sir2-like proteins to catalyze the deacetylation reaction . on the contrary , the conserved residues in the swirm amino oxidases are the same as other amino oxidases , suggesting that they share similar catalytic activities , as proposed above . the combination with the swirm domain , which is predicted to be a protein - protein interaction module , suggests that these enzymes may be part of a larger complex , like other chromatin - modifying enzymes . whereas in vertebrates kiaa0601 has been shown to interact with the histone - deacetylase - containing complexes , the presence of multiple proteins in this family could point to the formation of other distinct complexes . interestingly , the swirm amino - oxidase - type proteins are absent in s. cerevisiae but present in s. pombe and other crown - group eukaryotes . hence , it is likely to have been present in the ancestral crown - group eukaryote and secondarily lost in s. cerevisiae . we have shown previously that several functionally linked genes , encoding proteins involved in chromatin - structure dynamics , which are conserved in s. pombe and other crown - group eukaryotes , have been lost as a group in s. cerevisiae . this implies that the swirm amino - oxidase - type proteins may be functionally linked to these other genes that were co - eliminated along with it in s. cerevisiae . these include genes for proteins such as the set domain methyltransferase clr4p , the chromodomain protein swi6p , the phd finger protein mlo2p , the chromosomal actin - like protein spac23d3.09 , and the predicted prolylhydroxylase with the double - stranded -helix domain spac343.11c . some of these proteins may be part of a multiprotein chromatin - modifying catalytic complex of which the swirm amino oxidase proteins are a part . in vertebrates , the swirm domain is found fused along with a myb ( sant ) domain to a jab1/pad1 domain ( for example , in the human protein kiaa1915 , figure 2 ) . this latter domain is commonly associated with several proteasomal and signalosomal proteins and is involved in ubiquitin - mediated protein degradation . in this case , the swirm domain may serve to recruit proteasomal complexes to specific chromatin proteins to effect their degradation . in the course of our systematic survey of eukaryotic chromosomal proteins , we observed a conserved globular module shared by the swi3p and rsc8p proteins from the yeast saccharomyces cerevisiae that did not map to any previously characterized domain . these homologous proteins are parts of the multisubunit swi / snf and rsc complexes that drive chromatin remodeling through the snf2/swi2-like atpase subunit . the region of similarity encompassing this segment has been termed ' conserved region 1 ' in . both these proteins additionally contain a myb - related helix - turn - helix domain termed the sant domain at their carboxyl terminus and a further carboxy - terminal conserved -helical extension restricted to orthologs of rsc8p and swi3p . given that these subunits are key components of the respective chromatin - remodeling complexes , and mediate multiple interactions , we sought to further investigate the provenance of their conserved amino - terminal module using computational methods . a psi - blast search initiated with this region from rsc8p ( gi : 14318562 , region 80 - 177 ) not only recovered its paralogs and orthologs from other organisms such as moira from drosophila and baf155 and baf170 from vertebrates , but also several other uncharacterized proteins from diverse eukaryotes with statistically significant expected values ( e - values ) at the point of detection . for example , the search recovered the nuclear protein spac23e2.02 from schizosaccharomyces pombe in iteration 4 ( e = 2 10 ) , the human proteins kiaa1915 ( e = 10 ) and kiaa0601 ( e = 10 ) in iteration 2 and the arabidopsis thaliana protein at2g47620 in iteration 3 ( e = 10 ) . we prepared a multiple alignment of these regions from all the proteins detected in these searches ( figure 1 ) and used it to construct a psi - blast position - specific score matrix ( pssm ) and a hidden markov model for further searches against the complete or nearly complete proteome databases of individual eukaryotes . these searches allowed us to detect additional copies of this region in the carboxyl termini of the asynaptic 1 protein ( a hop1p homolog ) from plants . additionally these searches also recovered a conserved globular region in the extreme carboxyl terminus of the ada2-like proteins from eukaryotes , with moderate significance . while these newly detected proteins have a somewhat distinct conservation pattern from the originally detected set ( figure 1 ) , their secondary structure predicted using the jpred program closely matches that predicted for the originally detected set . this observation , together with the information that the ada2-like proteins are chromatinic proteins , just like the members of the original set , suggests that these domains are probably distant relatives of each other ( figure 1 ) . the conserved region described above occurred in various distinct domain - architecture contexts , suggesting that it is an evolutionarily mobile domain ( figure 2 ) . we named this domain the swirm domain after the proteins swi3p , rsc8p and moira in which it was first recognized . secondary - structure predictions , based on the multiple alignment , indicate the presence of four distinct a helices . hence , it is predicted that the swirm domain is likely to form a globular structure in the form of a tetrahelical bundle ( figure 1 ) . however , a direct comparison showed no specific relationship between the sequence conservation pattern of the swirm domain and those of other -helical bundles that are commonly encountered in chromosomal proteins such as the bromodomain , the myb or sant domains and other helical dna - binding domains . the differences in conservation pattern between the distantly related ada2p carboxy - terminal domains and the canonical swirm domains suggest that the former are likely to interact with a very distinct set of partners . rsc8p ( swh3p ) has been shown to mediate multiple interactions in the rsc complex : it undergoes dimerization via the carboxy - terminal coiled - coil segment , associates with the swi2/snf2 atpase sth1p by forming two distinct contacts , and it forms a complex with rsc6p subunit . deletion analyses have shown that the region of the protein that includes the swirm domain is probably required for at least one of the contacts with sth1p and perhaps even those with rsc6p . this suggests that the swirm domain is most likely to mediate protein - protein interactions . the rsc8p- and swi3p - related proteins have fairly complex architectures , implying that the different modules may mediate distinct interactions . rsc8p has an additional zz domain between the swirm and myb domains , whereas the animal and dictyostelium versions , like moira , have an additional amino - terminal chromodomain ( figure 2 ) . however , the common denominator in all these proteins are the swirm and myb domains , suggesting that they mediate the evolutionarily conserved interactions of these proteins , such as those with the swi2/snf2 atpase . the presence of a divergent swirm domain , linked to a horma domain , in asy1 , the plant ortholog of the yeast hop1p , is consistent with the role of these proteins in mediating protein - protein interactions in the process of chromosome synapsis . the ada2p - like proteins ( figure 2 ) are chromatin - associated proteins and are known to mediate protein - protein interactions . they contain a distinct carboxy - terminal globular domain that is distantly related to the swirm domain , suggesting that this domain could mediate a subset of their interactions with the transcriptional machinery . a striking group of proteins with the swirm domain is the one typified by spac23e2.02 and spbc146.09c from s. pombe . between one and four orthologs of this protein are encoded by different crown - group eukaryotes ( figures 1,3 ) and contain the swirm domain linked to a predicted fad - dependent oxidoreductase domain . a similar observation regarding the presence of a fad - binding domain has been reported for kiaa0601 , a vertebrate protein of this family . kiaa0601 , along with the co - repressor corest , is stably associated with the histone deacetylase complex . furthermore , spac23e2.02 has been shown to be a nuclear protein and along with its paralog spbc146.09c , contains at its extreme carboxyl terminus an hmg1 domain , which is a common dna - binding module found in diverse chromosomal proteins ( figures 2,3 ) . the closest relatives of the predicted oxidoreductase domains linked to the swirm domain are the polyamine oxidases and the monoamine oxidases ( figure 3 ) . these enzymes are involved in the oxidation of amino groups of polyamines such as spermine and spermidine and monoamines such as dopamine and serotonin . the availability of the crystal structure for the polyamine oxidase from maize allowed us to evaluate the sequence conservation of the oxidoreductase domain fused to the swirm domains . the alignment of this domain from the spac23e2.02-like proteins spans the entire length of the amino oxidase and contains the hallmark residues required to bind the fad cofactor ( figure 3 ) . these include the glycine - rich loop bounded by a -strand and an a helix that is typical of rossmann fold nucleotide - binding proteins , a conserved glutamate specific to the amino oxidase family , that interacts with ribose in fad and other residues of the substrate and cofactor - binding site . thus , these oxidoreductase domains are likely to function as amino oxidases ; this , together with their linkage within the same polypeptide to domains typical of chromatin proteins , and evidence for nuclear localization , suggests that they are novel chromatin - modifying enzymes . consistent with this , the complex containing kiaa0601 has been shown to contain fad and was suggested to be a chromatin - modifying enzyme . building on these observations , we suggest two functional possibilities for these proteins : first , they could act as novel protein - modifying enzymes that oxidize the amino groups of lysines or arginines present on histones or transcription factors ; alternatively they could affect chromatin structure by oxidizing basic polyamines present in the chromatin and thereby reducing the charge balance . it is also conceivable that the fad cofactor of these enzymes functions analogously to nad , in a deacetylation reaction of acetyllysines similar to that carried out by sir2 enzymes . whereas both these enzymes are derived versions of the rossmann fold and bind a dinucleotide cofactor , the swirm amino oxidase proteins do not possess equivalents of the unique inserts with residues that allow the sir2-like proteins to catalyze the deacetylation reaction . on the contrary , the conserved residues in the swirm amino oxidases are the same as other amino oxidases , suggesting that they share similar catalytic activities , as proposed above . the combination with the swirm domain , which is predicted to be a protein - protein interaction module , suggests that these enzymes may be part of a larger complex , like other chromatin - modifying enzymes . whereas in vertebrates kiaa0601 has been shown to interact with the histone - deacetylase - containing complexes , the presence of multiple proteins in this family could point to the formation of other distinct complexes . interestingly , the swirm amino - oxidase - type proteins are absent in s. cerevisiae but present in s. pombe and other crown - group eukaryotes . hence , it is likely to have been present in the ancestral crown - group eukaryote and secondarily lost in s. cerevisiae . we have shown previously that several functionally linked genes , encoding proteins involved in chromatin - structure dynamics , which are conserved in s. pombe and other crown - group eukaryotes , have been lost as a group in s. cerevisiae . this implies that the swirm amino - oxidase - type proteins may be functionally linked to these other genes that were co - eliminated along with it in s. cerevisiae . these include genes for proteins such as the set domain methyltransferase clr4p , the chromodomain protein swi6p , the phd finger protein mlo2p , the chromosomal actin - like protein spac23d3.09 , and the predicted prolylhydroxylase with the double - stranded -helix domain spac343.11c . some of these proteins may be part of a multiprotein chromatin - modifying catalytic complex of which the swirm amino oxidase proteins are a part . in vertebrates , the swirm domain is found fused along with a myb ( sant ) domain to a jab1/pad1 domain ( for example , in the human protein kiaa1915 , figure 2 ) . this latter domain is commonly associated with several proteasomal and signalosomal proteins and is involved in ubiquitin - mediated protein degradation . in this case , the swirm domain may serve to recruit proteasomal complexes to specific chromatin proteins to effect their degradation . we define a conserved domain of about 85 residues , predicted to participate in protein - protein interactions , in different eukaryotic chromatin proteins such as swi3p and rsc8p . homologs of these molecules , with the swirm domain , are found in all eukaryotes belonging to the crown group , as well as earlier - branching protists such as the apicomplexans . a version of the swirm domain is found linked to an amino - oxidase domain in a class of nuclear proteins that are represented in most crown - group eukaryotes . these proteins are present in multiple copies in plant proteomes and are entirely absent in the yeast s. cerevisiae . we predict that these proteins define a new class of chromatin - modifying enzymes that are likely to oxidize the amino groups of histones or other nuclear proteins . alternatively , they may oxidize polyamines in chromatin to alter the charge balance in the chromatin . in humans the swirm domain is found linked to a jab1/pad1 catalytic domain , suggesting that this protein may serve as a link in the regulation of chromatinic proteins through proteasomal degradation . the nonredundant ( nr ) database of protein sequences ( national center for biotechnology information , nih , bethesda ) was searched using the blastp program . profile searches were carried out using the psi - blast program with either a single sequence or an alignment used as the query , with a profile inclusion expectation ( e ) value threshold of 0.01 , and were iterated until convergence . previously known conserved protein domains were detected using the corresponding psi - blast - derived pssms . the pssms were prepared by choosing one or more starting queries ( seeds ) for a set of most frequently encountered domains ( see for details ) and run against the nr database until convergence with the -c option of psi - blast to save the pssm . it was ensured that at convergence no false positives were included in the profiles . this profile database can be downloaded from or used on the internet via the rps - blast program . all globular segments of proteins that did not map to domains with previously constructed pssms were searched individually using psi - blast to detect any additional domains that may have been overlooked . multiple alignments were constructed using the t_coffee program , followed by manual correction based on the psi - blast results . protein secondary structure was predicted using a multiple alignment as the input for the jpred program . proteins are designated by their gene names followed by the species abbreviations and genbank ( gi ) numbers . the coloring represents the conservation profile of amino - acid residues at 90% consensus distinguished by the following amino - acid classes : h , hydrophobic residues ( liyfmwacv ( in the single - letter amino - acid code ) ) , a , aromatic residues ( fhyw ) and l , aliphatic ( liav ) residues , all shaded yellow ; c , charged ( kerdh ) residues ( basic ( krh ) residues and acidic ( de ) residues ) colored magenta ; p , polar ( stedrkhnqc ) residues colored blue ; s , small ( sacgdnpvt ) residues colored green ; u , tiny ( gas ) residues shaded green ( in positions that are always glycine , this is indicated with a g ) ; b , big ( liyerfmwq ) residues shaded gray . the predicted secondary structure is shown above the alignment : h or h , helix ; e or e , strand . species abbreviations : at , arabidopsis thaliana ; bna , brassica napus ; ce , caenorhabditis elegans ; ddi , dictyostelium discoideum ; dm , drosophila melanogaster ; ecu , encephalitozoon cuniculi ; hs , homo sapiens ; osa , oryza sativa ; sc , saccharomyces cerevisiae ; sp , schizosaccharomyces pombe ; zm , zea mays . a subset of rsc8p orthologs is represented in the automatically generated , uncurated pfam - b entry 3680 . the phyletic distribution of a particular domain architecture is additionally given in brackets , where a represents animals ; p , plants ; f , fungi ; d , d. discoideum and pf , plasmodium falciparum . zz represents the zz - type of zinc finger , and ' helical ' designates the conserved -helical domain found at the carboxyl terminus of proteins with a myb ( sant ) and an rsc8-like swirm domain . the protein designations , coloring scheme and consensus abbreviations are as described in figure 1 ; + , acidic residues . the crystal - structure - derived secondary - structure assignments are shown above the alignment with positions involved in cofactor or substrate binding marked by an asterisk . species abbreviations : at , a. thaliana ; ce , c. elegans ; dm , d. melanogaster ; hs , homo sapiens ; mm , mus musculus ; mtu , mycobacterium tuberculosis ; rn , rattus norvegicus ; sp , s. pombe ; zma , z. mays .

using computational sequence - profile analysis methods , a previously uncharacterized , predicted -helical domain of about 85 residues was identified in chromosomal proteins such as swi3p , rsc8p , moira and several other uncharacterized proteins . this module , termed the swirm domain , is predicted to mediate specific protein - protein interactions in the assembly of chromatin - protein complexes .

Background Results and discussion Identification of the SWIRM domain Functional implications and domain architectures of the SWIRM domains Conclusions Materials and methods Figures and Tables

these dynamics are regulated both by a variety of protein - protein and protein - dna interactions and by catalytic modification and reorganization of proteins and nucleic acids that comprise the chromosomes . in recent years it has become clear that covalent modification of histones , transcription factors and other chromosomal proteins plays a major part in the dynamics of chromatin . other enzymes , such as deacetylases and phosphatases , remove covalent modifications from proteins and thus reverse their effects . the covalent modifications may change the local or global charge properties of chromosomal proteins and regulate their interactions with dna . besides covalent modifications , atp - dependent enzymes such as the swi2/snf2 atpases and other chaperone - like proteins remodel the chromatin by rearranging the binding pattern of histones and other chromosomal proteins . these covalent and non - covalent catalytic actions on the chromatin can result in condensation or decondensation of chromatin , either locally or on the chromosomal scale , and thereby regulate access of transcription factors and other proteins to the chromatin . well studied examples of these are the bromodomain that interacts with acetylated peptides , chromodomains that mediate specific interactions with proteins and rna , the phd finger mediating protein - protein interactions , the myb and sant domains that interact with both dna and proteins , and the sap and at - hook domains that interact with dna . these domains often occur together in large proteins linked to catalytic domains and may serve to tether these proteins to different components of chromatin and deliver catalytic activities to specific locations . the computational analysis of chromatin proteins has helped in the identification of a large number of conserved modules in the chromosomal proteins . using computational analysis , we also show that this domain is found linked to catalytic domains such as oxidoreductase and jun activating binding protein 1 ( jab ) domains and these proteins may define a novel class of chromatin - modifying enzymes . in the course of our systematic survey of eukaryotic chromosomal proteins , we observed a conserved globular module shared by the swi3p and rsc8p proteins from the yeast saccharomyces cerevisiae that did not map to any previously characterized domain . given that these subunits are key components of the respective chromatin - remodeling complexes , and mediate multiple interactions , we sought to further investigate the provenance of their conserved amino - terminal module using computational methods . a psi - blast search initiated with this region from rsc8p ( gi : 14318562 , region 80 - 177 ) not only recovered its paralogs and orthologs from other organisms such as moira from drosophila and baf155 and baf170 from vertebrates , but also several other uncharacterized proteins from diverse eukaryotes with statistically significant expected values ( e - values ) at the point of detection . these searches allowed us to detect additional copies of this region in the carboxyl termini of the asynaptic 1 protein ( a hop1p homolog ) from plants . we named this domain the swirm domain after the proteins swi3p , rsc8p and moira in which it was first recognized . hence , it is predicted that the swirm domain is likely to form a globular structure in the form of a tetrahelical bundle ( figure 1 ) . however , a direct comparison showed no specific relationship between the sequence conservation pattern of the swirm domain and those of other -helical bundles that are commonly encountered in chromosomal proteins such as the bromodomain , the myb or sant domains and other helical dna - binding domains . rsc8p ( swh3p ) has been shown to mediate multiple interactions in the rsc complex : it undergoes dimerization via the carboxy - terminal coiled - coil segment , associates with the swi2/snf2 atpase sth1p by forming two distinct contacts , and it forms a complex with rsc6p subunit . deletion analyses have shown that the region of the protein that includes the swirm domain is probably required for at least one of the contacts with sth1p and perhaps even those with rsc6p . this suggests that the swirm domain is most likely to mediate protein - protein interactions . however , the common denominator in all these proteins are the swirm and myb domains , suggesting that they mediate the evolutionarily conserved interactions of these proteins , such as those with the swi2/snf2 atpase . the presence of a divergent swirm domain , linked to a horma domain , in asy1 , the plant ortholog of the yeast hop1p , is consistent with the role of these proteins in mediating protein - protein interactions in the process of chromosome synapsis . the ada2p - like proteins ( figure 2 ) are chromatin - associated proteins and are known to mediate protein - protein interactions . they contain a distinct carboxy - terminal globular domain that is distantly related to the swirm domain , suggesting that this domain could mediate a subset of their interactions with the transcriptional machinery . a striking group of proteins with the swirm domain is the one typified by spac23e2.02 and spbc146.09c from s. pombe . between one and four orthologs of this protein are encoded by different crown - group eukaryotes ( figures 1,3 ) and contain the swirm domain linked to a predicted fad - dependent oxidoreductase domain . furthermore , spac23e2.02 has been shown to be a nuclear protein and along with its paralog spbc146.09c , contains at its extreme carboxyl terminus an hmg1 domain , which is a common dna - binding module found in diverse chromosomal proteins ( figures 2,3 ) . the closest relatives of the predicted oxidoreductase domains linked to the swirm domain are the polyamine oxidases and the monoamine oxidases ( figure 3 ) . these enzymes are involved in the oxidation of amino groups of polyamines such as spermine and spermidine and monoamines such as dopamine and serotonin . these include the glycine - rich loop bounded by a -strand and an a helix that is typical of rossmann fold nucleotide - binding proteins , a conserved glutamate specific to the amino oxidase family , that interacts with ribose in fad and other residues of the substrate and cofactor - binding site . thus , these oxidoreductase domains are likely to function as amino oxidases ; this , together with their linkage within the same polypeptide to domains typical of chromatin proteins , and evidence for nuclear localization , suggests that they are novel chromatin - modifying enzymes . consistent with this , the complex containing kiaa0601 has been shown to contain fad and was suggested to be a chromatin - modifying enzyme . building on these observations , we suggest two functional possibilities for these proteins : first , they could act as novel protein - modifying enzymes that oxidize the amino groups of lysines or arginines present on histones or transcription factors ; alternatively they could affect chromatin structure by oxidizing basic polyamines present in the chromatin and thereby reducing the charge balance . whereas both these enzymes are derived versions of the rossmann fold and bind a dinucleotide cofactor , the swirm amino oxidase proteins do not possess equivalents of the unique inserts with residues that allow the sir2-like proteins to catalyze the deacetylation reaction . on the contrary , the conserved residues in the swirm amino oxidases are the same as other amino oxidases , suggesting that they share similar catalytic activities , as proposed above . the combination with the swirm domain , which is predicted to be a protein - protein interaction module , suggests that these enzymes may be part of a larger complex , like other chromatin - modifying enzymes . interestingly , the swirm amino - oxidase - type proteins are absent in s. cerevisiae but present in s. pombe and other crown - group eukaryotes . hence , it is likely to have been present in the ancestral crown - group eukaryote and secondarily lost in s. cerevisiae . this implies that the swirm amino - oxidase - type proteins may be functionally linked to these other genes that were co - eliminated along with it in s. cerevisiae . these include genes for proteins such as the set domain methyltransferase clr4p , the chromodomain protein swi6p , the phd finger protein mlo2p , the chromosomal actin - like protein spac23d3.09 , and the predicted prolylhydroxylase with the double - stranded -helix domain spac343.11c . some of these proteins may be part of a multiprotein chromatin - modifying catalytic complex of which the swirm amino oxidase proteins are a part . in vertebrates , the swirm domain is found fused along with a myb ( sant ) domain to a jab1/pad1 domain ( for example , in the human protein kiaa1915 , figure 2 ) . in this case , the swirm domain may serve to recruit proteasomal complexes to specific chromatin proteins to effect their degradation . in the course of our systematic survey of eukaryotic chromosomal proteins , we observed a conserved globular module shared by the swi3p and rsc8p proteins from the yeast saccharomyces cerevisiae that did not map to any previously characterized domain . both these proteins additionally contain a myb - related helix - turn - helix domain termed the sant domain at their carboxyl terminus and a further carboxy - terminal conserved -helical extension restricted to orthologs of rsc8p and swi3p . given that these subunits are key components of the respective chromatin - remodeling complexes , and mediate multiple interactions , we sought to further investigate the provenance of their conserved amino - terminal module using computational methods . a psi - blast search initiated with this region from rsc8p ( gi : 14318562 , region 80 - 177 ) not only recovered its paralogs and orthologs from other organisms such as moira from drosophila and baf155 and baf170 from vertebrates , but also several other uncharacterized proteins from diverse eukaryotes with statistically significant expected values ( e - values ) at the point of detection . we named this domain the swirm domain after the proteins swi3p , rsc8p and moira in which it was first recognized . hence , it is predicted that the swirm domain is likely to form a globular structure in the form of a tetrahelical bundle ( figure 1 ) . however , a direct comparison showed no specific relationship between the sequence conservation pattern of the swirm domain and those of other -helical bundles that are commonly encountered in chromosomal proteins such as the bromodomain , the myb or sant domains and other helical dna - binding domains . rsc8p ( swh3p ) has been shown to mediate multiple interactions in the rsc complex : it undergoes dimerization via the carboxy - terminal coiled - coil segment , associates with the swi2/snf2 atpase sth1p by forming two distinct contacts , and it forms a complex with rsc6p subunit . deletion analyses have shown that the region of the protein that includes the swirm domain is probably required for at least one of the contacts with sth1p and perhaps even those with rsc6p . this suggests that the swirm domain is most likely to mediate protein - protein interactions . rsc8p has an additional zz domain between the swirm and myb domains , whereas the animal and dictyostelium versions , like moira , have an additional amino - terminal chromodomain ( figure 2 ) . however , the common denominator in all these proteins are the swirm and myb domains , suggesting that they mediate the evolutionarily conserved interactions of these proteins , such as those with the swi2/snf2 atpase . the presence of a divergent swirm domain , linked to a horma domain , in asy1 , the plant ortholog of the yeast hop1p , is consistent with the role of these proteins in mediating protein - protein interactions in the process of chromosome synapsis . the ada2p - like proteins ( figure 2 ) are chromatin - associated proteins and are known to mediate protein - protein interactions . they contain a distinct carboxy - terminal globular domain that is distantly related to the swirm domain , suggesting that this domain could mediate a subset of their interactions with the transcriptional machinery . a striking group of proteins with the swirm domain is the one typified by spac23e2.02 and spbc146.09c from s. pombe . between one and four orthologs of this protein are encoded by different crown - group eukaryotes ( figures 1,3 ) and contain the swirm domain linked to a predicted fad - dependent oxidoreductase domain . a similar observation regarding the presence of a fad - binding domain has been reported for kiaa0601 , a vertebrate protein of this family . furthermore , spac23e2.02 has been shown to be a nuclear protein and along with its paralog spbc146.09c , contains at its extreme carboxyl terminus an hmg1 domain , which is a common dna - binding module found in diverse chromosomal proteins ( figures 2,3 ) . the closest relatives of the predicted oxidoreductase domains linked to the swirm domain are the polyamine oxidases and the monoamine oxidases ( figure 3 ) . these enzymes are involved in the oxidation of amino groups of polyamines such as spermine and spermidine and monoamines such as dopamine and serotonin . these include the glycine - rich loop bounded by a -strand and an a helix that is typical of rossmann fold nucleotide - binding proteins , a conserved glutamate specific to the amino oxidase family , that interacts with ribose in fad and other residues of the substrate and cofactor - binding site . thus , these oxidoreductase domains are likely to function as amino oxidases ; this , together with their linkage within the same polypeptide to domains typical of chromatin proteins , and evidence for nuclear localization , suggests that they are novel chromatin - modifying enzymes . consistent with this , the complex containing kiaa0601 has been shown to contain fad and was suggested to be a chromatin - modifying enzyme . building on these observations , we suggest two functional possibilities for these proteins : first , they could act as novel protein - modifying enzymes that oxidize the amino groups of lysines or arginines present on histones or transcription factors ; alternatively they could affect chromatin structure by oxidizing basic polyamines present in the chromatin and thereby reducing the charge balance . whereas both these enzymes are derived versions of the rossmann fold and bind a dinucleotide cofactor , the swirm amino oxidase proteins do not possess equivalents of the unique inserts with residues that allow the sir2-like proteins to catalyze the deacetylation reaction . on the contrary , the conserved residues in the swirm amino oxidases are the same as other amino oxidases , suggesting that they share similar catalytic activities , as proposed above . the combination with the swirm domain , which is predicted to be a protein - protein interaction module , suggests that these enzymes may be part of a larger complex , like other chromatin - modifying enzymes . hence , it is likely to have been present in the ancestral crown - group eukaryote and secondarily lost in s. cerevisiae . we have shown previously that several functionally linked genes , encoding proteins involved in chromatin - structure dynamics , which are conserved in s. pombe and other crown - group eukaryotes , have been lost as a group in s. cerevisiae . these include genes for proteins such as the set domain methyltransferase clr4p , the chromodomain protein swi6p , the phd finger protein mlo2p , the chromosomal actin - like protein spac23d3.09 , and the predicted prolylhydroxylase with the double - stranded -helix domain spac343.11c . some of these proteins may be part of a multiprotein chromatin - modifying catalytic complex of which the swirm amino oxidase proteins are a part . in vertebrates , the swirm domain is found fused along with a myb ( sant ) domain to a jab1/pad1 domain ( for example , in the human protein kiaa1915 , figure 2 ) . in this case , the swirm domain may serve to recruit proteasomal complexes to specific chromatin proteins to effect their degradation . we define a conserved domain of about 85 residues , predicted to participate in protein - protein interactions , in different eukaryotic chromatin proteins such as swi3p and rsc8p . homologs of these molecules , with the swirm domain , are found in all eukaryotes belonging to the crown group , as well as earlier - branching protists such as the apicomplexans . a version of the swirm domain is found linked to an amino - oxidase domain in a class of nuclear proteins that are represented in most crown - group eukaryotes . these proteins are present in multiple copies in plant proteomes and are entirely absent in the yeast s. cerevisiae . we predict that these proteins define a new class of chromatin - modifying enzymes that are likely to oxidize the amino groups of histones or other nuclear proteins . alternatively , they may oxidize polyamines in chromatin to alter the charge balance in the chromatin . in humans the swirm domain is found linked to a jab1/pad1 catalytic domain , suggesting that this protein may serve as a link in the regulation of chromatinic proteins through proteasomal degradation . it was ensured that at convergence no false positives were included in the profiles . the coloring represents the conservation profile of amino - acid residues at 90% consensus distinguished by the following amino - acid classes : h , hydrophobic residues ( liyfmwacv ( in the single - letter amino - acid code ) ) , a , aromatic residues ( fhyw ) and l , aliphatic ( liav ) residues , all shaded yellow ; c , charged ( kerdh ) residues ( basic ( krh ) residues and acidic ( de ) residues ) colored magenta ; p , polar ( stedrkhnqc ) residues colored blue ; s , small ( sacgdnpvt ) residues colored green ; u , tiny ( gas ) residues shaded green ( in positions that are always glycine , this is indicated with a g ) ; b , big ( liyerfmwq ) residues shaded gray . zz represents the zz - type of zinc finger , and ' helical ' designates the conserved -helical domain found at the carboxyl terminus of proteins with a myb ( sant ) and an rsc8-like swirm domain .

the ability of music to cause emotional reactions and enjoyment in listeners is undoubtedly crucial to its existence . previous neuroimaging studies [ 14 ] focused on how the brain reacts to pleasant music , leaving open why people like the music they do . these differences can not be due solely to qualities inherent in the music itself , since the same piece may be a psychomusicological studies , for example , [ 5 , 6 ] have pointed to the mere exposure effect ( mee ) as an important part of the explanation . the mee means that simple exposure to a novel , neutral stimulus by itself increases liking for it [ 7 , 8 ] . accordingly , studies of music preference generally find that liking increases with exposure [ 5 , 6 ] , a phenomenon which is utilised in practice by the music and advertising industries . while the mee is a well - established behavioural phenomenon , we know little about its biological basis . only a single neuroimaging study , by elliott and dolan , has so far focused specifically on it . that study used subliminal visual stimuli , and it showed particular involvement of the right lateral prefrontal cortex in implicit memory expressed in preference judgements , indicating this as a key brain area related to the mee . as for the mee of music , our study is the first to examine its neural basis . we examined supraliminal ( rather than subliminal ) stimuli , which are probably most relevant to everyday music listening . a learning phase familiarised participants with a number of melodies , where the amount of exposure to subsets of the melodies was varied systematically in order to induce different levels of implicit learning . subsequently , they were scanned using functional magnetic resonance imaging ( fmri ) , while performing a liking judgement of the melodies , succeeded by a recognition test . we hypothesized that the right lateral prefrontal cortex would play a role in the mee for music , as it did for visual stimuli , that is , show differential activation as a function of prior exposure . however , given the absence of prior studies on the subject , additional brain areas could be involved . regarding perception of stimulus novelty , on the other hand , we expected involvement of the medial temporal ( including hippocampal ) region based on evidence from the study by elliott and dolan , as well as others , for example [ 2 , 1012 ] . concerning activations related to subjective liking , we hypothesized the participation of limbic and paralimbic areas previously linked to positive judgements of music , such as insula , striatum , anterior cingulate , and orbitofrontal cortex [ 14 ] . additionally , it was of interest to us if the results would support the valence lateralization model [ 2 , 13 , 14 ] , that is , that neural responses to positively judged melodies would be left - lateralized . 30 single - voice melodies of 13 s duration each were composed for the study , see figure 1 for two examples . melodies were digital sequences in both piano and guitar versions , quantised and generated in cubase sx , postprocessed in adobe audition . 10 melodies were in the key of a flat , 10 in e , and 10 in c. the 10 melodies in each key were further balanced between major and minor mode , resulting in a total of six different melody types ( see for the report on neural correlates of musical mode ) . other parameters of the melodies were kept identical , including ambitus ( a 10th ) , tempo ( 80 beats per minute ) , and amplitude . as we wished to study exposure effects , we used unknown melodies to eliminate the possibility of preexperimental knowledge of the stimuli . in composing these melodies melodies were composed adhering to the guidelines mentioned above , in order to render them comparable , thus avoiding differences in neural activation solely on the basis of variation of superficial features , such as timbre or tempo . on the other hand , it was also necessary to induce a certain level of variety and song - like qualities in the melodies , in order to make them stand out from each other , and be appreciated as individual pieces of music . we used supraliminal stimuli ( as opposed to for example elliott and dolan ) , since music naturally unfolds over seconds and minutes , rather than milliseconds , and since listeners in real life are in fact most often aware ( at least potentially ) of the music they are listening to . additionally , six melodies for the learning phase were made along identical guidelines as above , but incorporating a single note detuned by 50 cents ( half a semitone ) . the melodies containing a detuned note were not used in the scanning part of the experiment . an ascending chromatic scale of uniformly distributed intervals ( minor seconds ) , but otherwise with similar characteristics as the melodies , was also employed in the fmri part of the experiment . a preexperimental liking rating of the melodies was done by 49 volunteers , who did not participate in the fmri experiment itself . this rating was carried out in order to divide the melodies into five groups containing six melodies each , so that the mean liking for each melody group would be as equal as possible . the mean liking ratings for the five groups were 2.95 , 2.97 , 2.97 , 2.94 , and 2.98 , respectively , on a scale from 1 to 5 ( overall mean 2.96 , sd = 0.51 ) . 21 healthy right - handed ( by self - report ) volunteers , 12 female , mean age 27.3 ( range 2033 ) participated in the scanning experiment . participants were nonmusicians , but musically adept , as indicated by pitch and rhythm tests , as used at the royal academy of music , aarhus , denmark . in these tests , participants were asked to reproduce progressively more complex melodies and rhythms , by singing ( pitch ) or clapping ( rhythm ) . the ethical committee of aarhus county approved the study , and participants gave their informed consent prior to their inclusion in the study . for each participant , the experiment incorporated five phases ( a e ) ; two outside the scanner , followed by two in the scanner , and lastly a debriefing and musical ability test outside the scanner . phases a and b utilised five different pseudorandomised scenarios of stimulus sequences , four of these were used four times each , and the last one five times ( for a total of 21 , corresponding to the number of participants ) . total randomisation was avoided to ensure that melodies of the same key and mode would not succeed each other , and that the occurrence of different stimulus types were uniformly separated ( by employing a split - halves method ) . in the scanning phases c and d , stimulus sequences were pseudorandomised for each participant individually in order to avoid the amplification of possible sequence effects . ( a ) baseline rating and training phaseparticipants listened to 18 melodies over headphones , that they would hear again in phase b. they rated each melody immediately after listening to it , in a two - second gap of silence between melodies , on a 15 scale ( 1 : least liked , 5 : most liked ) . participants listened to 18 melodies over headphones , that they would hear again in phase b. they rated each melody immediately after listening to it , in a two - second gap of silence between melodies , on a 15 scale ( 1 : least liked , 5 : most liked ) . ( b ) learning phaseparticipants listened again to the 18 melodies , which were divided into three different groups of six . the melodies were played in pseudorandomised order , either once , seven or 31 times each , depending on which group they belonged to . this meant that participants would have heard a given melody either two , eight , or 32 times , at the conclusion of phase b , depending on scenario ( see above ) . due to the large number of stimulus presentations in this phase , resulting in a total duration of about an hour , three measures were taken to aid the concentration of participants . first , participants were asked to press a button as quickly as possible to the occurrence of detuned notes in the six special melodies that would appear five times each throughout phase b. second , instrument sound in this phase alternated between piano and guitar , in order to help differentiate one melody from the next ( the guitar versions of the melodies were not used for the scanning sessions ) . lastly , participants were allowed a five minute break halfway through the learning phase . participants listened again to the 18 melodies , which were divided into three different groups of six . the melodies were played in pseudorandomised order , either once , seven or 31 times each , depending on which group they belonged to . this meant that participants would have heard a given melody either two , eight , or 32 times , at the conclusion of phase b , depending on scenario ( see above ) . due to the large number of stimulus presentations in this phase , resulting in a total duration of about an hour , three measures were taken to aid the concentration of participants . first , participants were asked to press a button as quickly as possible to the occurrence of detuned notes in the six special melodies that would appear five times each throughout phase b. second , instrument sound in this phase alternated between piano and guitar , in order to help differentiate one melody from the next ( the guitar versions of the melodies were not used for the scanning sessions ) . lastly , participants were allowed a five minute break halfway through the learning phase . ( c ) liking scanning phasein the scanner , participants listened to these 18 melodies again once each , along with six further melodies that had not been heard previously ( for a total of 24 ) . the 24 melodies thus belonged to one of four categories designated f0 , f2 , f8 , and f32 , based on the number of previous repetitions in the given scenario . this resulted in a total of 126 trials ( melody presentations ) for each exposure category ( six melodies in each group , times 21 subjects ) . participants were instructed to visually fixate on a crosshair on a screen , listen carefully to the stimuli over the headphones , and immediately after each melody to rate how well they liked it on a scale from 1 ( least liked ) to 5 ( most liked ) , using a response box with one button for each finger . the scale and rating procedure was identical to the one in phase a , and thus well known to participants . stimulus sequences further included six repetitions of the chromatic scale , and a number of 28 s silences in between , yielding jittered interstimulus intervals to allow more exhaustive sampling of the fmri signal . in the scanner , participants listened to these 18 melodies again once each , along with six further melodies that had not been heard previously ( for a total of 24 ) . the 24 melodies thus belonged to one of four categories designated f0 , f2 , f8 , and f32 , based on the number of previous repetitions in the given scenario . this resulted in a total of 126 trials ( melody presentations ) for each exposure category ( six melodies in each group , times 21 subjects ) . participants were instructed to visually fixate on a crosshair on a screen , listen carefully to the stimuli over the headphones , and immediately after each melody to rate how well they liked it on a scale from 1 ( least liked ) to 5 ( most liked ) , using a response box with one button for each finger . the scale and rating procedure was identical to the one in phase a , and thus well known to participants . stimulus sequences further included six repetitions of the chromatic scale , and a number of 28 s silences in between , yielding jittered interstimulus intervals to allow more exhaustive sampling of the fmri signal . ( d ) memory phaseidentical to the preceding phase , except for two important differences . first , a further six new melodies were introduced ( bringing the total to 30 in five categories , designated f0 , f1 , f2 , f8 , f32 ) . second , the task for the participants was to judge their certainty of having heard the melody before ( 1 : certain that melody has not been played , 5 : certain that melody has been played ) . the rating was done with participants in the scanner , as for phase c , in order to ensure that the recognition rating was done in exactly the same physical context as the previous liking rating , and to have the two ratings as close to one another as possible in time . first , a further six new melodies were introduced ( bringing the total to 30 in five categories , designated f0 , f1 , f2 , f8 , f32 ) . second , the task for the participants was to judge their certainty of having heard the melody before ( 1 : certain that melody has not been played , 5 : certain that melody has been played ) . the rating was done with participants in the scanner , as for phase c , in order to ensure that the recognition rating was done in exactly the same physical context as the previous liking rating , and to have the two ratings as close to one another as possible in time . ( e ) debriefing interview and musical tests phasefollowing the fmri scan , participants were debriefed , interviewed about their experiences during the experiment , and pitch and rhythm tests were performed to confirm a basic level of musical ability ( a 22nd subject was excluded from the data analysis on these grounds ) . following the fmri scan , participants were debriefed , interviewed about their experiences during the experiment , and pitch and rhythm tests were performed to confirm a basic level of musical ability ( a 22nd subject was excluded from the data analysis on these grounds ) . scanning was performed on a signa excite 1.5 tesla mr scanner ( general electric medical systems , milwaukee , wi , usa ) . t1-weighted anatomical images were acquired , and functional images were obtained using a gradient - echo echoplanar imaging sequence ; tr = 2700 ms , te = 40 ms . 34 axial slices covering the whole brain were acquired ; 5 mm thickness ( no gap ) with an in - plane resolution of 3.44 3.44 mm ( 64 64 matrix ) . fmri data analysis of the data from the scanning phase c was done with spm5 ( institute of neurology , london , uk ) . data preprocessing consisted in realignment to the first image and unwarping , coregistration with the t1-weighted images , and slice timing . subsequently , segmentation of gray and white matter , together with spatial normalisation , was performed with the unified segmentation approach . during the spatial normalisation process images images were spatially smoothed using a 10 mm full width at half maximum gaussian kernel . periods with acoustic stimuli were modelled with a hemodynamic response function , whereas periods of subjective ratings after each stimulus were not modelled . the subjective liking rating , and the objective exposure frequency category ( f0 , f2 , f8 , or f32 ) , pertaining to each melody , were entered into the model as parametric modulations . contrasts were set up to test effects of subjective liking , based on individual subject ratings , and for effects of stimulus exposure frequency . only clusters of more than 10 voxels were reported in order to minimise the risk of including spurious activations . anatomical locations were established using mainly talairach and tournoux and the talairach client software . to analyse the effects of exposure on liking and recognition , respectively , one - factor repeated measures analyses of variance ( anova ) were carried out on the data from phase c and d ( see table 1 and section 2.2.1 ) . f32 , as described in subsection 2.2.1 . to ascertain which conditions prompted any significant effect observed , the mean liking ratings for melodies heard 0 , 2 , 8 , and 32 times by the end of the learning phase , are shown in figure 2 ( the groups are termed f0 ( frequency 0 ) , f2 , etc . ) . the ratings were obtained during the fmri scan ( i.e. , phase c ; refer to section 4 for experimental procedure ) . repeated measures anova showed a statistically significant effect of exposure on liking rating , f ( 3,60 ) = 2.784 , p = 0.048 . pairwise comparisons of means showed that liking for the f32 group was significantly higher than for the f0 group , t ( 20 ) = 2.312 , p = 0.032 . none of the other pairwise comparisons was statistically significant ( although the f8 to f32 difference was borderline significant , p = 0.051 ) . adjusting for the baseline ( phase a ) rating of each melody did not change this pattern of findings . reaction times ( rts ) for the liking ratings differed as a function of presentation frequency , as shown by a repeated measures anova , f ( 3,60 ) = 3.842 , p = 0.014 . pairwise comparisons of means revealed that subjects responded faster to the f32 group than to the other groups , f0 versus f32 : t ( 20 ) = 3.08 , p = 0.006 ; f2 versus f32 : t ( 20 ) = 2.103 , p = 0.048 ; f8 versus f32 : t ( 20 ) = 2.541 , p = 0.019 . the actual temporal difference was small ; 1.65 secs . for the f32 group versus 1.80 secs on average for the three other groups , yielding a 0.15 secs . rt also differed slightly as a function of subjective liking rating , f ( 4,44 ) = 3.278 , p = 0.019 . rt for 1 and 5 ratings were on average respectively 0.09 secs and 0.13 secs faster than the overall mean for liking ratings . mean recognition ratings for melodies heard 0 , 1 , 2 , 8 , and 32 times before are shown in figure 3 . repeated measures anova showed a statistically highly significant effect of exposure on recognition rating , f ( 3.42,68.42 ) = 71.734 , p < 0.001 ( the sphericity assumption was not met , so huynh - feldt correction was applied ) . pairwise comparisons of means revealed that every pair of means differed significantly ; paired samples t - test , p < 0.05 . rt for memory ratings did not differ significantly as a function of exposure , f ( 3.06,61.20 ) = 1.098 , p = 0.358 ( the sphericity assumption was not met , so huynh - feldt correction was applied ) . brain regions showing increased activity as a function of subjective liking rating ( irrespective of presentation frequency ) were all located in the left hemisphere and included a cluster in the anterior part of the insula ( ba 13 ) , extending into the rolandic operculum ( table 2 and figure 4 ) . further activations were found in the dorsal striatum , namely , in the putamen , and in the body of the caudate nucleus . the opposite contrast , of areas showing decreased activity as a function of liking rating , did not result in any significant activation . the contrast based on the amount of previous exposure to the melodies showed activity increases in dorsolateral prefrontal cortex ( dlpfc ) , namely , in bilateral middle frontal gyri ( ba 9 ) , and neighbouring inferior middle / frontal gyri ( ba 46 ) . differential activations of the left hemisphere further incorporated activations of the postcentral gyrus ( ba 2 ) , and the supramarginal gyrus ( smg ) ( ba 40 ) . ba 40 of the right inferior parietal lobe was also implicated , as was ba 6 of the left middle frontal gyrus . the opposite contrast , corresponding to an increase in the fmri signal as a function of stimulus novelty , did not result in any significant activation . we have shown that listening to melodies rated as likeable differentially activates deeper brain structures related to emotion processing , all in the left hemisphere , including the anterior insula and the dorsal striatum . a supraliminal mere exposure effect was a contributing factor to how positively melodies were judged , and brain regions subserving this effect , that is , exhibiting increased activation as a function of prior exposure , included the dorsolateral prefrontal cortex ( bilateral ba 9 and ba 46 ) . we would like to caution that the results discussed here are based on an analysis uncorrected for multiple comparisons , ( see section 2.2.2 ) and would therefore benefit from further empirical substantiation . preceding neuroimaging studies of liking effects in music listening have highlighted the contribution of limbic structures , consistent with the knowledge of their involvement in processing of emotions in general ( e.g. , ) . a pet study found several limbic and paralimbic structures to be involved when listeners experienced so - called chills from the music . of these areas , some in particular have been confirmed by one or more of the subsequent studies on responses to pleasant music with pet and fmri , including the ventral striatum ( nucleus accumbens ) , anterior cingulate , orbitofrontal cortex , and insula [ 24 ] . there is also evidence suggesting that neural responses to pleasant music in general tend to be more lateralized to the left hemisphere , an effect often termed the valence lateralization model [ 2 , 13 , 14 ] . of the brain regions that have been previously implicated in the liking - related network , we also found differential activation of the striatum , more precisely its dorsal part , rather than the ventral part often found in previous studies ( see below ) . this only partial replication of the previously observed liking - related network may relate to the fact , that the melodies composed for our study were relatively short , unknown , computer - recorded single - voice melodies , in order to maximize experimental control . previous studies [ 14 ] were based on recorded music excerpts , mostly from the classical repertoire . activation of the entire liking - related network may be contingent upon using music excerpts varying on a wider range of parameters , such as instrumentation and dynamics , although this could potentially jeopardise stimulus comparability . the involvement of the insula , and more specifically its anterior part , was expected based on prior findings , although in our study it was left - lateralized , compared to mostly bilateral in previous studies on pleasant music ( ibid . ) . menon and levitin used effective connectivity analysis in their fmri study to show a strong ventral tegmental area - mediated interaction of nucleus accumbens and the left anterior insula , forming a mesolimbic dopaminergic system that is central to reward processing . the insula is furthermore known to be one of the most important nodal points ( together with the hypothalamus ) in neural pathways concerned with autonomic , somatic , and emotional functions [ 21 , 22 ] . on these grounds , menon and levitin attributed insula activation during music listening to its role in regulating autonomic and physiological responses to a rewarding and emotionally salient stimulus . while this explanation remains plausible in our study as well , we can not completely discount a motor response influence , given the slightly faster rt for both the highest and lowest rated melodies ( 0.09 secs and 0.13 secs faster than the mean , resp . ) . although it is not ideal that rt differs across stimulus categories , at least four points indicate that the problem was not of an alarming magnitude in the context of our study . second , the motor response ( button press ) , occuring after the melody itself , was not included in the data design model . third , the faster reaction times concerned both extremes of the rating scale , therefore not affecting the subjective liking contrast in a unidirectional way . fourth , and perhaps most importantly , the fmri activation map did not appear to be heavily influenced by finger movements , since neither the primary motor nor somatosensory cortices were found to be differentially involved . moreover , the anterior insula , especially of the left hemisphere has also been implicated in preference judgements of nonauditory stimuli , such as visual ( e.g. , [ 23 , 24 ] ) , and food stimuli . consequently , with a cautionary note on possible motor - related contamination , the most likely explanation for the anterior insula activity as a function of liking remains its role as a paralimbic emotion- and reward - related region . the rolandic operculum also showed liking - related activation , although the exact location of the activation peak proved difficult , given the relative proximity of functional areas in this region , in conjunction with the spatial resolution limitations of the fmri method . it is noteworthy that a previous study also indicated rolandic operculum involvement in the perception of pleasant music . in that study , as well as our own , it is possible that the activity of this region was related to sound production planning ( wanting to hum along ) , since rolandic opercular areas have been found to be implicated in both overt and covert singing and speaking in previous neuroimaging studies [ 2629 ] . the subjects in our study were instructed to lie still , and consequently the rolandic opercular activity would more likely be related to covert than overt vocalisation . we found activation of the dorsal part of the striatum , namely , the putamen and caudate nucleus . the dorsal striatum has been associated with music before in fmri studies ; the caudate nucleus in a contrast of happy versus neutral music , and the putamen in connection with melodic processing . the dorsal striatum has also been implicated in reward processing of various types of stimuli , especially food and drink , for example [ 25 , 32 , 33 ] . small et al . demonstrated with pet a feeding - induced increase in dopamine release in the dorsal striatum , which at the same time correlated with pleasantness ratings of the food . as for the insula activation , a cautionary note on a possible motor response contamination applies here as well , because the basal ganglia , of which the putamen and caudate nucleus are constituents , are known to play a key role in the planning and modulation of movement . we did not find any differential activation in the opposite contrast , that is , related to disliking . other studies have shown possible neural correlates of unpleasant , dissonant music ( e.g. , [ 3 , 34 , 35 ] ) . a plausible reason for our negative finding is that our stimuli were not sufficiently unpleasant to cause significant differential activation based on low subjective liking , as opposed to the dissonant stimuli used in the studies mentioned , which is a musical property known to cause a high degree of unpleasantness . apart from the particular brain structures involved , we also wished to examine whether our results would support the finding of some researchers ( e.g. , [ 2 , 13 , 14 ] ) , that neural responses to positively judged melodies are more left - lateralized . the distribution of voxels in our subjective liking contrast was indeed exclusively left - lateralized . this lends some support to a valence lateralization model , but it should be noted that at least some versions of the lateralization model focus on cortical , and more specifically frontal and anterior temporal areas , for example , with empirical support concerning music perception by for example an eeg study by altenmller et al . . however , later developments of this type of model have included subcortical ( limbic and paralimbic ) areas as well , for example . evidence from our own as well as previous studies notwithstanding , there does seem to be a need for further empirical work concerning the lateralization effects of positive judgement of music , as not all studies seem to corroborate such findings , for example [ 3 , 38 ] . moreover , it is conceivable that there is a connection between the left lateralization associated with pleasurable melodies , and vocalisation , which in itself could be an explanatory factor behind the observed lateralization effect in this and previous studies ( see also the discussion above regarding vocalisation ) . the prefrontal cortex , in particular ba 9 of the middle frontal gyrus , which was also a central finding in the sole prior neuroimaging study of the mee , was one of the two most prominent activation sites in our study . the other was ba 46 , which together with ba 9 forms the dlpfc [ 39 , 40 ] . we also found additional areas of activation ( bilateral ba 40 , and left ba 2 and 6 ) , which will be discussed afterwards . the dlpfc is central to higher - level cognitive operations , such as executive function and memory functions , including retrieval and working memory ( e.g. , ) . working memory plays a crucial role in tasks related to the perception of music , which unfolds over time , since listeners need to maintain the auditory stimuli in short - term memory and relate them to subsequent input , see also . accordingly , studies on working memory in music perception have shown its relation to dorsolateral and inferior frontal regions , see for example [ 41 , 42 ] for a review . when listening to a familiar melody , it is conceivable that stored information about it will continuously be compared with the current auditory input , in the working memory system . platel et al . thus found bilateral activation of mainly ba 9 and 10 of the middle frontal gyri in an episodic music memory task , using pet , and they ascribed this frontal activity to perceptual analysis of the melodies in working memory . parallel to its role in working memory , the dlpfc has also been widely implicated in memory retrieval , especially when some kind of evaluation of the retrieved information is needed ; see for a review . this function of the dlpfc could well have played a role in the observed activity pattern in our study ( discussed below in the section on the mee ) . it should be noted that dlpfc activation as a function of familiarity is not exclusive to music perception . for example , a pet study by fletcher and dolan found that ba 9 of the middle frontal gyrus , extending inferiorly into ba 46 , responded to familiar versus novel visually presented words ( see also for a review ) . in sum , the observed dlpfc activation as a function of exposure was expected , based on prior research , and it can best be explained by an increase in automatic retrieval and working memory processes when listening to familiar melodies . the parietal activations as a function of exposure can also be tied to memory - related processes , given the evidence on parietal involvement ( including ba 40 ) in memory functions [ 4549 ] . moreover , the inferior part of ba 40 , that is , in particular the smg , has been described as especially implicated in successful memory retrieval [ 47 , 50 ] . subjects in our study certainly exhibited stronger memory traces for melodies heard more often , as shown by the recognition ratings , and this fact may have contributed to the observed inferior parietal activations . ( note , however , that subjects were not asked to explicitly recall melodies for the scanning session reported here , a topic which is discussed below . ) similarly , in the study of the mee by elliott and dolan , the left inferior parietal cortex ( ba 7 and 40 ) was associated with retrieval attempt . furthermore , other neuroimaging evidence has related activation of especially the smg part of the inferior parietal cortex to music perception [ 5154 ] . memory functions could very well have played a role in the smg activity found in those studies . from a connectivity perspective , it is interesting to note that the inferior parietal activations could be connected to the dlpfc activity discussed above , since these two groups of cortical areas form a frontoparietal functional network , which numerous studies have found to be involved in memory and working memory processes in a wide range of cognitive tasks ; see for example [ 46 , 5557 ] . the involvement of the left postcentral gyrus ( ba 2 ) , a primary somatosensory area , in conjunction with the premotor cortex ( ba 6 ) activation of the left middle frontal gyrus , could possibly have been related to the button press response itself ( which was done using the right hand ) . rts differed marginally between melodies heard most often and the rest , and it can not be completely discounted that this difference had an observable impact on the fmri signal in this exposure - related contrast . we had expected to find participation of medial temporal lobe regions ( particularly the hippocampus ) in the contrast pertaining to the neural basis of the effect of stimulus novelty based on findings from studies on both music as well as other stimulus types [ 2 , 912 ] . however , no significant differential activation was observed . a possible reason for this negative finding hinges on the prolonged learning phase , which thoroughly familiarised participants with the general style of the melodic material . participants may consequently not have perceived previously unheard melodies as sufficiently novel for the medial temporal lobe system to become differentially activated . previous exposure to melodies did produce an mee , meaning that participants preferred melodies heard the most often during the learning phase b ( figure 2 ) . since memory ratings confirmed that previous exposure had a substantial positive impact on the recognition of the melodies as well ( figure 2 ) , it seems highly probable that memory effects leading to this recognition facilitation also played a leading role in the observed liking increase . this interpretation of the behavioural results was supported by the fmri data , which showed a differential activation increase in memory - related dlpfc and parietal regions , as a function of exposure . importantly , participants were not actually queried about recognition , or other memory - related tasks , until after the learning phase and scanning ( i.e. , not until phase d , see section 2 ) . participants were thus not aware until this late stage of the experiment that their memory of the melodies would be tested . during the scan ( phase c ) , the reported dlpfc and inferior parietal activity therefore most likely reflect unintentional retrieval processes , drawing upon the exposure to the melodies during phase b. hearing more familiar melodies seems to have automatically engaged the memory system , even though participants did not actively try to remember them . previous behavioural research and theoretical work have often described the mee as essentially an implicit memory phenomenon , for example [ 5 , 58 ] . the sole previous neuroimaging study of the mee similarly related the effect to implicit retrieval . elliott and dolan ( ibid . ) incidentally showed a similar neural activation pattern to the one we find , including ba 9 . however , while the memory processes of the participants in our study may well have been initiated unintentionally , as well as learned incidentally , these processes can not be termed implicit , given that the explicit recognition test showed a clear learning effect . this result relates to the fact that we used supraliminal , and not the subliminal stimuli often employed in mee studies . it is therefore a significant result of the present study that some of the findings from based on subliminal visual stimuli , have now been replicated and expanded upon with relation to supraliminal auditory stimuli . however , the supraliminal nature of our stimuli , resulting in participants being able to consciously recognise melodies when asked to do so ( phase d ) , may in fact have diminished the mee , since evidence have supported that it is usually stronger for subliminal stimuli [ 59 , 60 ] . nonetheless , our stimuli had the benefit of being more relevant to how music is enjoyed in everyday listening . moreover , some researchers have suggested that explanations of the mee should not rely crucially on the distinction between explicit and implicit memory . a remaining question concerns the underlying reasons for the mee in music . the perceptual fluency / attributional model [ 62 , 63 ] argues that the easy , fluent processing associated with familiar stimuli is misattributed to a positive disposition toward the stimulus itself . zajonc supports instead the notion that an absence of adverse effects when a stimulus is presented will generate a positive inclination towards it , through the basic mechanism of conditioning . the merits of these theories notwithstanding , we would like to suggest that , especially in the case of music , which is a temporally extended stimulus , exposure effects on liking are highly dependent on expectancy ( sometimes termed anticipation or prediction ) . it has been observed in psychomusicological research [ 6567 ] by meyer and others that the degree of fulfillment of listeners ' expectations towards the music greatly influences how it is perceived and appraised . huron argues that the mere exposure effect should more aptly be considered a prediction effect . behavioural data have supported the notion that implicit learning of melodies does generate expectancy effects in listeners [ 68 , 69 ] . apart from these theoretical and behavioural empirical insights about expectancy in music , a magnetoencephalography study , see also , showed brain networks engaged in so - called predictive coding , see [ 72 , 73 ] , when perceiving music . future studies will hopefully elucidate the relation between exposure , liking , and expectancy , as well as its neural basis . this study investigated the neural correlates of liking and exposure in music perception , as well as their interrelation . listening to subjectively likeable melodies recruited part of the limbic / paralimbic system also shown by earlier studies . brain areas showing liking - related activation included the anterior insula , and the dorsal striatum ( putamen and caudate nucleus ) , all left - lateralized , thus providing some support of a valence lateralization model . the mere exposure effect was a contributing factor to how positively melodies were judged , since melodies heard most often during a prescan learning phase were rated as most liked , as well as most recognisable . the brain region subserving this exposure effect was mainly the dorsolateral prefrontal cortex , consistent with the main finding of the only prior neuroimaging study of the mere exposure effect . our study expanded on the previous by showing the effect for supraliminal auditory stimuli , as opposed to subliminal visual stimuli . furthermore , our study for the first time provided functional neuroimaging evidence of the mere exposure effect in music , pointing to underlying automatic memory processes being involved . we suggested expectancy as a potentially important factor in these phenomena , requiring further investigation by future studies .

we used functional magnetic resonance imaging to investigate the neural basis of the mere exposure effect in music listening , which links previous exposure to liking . prior to scanning , participants underwent a learning phase , where exposure to melodies was systematically varied . during scanning , participants rated liking for each melody and , later , their recognition of them . participants showed learning effects , better recognising melodies heard more often . melodies heard most often were most liked , consistent with the mere exposure effect . we found neural activations as a function of previous exposure in bilateral dorsolateral prefrontal and inferior parietal cortex , probably reflecting retrieval and working memory - related processes . this was despite the fact that the task during scanning was to judge liking , not recognition , thus suggesting that appreciation of music relies strongly on memory processes . subjective liking per se caused differential activation in the left hemisphere , of the anterior insula , the caudate nucleus , and the putamen .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

these differences can not be due solely to qualities inherent in the music itself , since the same piece may be a psychomusicological studies , for example , [ 5 , 6 ] have pointed to the mere exposure effect ( mee ) as an important part of the explanation . that study used subliminal visual stimuli , and it showed particular involvement of the right lateral prefrontal cortex in implicit memory expressed in preference judgements , indicating this as a key brain area related to the mee . a learning phase familiarised participants with a number of melodies , where the amount of exposure to subsets of the melodies was varied systematically in order to induce different levels of implicit learning . subsequently , they were scanned using functional magnetic resonance imaging ( fmri ) , while performing a liking judgement of the melodies , succeeded by a recognition test . we hypothesized that the right lateral prefrontal cortex would play a role in the mee for music , as it did for visual stimuli , that is , show differential activation as a function of prior exposure . concerning activations related to subjective liking , we hypothesized the participation of limbic and paralimbic areas previously linked to positive judgements of music , such as insula , striatum , anterior cingulate , and orbitofrontal cortex [ 14 ] . as we wished to study exposure effects , we used unknown melodies to eliminate the possibility of preexperimental knowledge of the stimuli . on the other hand , it was also necessary to induce a certain level of variety and song - like qualities in the melodies , in order to make them stand out from each other , and be appreciated as individual pieces of music . we used supraliminal stimuli ( as opposed to for example elliott and dolan ) , since music naturally unfolds over seconds and minutes , rather than milliseconds , and since listeners in real life are in fact most often aware ( at least potentially ) of the music they are listening to . a preexperimental liking rating of the melodies was done by 49 volunteers , who did not participate in the fmri experiment itself . this rating was carried out in order to divide the melodies into five groups containing six melodies each , so that the mean liking for each melody group would be as equal as possible . the ethical committee of aarhus county approved the study , and participants gave their informed consent prior to their inclusion in the study . for each participant , the experiment incorporated five phases ( a e ) ; two outside the scanner , followed by two in the scanner , and lastly a debriefing and musical ability test outside the scanner . total randomisation was avoided to ensure that melodies of the same key and mode would not succeed each other , and that the occurrence of different stimulus types were uniformly separated ( by employing a split - halves method ) . in the scanning phases c and d , stimulus sequences were pseudorandomised for each participant individually in order to avoid the amplification of possible sequence effects . ( a ) baseline rating and training phaseparticipants listened to 18 melodies over headphones , that they would hear again in phase b. they rated each melody immediately after listening to it , in a two - second gap of silence between melodies , on a 15 scale ( 1 : least liked , 5 : most liked ) . participants listened to 18 melodies over headphones , that they would hear again in phase b. they rated each melody immediately after listening to it , in a two - second gap of silence between melodies , on a 15 scale ( 1 : least liked , 5 : most liked ) . first , participants were asked to press a button as quickly as possible to the occurrence of detuned notes in the six special melodies that would appear five times each throughout phase b. second , instrument sound in this phase alternated between piano and guitar , in order to help differentiate one melody from the next ( the guitar versions of the melodies were not used for the scanning sessions ) . lastly , participants were allowed a five minute break halfway through the learning phase . first , participants were asked to press a button as quickly as possible to the occurrence of detuned notes in the six special melodies that would appear five times each throughout phase b. second , instrument sound in this phase alternated between piano and guitar , in order to help differentiate one melody from the next ( the guitar versions of the melodies were not used for the scanning sessions ) . the 24 melodies thus belonged to one of four categories designated f0 , f2 , f8 , and f32 , based on the number of previous repetitions in the given scenario . participants were instructed to visually fixate on a crosshair on a screen , listen carefully to the stimuli over the headphones , and immediately after each melody to rate how well they liked it on a scale from 1 ( least liked ) to 5 ( most liked ) , using a response box with one button for each finger . the 24 melodies thus belonged to one of four categories designated f0 , f2 , f8 , and f32 , based on the number of previous repetitions in the given scenario . participants were instructed to visually fixate on a crosshair on a screen , listen carefully to the stimuli over the headphones , and immediately after each melody to rate how well they liked it on a scale from 1 ( least liked ) to 5 ( most liked ) , using a response box with one button for each finger . stimulus sequences further included six repetitions of the chromatic scale , and a number of 28 s silences in between , yielding jittered interstimulus intervals to allow more exhaustive sampling of the fmri signal . second , the task for the participants was to judge their certainty of having heard the melody before ( 1 : certain that melody has not been played , 5 : certain that melody has been played ) . the rating was done with participants in the scanner , as for phase c , in order to ensure that the recognition rating was done in exactly the same physical context as the previous liking rating , and to have the two ratings as close to one another as possible in time . second , the task for the participants was to judge their certainty of having heard the melody before ( 1 : certain that melody has not been played , 5 : certain that melody has been played ) . the rating was done with participants in the scanner , as for phase c , in order to ensure that the recognition rating was done in exactly the same physical context as the previous liking rating , and to have the two ratings as close to one another as possible in time . the subjective liking rating , and the objective exposure frequency category ( f0 , f2 , f8 , or f32 ) , pertaining to each melody , were entered into the model as parametric modulations . contrasts were set up to test effects of subjective liking , based on individual subject ratings , and for effects of stimulus exposure frequency . to ascertain which conditions prompted any significant effect observed , the mean liking ratings for melodies heard 0 , 2 , 8 , and 32 times by the end of the learning phase , are shown in figure 2 ( the groups are termed f0 ( frequency 0 ) , f2 , etc . ) reaction times ( rts ) for the liking ratings differed as a function of presentation frequency , as shown by a repeated measures anova , f ( 3,60 ) = 3.842 , p = 0.014 . rt also differed slightly as a function of subjective liking rating , f ( 4,44 ) = 3.278 , p = 0.019 . rt for memory ratings did not differ significantly as a function of exposure , f ( 3.06,61.20 ) = 1.098 , p = 0.358 ( the sphericity assumption was not met , so huynh - feldt correction was applied ) . brain regions showing increased activity as a function of subjective liking rating ( irrespective of presentation frequency ) were all located in the left hemisphere and included a cluster in the anterior part of the insula ( ba 13 ) , extending into the rolandic operculum ( table 2 and figure 4 ) . further activations were found in the dorsal striatum , namely , in the putamen , and in the body of the caudate nucleus . the opposite contrast , of areas showing decreased activity as a function of liking rating , did not result in any significant activation . the contrast based on the amount of previous exposure to the melodies showed activity increases in dorsolateral prefrontal cortex ( dlpfc ) , namely , in bilateral middle frontal gyri ( ba 9 ) , and neighbouring inferior middle / frontal gyri ( ba 46 ) . differential activations of the left hemisphere further incorporated activations of the postcentral gyrus ( ba 2 ) , and the supramarginal gyrus ( smg ) ( ba 40 ) . ba 40 of the right inferior parietal lobe was also implicated , as was ba 6 of the left middle frontal gyrus . the opposite contrast , corresponding to an increase in the fmri signal as a function of stimulus novelty , did not result in any significant activation . we have shown that listening to melodies rated as likeable differentially activates deeper brain structures related to emotion processing , all in the left hemisphere , including the anterior insula and the dorsal striatum . a supraliminal mere exposure effect was a contributing factor to how positively melodies were judged , and brain regions subserving this effect , that is , exhibiting increased activation as a function of prior exposure , included the dorsolateral prefrontal cortex ( bilateral ba 9 and ba 46 ) . preceding neuroimaging studies of liking effects in music listening have highlighted the contribution of limbic structures , consistent with the knowledge of their involvement in processing of emotions in general ( e.g. of these areas , some in particular have been confirmed by one or more of the subsequent studies on responses to pleasant music with pet and fmri , including the ventral striatum ( nucleus accumbens ) , anterior cingulate , orbitofrontal cortex , and insula [ 24 ] . there is also evidence suggesting that neural responses to pleasant music in general tend to be more lateralized to the left hemisphere , an effect often termed the valence lateralization model [ 2 , 13 , 14 ] . of the brain regions that have been previously implicated in the liking - related network , we also found differential activation of the striatum , more precisely its dorsal part , rather than the ventral part often found in previous studies ( see below ) . this only partial replication of the previously observed liking - related network may relate to the fact , that the melodies composed for our study were relatively short , unknown , computer - recorded single - voice melodies , in order to maximize experimental control . the involvement of the insula , and more specifically its anterior part , was expected based on prior findings , although in our study it was left - lateralized , compared to mostly bilateral in previous studies on pleasant music ( ibid . ) menon and levitin used effective connectivity analysis in their fmri study to show a strong ventral tegmental area - mediated interaction of nucleus accumbens and the left anterior insula , forming a mesolimbic dopaminergic system that is central to reward processing . the insula is furthermore known to be one of the most important nodal points ( together with the hypothalamus ) in neural pathways concerned with autonomic , somatic , and emotional functions [ 21 , 22 ] . third , the faster reaction times concerned both extremes of the rating scale , therefore not affecting the subjective liking contrast in a unidirectional way . moreover , the anterior insula , especially of the left hemisphere has also been implicated in preference judgements of nonauditory stimuli , such as visual ( e.g. consequently , with a cautionary note on possible motor - related contamination , the most likely explanation for the anterior insula activity as a function of liking remains its role as a paralimbic emotion- and reward - related region . the rolandic operculum also showed liking - related activation , although the exact location of the activation peak proved difficult , given the relative proximity of functional areas in this region , in conjunction with the spatial resolution limitations of the fmri method . we found activation of the dorsal part of the striatum , namely , the putamen and caudate nucleus . the dorsal striatum has been associated with music before in fmri studies ; the caudate nucleus in a contrast of happy versus neutral music , and the putamen in connection with melodic processing . demonstrated with pet a feeding - induced increase in dopamine release in the dorsal striatum , which at the same time correlated with pleasantness ratings of the food . as for the insula activation , a cautionary note on a possible motor response contamination applies here as well , because the basal ganglia , of which the putamen and caudate nucleus are constituents , are known to play a key role in the planning and modulation of movement . we did not find any differential activation in the opposite contrast , that is , related to disliking . a plausible reason for our negative finding is that our stimuli were not sufficiently unpleasant to cause significant differential activation based on low subjective liking , as opposed to the dissonant stimuli used in the studies mentioned , which is a musical property known to cause a high degree of unpleasantness . moreover , it is conceivable that there is a connection between the left lateralization associated with pleasurable melodies , and vocalisation , which in itself could be an explanatory factor behind the observed lateralization effect in this and previous studies ( see also the discussion above regarding vocalisation ) . the prefrontal cortex , in particular ba 9 of the middle frontal gyrus , which was also a central finding in the sole prior neuroimaging study of the mee , was one of the two most prominent activation sites in our study . the dlpfc is central to higher - level cognitive operations , such as executive function and memory functions , including retrieval and working memory ( e.g. working memory plays a crucial role in tasks related to the perception of music , which unfolds over time , since listeners need to maintain the auditory stimuli in short - term memory and relate them to subsequent input , see also . accordingly , studies on working memory in music perception have shown its relation to dorsolateral and inferior frontal regions , see for example [ 41 , 42 ] for a review . when listening to a familiar melody , it is conceivable that stored information about it will continuously be compared with the current auditory input , in the working memory system . thus found bilateral activation of mainly ba 9 and 10 of the middle frontal gyri in an episodic music memory task , using pet , and they ascribed this frontal activity to perceptual analysis of the melodies in working memory . parallel to its role in working memory , the dlpfc has also been widely implicated in memory retrieval , especially when some kind of evaluation of the retrieved information is needed ; see for a review . this function of the dlpfc could well have played a role in the observed activity pattern in our study ( discussed below in the section on the mee ) . it should be noted that dlpfc activation as a function of familiarity is not exclusive to music perception . in sum , the observed dlpfc activation as a function of exposure was expected , based on prior research , and it can best be explained by an increase in automatic retrieval and working memory processes when listening to familiar melodies . the parietal activations as a function of exposure can also be tied to memory - related processes , given the evidence on parietal involvement ( including ba 40 ) in memory functions [ 4549 ] . subjects in our study certainly exhibited stronger memory traces for melodies heard more often , as shown by the recognition ratings , and this fact may have contributed to the observed inferior parietal activations . similarly , in the study of the mee by elliott and dolan , the left inferior parietal cortex ( ba 7 and 40 ) was associated with retrieval attempt . furthermore , other neuroimaging evidence has related activation of especially the smg part of the inferior parietal cortex to music perception [ 5154 ] . from a connectivity perspective , it is interesting to note that the inferior parietal activations could be connected to the dlpfc activity discussed above , since these two groups of cortical areas form a frontoparietal functional network , which numerous studies have found to be involved in memory and working memory processes in a wide range of cognitive tasks ; see for example [ 46 , 5557 ] . the involvement of the left postcentral gyrus ( ba 2 ) , a primary somatosensory area , in conjunction with the premotor cortex ( ba 6 ) activation of the left middle frontal gyrus , could possibly have been related to the button press response itself ( which was done using the right hand ) . rts differed marginally between melodies heard most often and the rest , and it can not be completely discounted that this difference had an observable impact on the fmri signal in this exposure - related contrast . we had expected to find participation of medial temporal lobe regions ( particularly the hippocampus ) in the contrast pertaining to the neural basis of the effect of stimulus novelty based on findings from studies on both music as well as other stimulus types [ 2 , 912 ] . a possible reason for this negative finding hinges on the prolonged learning phase , which thoroughly familiarised participants with the general style of the melodic material . previous exposure to melodies did produce an mee , meaning that participants preferred melodies heard the most often during the learning phase b ( figure 2 ) . since memory ratings confirmed that previous exposure had a substantial positive impact on the recognition of the melodies as well ( figure 2 ) , it seems highly probable that memory effects leading to this recognition facilitation also played a leading role in the observed liking increase . this interpretation of the behavioural results was supported by the fmri data , which showed a differential activation increase in memory - related dlpfc and parietal regions , as a function of exposure . importantly , participants were not actually queried about recognition , or other memory - related tasks , until after the learning phase and scanning ( i.e. during the scan ( phase c ) , the reported dlpfc and inferior parietal activity therefore most likely reflect unintentional retrieval processes , drawing upon the exposure to the melodies during phase b. hearing more familiar melodies seems to have automatically engaged the memory system , even though participants did not actively try to remember them . however , while the memory processes of the participants in our study may well have been initiated unintentionally , as well as learned incidentally , these processes can not be termed implicit , given that the explicit recognition test showed a clear learning effect . this result relates to the fact that we used supraliminal , and not the subliminal stimuli often employed in mee studies . the merits of these theories notwithstanding , we would like to suggest that , especially in the case of music , which is a temporally extended stimulus , exposure effects on liking are highly dependent on expectancy ( sometimes termed anticipation or prediction ) . huron argues that the mere exposure effect should more aptly be considered a prediction effect . future studies will hopefully elucidate the relation between exposure , liking , and expectancy , as well as its neural basis . this study investigated the neural correlates of liking and exposure in music perception , as well as their interrelation . brain areas showing liking - related activation included the anterior insula , and the dorsal striatum ( putamen and caudate nucleus ) , all left - lateralized , thus providing some support of a valence lateralization model . the mere exposure effect was a contributing factor to how positively melodies were judged , since melodies heard most often during a prescan learning phase were rated as most liked , as well as most recognisable . the brain region subserving this exposure effect was mainly the dorsolateral prefrontal cortex , consistent with the main finding of the only prior neuroimaging study of the mere exposure effect . furthermore , our study for the first time provided functional neuroimaging evidence of the mere exposure effect in music , pointing to underlying automatic memory processes being involved .

systemic lupus erythematosus ( sle ) is an autoimmune disease characterized by chronic inflammation in multiple organs such as kidney , lung , heart , joint , and so forth [ 15 ] . one of the most severe manifestations of sle is lupus nephritis , which is a potentially fatal complication [ 68 ] . many researchers have reported the morbidity and mortality of sle extensively ; however , the pathogenic mechanism of sle remains still elusive . reports have indicated that autoantibody - mediated immune response can trigger tissue damage , and thus contributes to the pathogenesis of sle [ 1 , 9 , 10 ] . in recent years , accumulating evidence indicates that deregulated production of proinflammatory cytokines such as tnf- and il-6 may play a critical role in immune dysfunction and mediate tissue inflammation and organ damage in sle [ 1 , 10 ] . it is reported that tnf- levels are significantly increased and correlated with sle disease activity , and blocking tnf- function has been found to decrease disease activity in clinical patients [ 1119 ] . additionally , reports also indicate that il-6 promotes autoantibody production in humans and mice with lupus nephritis [ 5 , 20 ] . therefore , the understanding of the detailed mechanism of inflammatory response would facilitate the advance of efficient therapies toward sle . recent evidence indicates that hmgb1 , a well - established damage associated molecular pattern ( damp ) , is responsible for the production of proinflammatory cytokines [ 2123 ] . hmgb1 is likely to be released from activated immune cells such as macrophages in the area of inflammation or injure [ 2429 ] . when released , hmgb1 participates in the secretion of downstream proinflammatory cytokines via binding to cell surface receptors such as receptor of advanced glycation end products ( rage ) , tlr2 and tlr4 , thus contributing to the occurrence and development of diverse inflammatory diseases and autoimmune diseases [ 2530 ] . proinflammatory and immune - stimulatory function of hmgb1 indicate its association with autoimmune diseases including rheumatoid arthritis and sle [ 29 , 31 ] . furthermore , hmgb1 has been found to be significantly elevated in lupus sera and identified as one of the components in dna - containing immune complexes that enhance proinflammatory cytokine production . all these data indicate that hmgb1 might act as a new inflammation - related factor in sle ; however , the precise role of hmgb1 in the inflammatory response during the pathogenesis of sle still remains unclear . our previous study has demonstrated that activated lymphocyte derived - dna ( ald - dna ) could induce sle syndrome including high levels of anti - dsdna antibody , glomerulonephritis , and proteinuria in healthy mice with conventional genetic background [ 3339 ] . in this study we investigated the potential role of hmgb1 in the pathogenesis of sle and its underlying mechanism . we found that hmgb1 levels were elevated and correlated with sle disease activity both in clinical patients and murine model . gain- and loss - of - function analysis revealed that hmgb1 aggravated the severity of sle , which might be due to its effect on macrophage inflammatory response . a total of 32 sle patients were recruited , and all of the peripheral blood samples were collected from these sle patients after obtaining informed consent . the diagnosis of sle was established according to the four of the american college of rheumatology ( acr ) revised criteria for the diagnosis of sle . the mean age of the patients was 32 ( range 19 to 54 ) years ( y ) , and 24 healthy individuals matched for gender and age were recruited as controls . six - week - old female balb / c mice were purchased from the experimental animal center of chinese academy of sciences ( shanghai , china ) . this study was strictly carried out according to the guide for the care and use of medical laboratory animals ( ministry of health , china , 1998 ) and with the ethical approval of the shanghai medical laboratory animal care and use committee as well as the ethical committee of fudan university . all surgery was performed under sodium pentobarbital anesthesia , and all animal procedures in this study were strictly performed in a manner to minimize suffering of laboratory mice . raw264.7 cells were maintained in dmem ( invitrogen life technologies ) supplemented with 10% fbs ( invitrogen life technologies ) in a 5% co2 incubator at 37c . pcaggs - hmgb1 ( phmgb1 ) and pcaggs ( empty vector ) were kindly provided by professor tadatsugu taniguchi ( university of tokyo , tokyo , japan ) . macrophages were transfected with 200 nm of indicated sirnas by mouse macrophage nucleofector kit ( lonza ) according to the manufacturer 's instructions . hmgb1 and rage antibody were obtained from cell signaling technology and gapdh antibody from santa cruz biotechnology . the extraction and purification of activated lymphocyte - derived dna ( ald - dna ) and unactivated lymphocyte - derived dna ( unald - dna ) were performed according to our previously described methods [ 3339 ] . to generate the murine model of sle , six - week - old female balb / c mice were immunized s.c . with ald - dna ( 50 g / mouse ) plus cfa ( sigma - aldrich ) on day 1 , followed by s.c . injection of ald - dna ( 50 g / mouse ) emulsified with cfa ( sigma - aldrich ) on days 14 and 28 for total of three times as described previously [ 3339 ] . mice in each group received an equal volume of pbs plus cfa or ifa , or unald - dna ( 50 mg / mouse ) plus cfa or ifa were used as controls . to investigate the effect of hmgb1 in the pathogenic process of sle mice were divided into six groups as follows : pbs plus vector , un - ald - dna plus vector , ald - dna plus vector , pbs plus phmgb1 , un - ald - dna plus phmgb1 and ald - dna plus phmgb1 . to further confirm the significance of hmgb1 , mice were treated with hmgb1 inhibitor glycyrrhizin ( 0.5 mg / mice ) every day . mice were divided into six groups as follows : pbs , un - ald - dna plus pbs , ald - dna plus pbs , pbs plus glycyrrhizin , un - ald - dna plus glycyrrhizin , and ald - dna plus glycyrrhizin . eight weeks later , mice were sacrificed , and surgically resected kidneys were collected for further cellular function and tissue histology analysis . to examine the potential role of hmgb1 in sle , 8 mice in each group were intramuscularly injected with 100 g of phmgb1 or empty vector per mouse 72 h earlier before injection with ald - dna . mice were then injected with phmgb1 every 2 weeks for total 6 times [ 35 , 36 ] . to block the function of hmgb1 in sle mice , mice were randomized to intramuscularly injection 0.5 mg per mouse glycyrrhizin every day for 2 months . twenty - four hours after the initial glycyrrhizin treatment , the mice were immunized with ald - dna ( 50 g / mouse ) three times in 4 week as previously described . serum anti - dsdna antibody levels in the mice were determined by elisa analysis as described previously [ 3339 ] . proteinuria of the mice was measured with the bca method ( thermo fisher scientific ) as previously described [ 3339 ] . murine renal tissues were surgically resected and dispersed in rpmi 1640 containing 5% fbs and 0.1% collagenase ( sigma - aldrich ) at 37c for 30 min , followed by progressive sieving to obtain single - cell suspensions . to analyze the inflammatory response of renal macrophages , cd11b / f4/80 renal macrophages were sorted from nephritic single - cell suspensions using a facsaria ( bd biosciences ) with fitc - labeled anti - f4/80 and pe - labeled anti - cd11b ( bd biosciences ) . for histology analysis , murine renal tissues were surgically resected and fixed in 4% paraformaldehyde ( sigma - aldrich ) , processed , and embedded in paraffin . h&e staining of renal tissue sections were performed according to the manufacturer 's instructions and assessed by two pathologists blinded to treatment group as previously described [ 3339 ] . the kidney score of glomerulonephritis pictures were acquired with nikon sclipss te2000-s microscope ( nikon ) equipped with act-1 software ( nikon ) . plasma was collected by edta as an anticoagulant , aliquoted , and stored at 80c . to assess the levels of hmgb1 , anti - dsdna antibody , tnf- , and il-6 in the plasma and supernatant of cell culture , elisa ( shino - test , sagamihara - shi , kanagawa , japan for hmgb1 ; ebioscience for tnf- and il-6 ; alpha diagnostic international for anti - dsdna ) was performed according to the manufacturer 's instructions . antibodies used here were anti - gapdh ( santa cruz biotechnology ) , hmgb1 ( cell signal technology ) , goat anti - mouse igg - hrp ( santa cruz biotechnology ) , and goat anti - rabbit igg - hrp ( santa cruz biotechnology ) . all analyses were performed by graphpad prism 5 ( graphpad software , la jolla , ca ) . a two - tailed p value < 0.05 was considered as statistically significant unless otherwise noted . to investigate whether hmgb1 was involved in the pathogenesis of sle , we first examined the levels of hmgb1 in sle patients . a total of 32 sle patients were recruited to our research and the general characteristics of patients were shown in table 1 . we determined the serum concentrations of hmgb1 in sle patients and healthy controls ( hc ) by elisa . ng / ml ) were significantly higher than those in hc ( 5.0877 2.7921 ng / ml , p < 0.05 ) ( figure 1(a ) ) . furthermore , serum hmgb1 levels in sle patients with active renal disease ( 42.5672 21.0052 ng / ml ) were significantly higher than those in patients without active renal disease ( 15.7279 8.8412 ng / ml , p < 0.05 , figure 1(b ) ) . moreover , we found that serum hmgb1 concentrations showed a highly significant correlation with sle disease activity index score ( sledai ) ( r = 0.4715 , p = 0.0064 , figure 1(c ) ) and anti - dsdna antibody levels ( r = 0.6257 , p = 0.0001 , figure 1(d ) ) . we also analyzed the expression of hmgb1 in peripheral blood mononuclear cells ( pbmcs ) from sle patients ( s1 , s2 , s3 , and s4 ) and hc ( h1 , h2 , h3 , and h4 ) using western blot . as shown in figure 1(e ) , the expression of hmgb1 was increased in pbmcs from sle patient compared to hc . we further analyzed the serum levels of hmgb1 in murine model of sle which were generated according to our previously reported procedures [ 3339 ] . ald - dna could successfully induce the sle syndrome manifested by high levels of anti - dsdna antibody ( see figure s1(a ) in supplementary material available online at http://dx.doi.org/10.1155/2015/946748 ) , proteinuria ( figure s1(b ) ) as well as glomerulonephritis ( figures s1(c ) and s1(d ) ) . of interest , we found that serum hmgb1 levels were significantly increased in murine model of sle compared with those in control mice ( figure 1(f ) ) . pearson correlation analysis showed that the serum hmgb1 levels were positively correlated with kidney score ( r = 0.6583 , p = 0.0022 , figure 1(g ) ) , indicating that hmgb1 levels were significantly associated with the severity of lupus nephritis . similarly , we observed that serum hmgb1 levels were related with the levels of anti - dsdna antibody ( r = 0.7278 , p = 0.0004 , figure 1(h ) ) and urine protein ( r = 0.6652 , p = 0.0019 , figure 1(i ) ) in ald - dna - induced murine model of sle . taken together , these data indicated that hmgb1 expression was upregulated and correlated with the severity of sle both in clinical patients and murine model . above data showed that hmgb1 levels were elevated and correlated with the severity of sle . to further evaluate whether hmgb1 was involved in the pathogenesis of sle , we upregulated the expression of hmgb1 by injecting balb / c mice intramuscularly with a hmgb1 overexpression plasmid ( phmgb1 ) . results showed that the injection of phmgb1 led to the elevation of serum hmgb1 levels in sle mice ( figure 2(a ) ) . to investigate the effect of increased hmgb1 levels on the progression of sle , we analyzed anti - dsdna antibody , proteinuria , renal pathology , and kidney score in phmgb1- or empty vector - treated mice . results showed that hmgb1 overexpression exacerbated renal pathology as revealed by the increased infiltration of proinflammatory cells into glomerular mesanguim and thickened basement membrane , as well as the atrophy of glomerular mesanguim ( figure 2(b ) ) , and upregulated the kidney score of sle mice ( figure 2(c ) ) . we also found that the levels of anti - dsdna antibody ( figure 2(d ) ) and urine protein ( figure 2(e ) ) in sle mice treated with phmgb1 were notably elevated compared with that in empty vector - treated sle mice . to further confirm the effect of hmgb1 on the progression of sle , we inhibited the function of hmgb1 in vivo by injecting balb / c mice intramuscularly with glycyrrhizin which has been demonstrated to be the blocker of hmgb1 [ 4143 ] . as shown in figure 3(a ) , glycyrrhizin administration significantly decreased the serum hmgb1 levels in sle mice . of note , glycyrrhizin treatment was found to efficiently ameliorate renal pathology as demonstrated by decreased infiltration of proinflammatory cells into glomerular mesanguim , recovery from thicken basement membrane and the atrophic glomerular mesanguim ( figure 3(b ) ) , and decrease the kidney score in sle mice ( figure 3(c ) ) . furthermore , we revealed that glycyrrhizin treatment reduced levels of anti - dsdna antibody ( figure 3(d ) ) and urine protein ( figure 3(e ) ) in sle mice . taken together , these data demonstrated that hmgb1 played a crucial role in modulating the severity of sle . it is well established that macrophages are prominent within the inflamed kidneys and are key mediators in lupus nephritis [ 4449 ] . our previous studies have also confirmed that macrophage is the central mediator in ald - dna - induced sle [ 3336 ] . to study whether hmgb1 was involved in macrophage inflammatory response , we detected the production of hmgb1 in raw264.7 cells stimulated with ald - dna . the results showed that the ald - dna administration led to the upregulation of hmgb1 levels in raw264.7 cells ( figures 4(a ) and 4(b ) ) . we then investigated the role of hmgb1 in ald - dna - induced macrophage inflammatory response by transfecting phmgb1 into raw264.7 cells . results showed that transfection of phmgb1 notably increased hmgb1 levels in raw264.7 cells ( figure 4(c ) ) . of importance , hmgb1 overexpression in raw264.7 cells was found to aggravate the secretion of tnf- ( figure 4(e ) ) and il-6 ( figure 4(f ) ) induced by ald - dna . we further downregulated hmgb1 expression by specific sirna against hmgb1 ( sihmgb1 ) in raw264.7 cells . western blot showed that transfection of sihmgb1 efficiently decreased hmgb1 levels in raw264.7 cells ( figure 4(d ) ) . we stimulated sihmgb1-treated raw264.7 cells with ald - dna , followed by detecting the concentrations of proinflammatory cytokines in the supernatants . results showed that the sihmgb1-mediated downregulation of hmgb1 levels significantly inhibited the secretion of tnf- ( figure 4(g ) ) and il-6 ( figure 4(h ) ) in ald - dna - stimulated macrophages . moreover , we isolated cd11b / f4/80 renal macrophages from phmgb1- or empty vector - treated sle mice , glycyrrhizin- or pbs - treated sle mice and stimulated these cells with ald - dna , followed by detecting the production of tnf- and il-6 . as shown in figure 4(i ) , renal macrophages secreted much higher levels of tnf- and il-6 in phmgb1-treated sle mice than those from empty vector - treated sle mice , whereas renal macrophages from glycyrrhizin - treated sle mice secreted lower levels of tnf- and il-6 than those pbs - treated sle mice ( figure 4(j ) ) . to study the relationship between hmgb1 and proinflammatory cytokines , we analyzed the correlation between hmgb1 and proinflammatory cytokines ( tnf- and il-6 ) in sle patients . we first detected the levels of serum tnf- and il-6 in sle patients , and the results demonstrated that the concentrations of tnf- ( figure 5(a ) ) and il-6 ( figure 5(b ) ) in sle patients were significantly higher than those in hc ( p < 0.05 ) . further study was conducted to analyze the correlation between hmgb1 and proinflammatory cytokines ( tnf- and il-6 ) . we observed that hmgb1 levels were associated with tnf- ( r = 0.3559 , p = 0.0456 , figure 5(c ) ) and il-6 levels ( r = 0.3597 , p = 0.0432 , figure 5(d ) ) in sle patients . taken together , these data indicated that hmgb1 was pivotal for ald - dna - induced macrophage inflammatory response and correlated with the levels of tnf- and il-6 in sle . previous studies indicate that hmgb1 is an endogenous ligand of rage , tlr2 , and tlr4 [ 5052 ] . to evaluate which receptor might be involved in the hmgb1-mediated inflammatory effect in ald - dna - stimulated macrophages , we first upregulated hmgb1 expression in raw264.7 cells and then stimulated these cells with ald - dna in the presence of tlr2/4 inhibitor ( oxpapc ) or rage inhibitor ( rage - fc ) . the results showed that the production of tnf- ( figure 6(a ) ) and il-6 ( figure 6(b ) ) from raw264.7 cells exposed to tlr2/4 inhibitor was not impaired in the process of hmgb1-enhanced macrophage inflammatory response , whereas blocking the function of rage with rage - fc effectively reduced the secretion of tnf- ( figure 6(a ) ) and il-6 ( figure 6(b ) ) . to further validate the importance of rage receptor , we downregulated the expression of rage by sirna and then evaluated the effect of hmgb1 on the production of proinflammatory cytokines in ald - dna - stimulated raw264.7 cells . western blot analysis confirmed that transfection of sirage could notably decrease rage levels in raw264.7 cells ( figure 6(c ) ) . of importance , it was found that sirna - mediated downregulation of rage significantly inhibited hmgb1-enhanced release of tnf- ( figure 6(d ) ) and il-6 ( figure 6(e ) ) in ald - dna - stimulated macrophages . collectively , these data indicated that rage might be critical for proinflammatory signaling during the process of hmgb1-aggravated macrophages inflammatory response induced by ald - dna , whereas tlr2 and tlr4 appeared to be dispensable . sle , a chronic inflammatory autoimmune disorder , is a potentially fatal disease characterized by immune complex deposition and the subsequent inflammation that contributes to severe tissue damage [ 3 , 5 ] . recent reports show that some multifunctional proteins such as hmgb1 might be involved in autoimmune and inflammatory diseases including sle [ 31 , 5255 ] . our previous study revealed that hmgb1 was required for autoantibody induction by dna - containing immune complexes in sle patients , implying that the critical role of hmgb1 in the pathogenesis of sle . in the present investigation , we further explored the role and mechanism of hmgb1 in the pathogenesis of sle . our results demonstrated that hmgb1 levels were increased and correlated with the severity of sle in both clinical patients and murine model , consistent with previous findings [ 5760 ] . to evaluate whether hmgb1 was involved in the pathogenesis of sle , we overexpressed hmgb1 in ald - dna - immunized mice and found that hmgb1 overexpression significantly enhanced the severity of sle . to further confirm the role of hmgb1 in sle glycyrrhizin is commonly used in clinical application for its anti - inflammatory activity , and it binds to hmgb1 directly , thereby inhibiting the extracellular hmgb1 secretion . evidence also shows that glycyrrhizin administration could suppress hmgb1 function resulting in the amelioration of ischemic spinal cord injury and damage caused by cerebral hemorrhage [ 42 , 43 ] . our results showed that blockade of hmgb1 function by glycyrrhizin led to dramatic downregulation of serum hmgb1 levels , and thus decreased the severity of sle . these data suggested that hmgb1 played a crucial role in the pathogenesis of sle , implicating a promising hmgb1-based therapeutic strategy against sle . the increasing number of evidence has emerged to suggest the crucial role of proinflammatory cytokines in the pathogenesis of sle . the consequence of disorder of proinflammatory cytokines would be an immune dysregulation followed by local inflammatory processes and tissue damage [ 61 , 62 ] . circumstantial data suggests that tnf- may serve as an important autocrine and paracrine factor in glomerular injury [ 1113 ] . in addition , il-6 is produced in many cell types like monocytes , fibroblasts , endothelial cells , and also t and b lymphocytes and has a range of biological activities on various target cells . considering the importance of hmgb1 in the regulation of inflammatory response , we analyzed the correlation between hmgb1 and proinflammatory cytokines expression in sle . our studies found that the concentrations of tnf- and il-6 in sle patients were significantly higher than those in hc , consistent with previous study . further studies showed that hmgb1 levels were correlated with the levels of tnf- and il-6 in sle patients . moreover , our data found that hmgb1 promoted inflammatory response of renal macrophages in sle mice . these results suggested that hmgb1 might be involved in the pathogenesis of sle via regulating macrophage inflammatory response , however , the definite relationship between hmgb1 and macrophage inflammatory response needs further investigation . accumulating data demonstrate that activated macrophages that infiltrate kidneys mediate the onset of an aggressive adaptive immune response leading to the pathogenesis of sle in mice [ 6570 ] . these reports give a clue that macrophages play a crucial pathogenic role in the development of sle . our previous study has also indicated that ald - dna immunization lead to macrophage infiltration and aberrant activation , which mediate the onset and aggravation of sle , indicating that aberrant activation of macrophage plays a crucial pathogenic role in ald - dna - induced sle [ 3339 ] . here , we found that hmgb1 enhanced ald - dna - induced macrophage inflammatory response both in vivo and in vitro . hmgb1 is a ubiquitously expressed , abundant architectural chromosomal protein of 215 amino acids , with a highly conserved sequence across species . at least three receptors are reported to mediate the proinflammatory and immune - activate effects of extracellular hmgb1 : rage , tlr2 , and tlr4 [ 2528 ] . our results suggested that rage might be critical for proinflammatory signaling during the process of hmgb1-aggravated macrophage inflammatory response induced by ald - dna , whereas tlr2 and tlr4 seemed to be dispensable . collectively , it seemed that hmgb1 was a crucial cofactor that could modify the stimulatory activity of macrophage . in summary , our research reported that hmgb1 levels were significantly increased and correlated with sle disease activity in both clinical patients and murine model . further study suggested that hmgb1 aggravated the severity of sle via facilitating macrophage inflammatory response . moreover , rage might be critical for proinflammatory signaling during the process of hmgb1-aggravated macrophages inflammatory response . these findings may help to develop anti - inflammatory therapeutics which blunted macrophage activation by blocking hmgb1 function in sle .

background / purpose . hmgb1 , which may act as a proinflammatory mediator , has been proposed to contribute to the pathogenesis of multiple chronic inflammatory and autoimmune diseases including systemic lupus erythematosus ( sle ) ; however , the precise mechanism of hmgb1 in the pathogenic process of sle remains obscure . method . the expression of hmgb1 was measured by elisa and western blot . the elisa was also applied to detect proinflammatory cytokines levels . furthermore , nephritic pathology was evaluated by h&e staining of renal tissues . results . in this study , we found that hmgb1 levels were significantly increased and correlated with sle disease activity in both clinical patients and murine model . furthermore , gain- and loss - of - function analysis showed that hmgb1 exacerbated the severity of sle . of note , the hmgb1 levels were found to be associated with the levels of proinflammatory cytokines such as tnf- and il-6 in sle patients . further study demonstrated that increased hmgb1 expression deteriorated the severity of sle via enhancing macrophage inflammatory response . moreover , we found that receptor of advanced glycation end products played a critical role in hmgb1-mediated macrophage inflammatory response . conclusion . these findings suggested that hmgb1 might be a risk factor for sle , and manipulation of hmgb1 signaling might provide a therapeutic strategy for sle .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

systemic lupus erythematosus ( sle ) is an autoimmune disease characterized by chronic inflammation in multiple organs such as kidney , lung , heart , joint , and so forth [ 15 ] . one of the most severe manifestations of sle is lupus nephritis , which is a potentially fatal complication [ 68 ] . many researchers have reported the morbidity and mortality of sle extensively ; however , the pathogenic mechanism of sle remains still elusive . reports have indicated that autoantibody - mediated immune response can trigger tissue damage , and thus contributes to the pathogenesis of sle [ 1 , 9 , 10 ] . in recent years , accumulating evidence indicates that deregulated production of proinflammatory cytokines such as tnf- and il-6 may play a critical role in immune dysfunction and mediate tissue inflammation and organ damage in sle [ 1 , 10 ] . it is reported that tnf- levels are significantly increased and correlated with sle disease activity , and blocking tnf- function has been found to decrease disease activity in clinical patients [ 1119 ] . therefore , the understanding of the detailed mechanism of inflammatory response would facilitate the advance of efficient therapies toward sle . recent evidence indicates that hmgb1 , a well - established damage associated molecular pattern ( damp ) , is responsible for the production of proinflammatory cytokines [ 2123 ] . hmgb1 is likely to be released from activated immune cells such as macrophages in the area of inflammation or injure [ 2429 ] . when released , hmgb1 participates in the secretion of downstream proinflammatory cytokines via binding to cell surface receptors such as receptor of advanced glycation end products ( rage ) , tlr2 and tlr4 , thus contributing to the occurrence and development of diverse inflammatory diseases and autoimmune diseases [ 2530 ] . proinflammatory and immune - stimulatory function of hmgb1 indicate its association with autoimmune diseases including rheumatoid arthritis and sle [ 29 , 31 ] . furthermore , hmgb1 has been found to be significantly elevated in lupus sera and identified as one of the components in dna - containing immune complexes that enhance proinflammatory cytokine production . all these data indicate that hmgb1 might act as a new inflammation - related factor in sle ; however , the precise role of hmgb1 in the inflammatory response during the pathogenesis of sle still remains unclear . our previous study has demonstrated that activated lymphocyte derived - dna ( ald - dna ) could induce sle syndrome including high levels of anti - dsdna antibody , glomerulonephritis , and proteinuria in healthy mice with conventional genetic background [ 3339 ] . in this study we investigated the potential role of hmgb1 in the pathogenesis of sle and its underlying mechanism . we found that hmgb1 levels were elevated and correlated with sle disease activity both in clinical patients and murine model . gain- and loss - of - function analysis revealed that hmgb1 aggravated the severity of sle , which might be due to its effect on macrophage inflammatory response . the diagnosis of sle was established according to the four of the american college of rheumatology ( acr ) revised criteria for the diagnosis of sle . this study was strictly carried out according to the guide for the care and use of medical laboratory animals ( ministry of health , china , 1998 ) and with the ethical approval of the shanghai medical laboratory animal care and use committee as well as the ethical committee of fudan university . all surgery was performed under sodium pentobarbital anesthesia , and all animal procedures in this study were strictly performed in a manner to minimize suffering of laboratory mice . to generate the murine model of sle , six - week - old female balb / c mice were immunized s.c . to investigate the effect of hmgb1 in the pathogenic process of sle mice were divided into six groups as follows : pbs plus vector , un - ald - dna plus vector , ald - dna plus vector , pbs plus phmgb1 , un - ald - dna plus phmgb1 and ald - dna plus phmgb1 . to examine the potential role of hmgb1 in sle , 8 mice in each group were intramuscularly injected with 100 g of phmgb1 or empty vector per mouse 72 h earlier before injection with ald - dna . to block the function of hmgb1 in sle mice , mice were randomized to intramuscularly injection 0.5 mg per mouse glycyrrhizin every day for 2 months . proteinuria of the mice was measured with the bca method ( thermo fisher scientific ) as previously described [ 3339 ] . for histology analysis , murine renal tissues were surgically resected and fixed in 4% paraformaldehyde ( sigma - aldrich ) , processed , and embedded in paraffin . h&e staining of renal tissue sections were performed according to the manufacturer 's instructions and assessed by two pathologists blinded to treatment group as previously described [ 3339 ] . to assess the levels of hmgb1 , anti - dsdna antibody , tnf- , and il-6 in the plasma and supernatant of cell culture , elisa ( shino - test , sagamihara - shi , kanagawa , japan for hmgb1 ; ebioscience for tnf- and il-6 ; alpha diagnostic international for anti - dsdna ) was performed according to the manufacturer 's instructions . to investigate whether hmgb1 was involved in the pathogenesis of sle , we first examined the levels of hmgb1 in sle patients . we determined the serum concentrations of hmgb1 in sle patients and healthy controls ( hc ) by elisa . furthermore , serum hmgb1 levels in sle patients with active renal disease ( 42.5672 21.0052 ng / ml ) were significantly higher than those in patients without active renal disease ( 15.7279 8.8412 ng / ml , p < 0.05 , figure 1(b ) ) . moreover , we found that serum hmgb1 concentrations showed a highly significant correlation with sle disease activity index score ( sledai ) ( r = 0.4715 , p = 0.0064 , figure 1(c ) ) and anti - dsdna antibody levels ( r = 0.6257 , p = 0.0001 , figure 1(d ) ) . we also analyzed the expression of hmgb1 in peripheral blood mononuclear cells ( pbmcs ) from sle patients ( s1 , s2 , s3 , and s4 ) and hc ( h1 , h2 , h3 , and h4 ) using western blot . as shown in figure 1(e ) , the expression of hmgb1 was increased in pbmcs from sle patient compared to hc . we further analyzed the serum levels of hmgb1 in murine model of sle which were generated according to our previously reported procedures [ 3339 ] . of interest , we found that serum hmgb1 levels were significantly increased in murine model of sle compared with those in control mice ( figure 1(f ) ) . pearson correlation analysis showed that the serum hmgb1 levels were positively correlated with kidney score ( r = 0.6583 , p = 0.0022 , figure 1(g ) ) , indicating that hmgb1 levels were significantly associated with the severity of lupus nephritis . similarly , we observed that serum hmgb1 levels were related with the levels of anti - dsdna antibody ( r = 0.7278 , p = 0.0004 , figure 1(h ) ) and urine protein ( r = 0.6652 , p = 0.0019 , figure 1(i ) ) in ald - dna - induced murine model of sle . taken together , these data indicated that hmgb1 expression was upregulated and correlated with the severity of sle both in clinical patients and murine model . above data showed that hmgb1 levels were elevated and correlated with the severity of sle . to further evaluate whether hmgb1 was involved in the pathogenesis of sle , we upregulated the expression of hmgb1 by injecting balb / c mice intramuscularly with a hmgb1 overexpression plasmid ( phmgb1 ) . results showed that the injection of phmgb1 led to the elevation of serum hmgb1 levels in sle mice ( figure 2(a ) ) . to investigate the effect of increased hmgb1 levels on the progression of sle , we analyzed anti - dsdna antibody , proteinuria , renal pathology , and kidney score in phmgb1- or empty vector - treated mice . results showed that hmgb1 overexpression exacerbated renal pathology as revealed by the increased infiltration of proinflammatory cells into glomerular mesanguim and thickened basement membrane , as well as the atrophy of glomerular mesanguim ( figure 2(b ) ) , and upregulated the kidney score of sle mice ( figure 2(c ) ) . we also found that the levels of anti - dsdna antibody ( figure 2(d ) ) and urine protein ( figure 2(e ) ) in sle mice treated with phmgb1 were notably elevated compared with that in empty vector - treated sle mice . to further confirm the effect of hmgb1 on the progression of sle , we inhibited the function of hmgb1 in vivo by injecting balb / c mice intramuscularly with glycyrrhizin which has been demonstrated to be the blocker of hmgb1 [ 4143 ] . of note , glycyrrhizin treatment was found to efficiently ameliorate renal pathology as demonstrated by decreased infiltration of proinflammatory cells into glomerular mesanguim , recovery from thicken basement membrane and the atrophic glomerular mesanguim ( figure 3(b ) ) , and decrease the kidney score in sle mice ( figure 3(c ) ) . furthermore , we revealed that glycyrrhizin treatment reduced levels of anti - dsdna antibody ( figure 3(d ) ) and urine protein ( figure 3(e ) ) in sle mice . taken together , these data demonstrated that hmgb1 played a crucial role in modulating the severity of sle . to study whether hmgb1 was involved in macrophage inflammatory response , we detected the production of hmgb1 in raw264.7 cells stimulated with ald - dna . the results showed that the ald - dna administration led to the upregulation of hmgb1 levels in raw264.7 cells ( figures 4(a ) and 4(b ) ) . we then investigated the role of hmgb1 in ald - dna - induced macrophage inflammatory response by transfecting phmgb1 into raw264.7 cells . results showed that transfection of phmgb1 notably increased hmgb1 levels in raw264.7 cells ( figure 4(c ) ) . of importance , hmgb1 overexpression in raw264.7 cells was found to aggravate the secretion of tnf- ( figure 4(e ) ) and il-6 ( figure 4(f ) ) induced by ald - dna . western blot showed that transfection of sihmgb1 efficiently decreased hmgb1 levels in raw264.7 cells ( figure 4(d ) ) . we stimulated sihmgb1-treated raw264.7 cells with ald - dna , followed by detecting the concentrations of proinflammatory cytokines in the supernatants . results showed that the sihmgb1-mediated downregulation of hmgb1 levels significantly inhibited the secretion of tnf- ( figure 4(g ) ) and il-6 ( figure 4(h ) ) in ald - dna - stimulated macrophages . moreover , we isolated cd11b / f4/80 renal macrophages from phmgb1- or empty vector - treated sle mice , glycyrrhizin- or pbs - treated sle mice and stimulated these cells with ald - dna , followed by detecting the production of tnf- and il-6 . as shown in figure 4(i ) , renal macrophages secreted much higher levels of tnf- and il-6 in phmgb1-treated sle mice than those from empty vector - treated sle mice , whereas renal macrophages from glycyrrhizin - treated sle mice secreted lower levels of tnf- and il-6 than those pbs - treated sle mice ( figure 4(j ) ) . to study the relationship between hmgb1 and proinflammatory cytokines , we analyzed the correlation between hmgb1 and proinflammatory cytokines ( tnf- and il-6 ) in sle patients . we first detected the levels of serum tnf- and il-6 in sle patients , and the results demonstrated that the concentrations of tnf- ( figure 5(a ) ) and il-6 ( figure 5(b ) ) in sle patients were significantly higher than those in hc ( p < 0.05 ) . further study was conducted to analyze the correlation between hmgb1 and proinflammatory cytokines ( tnf- and il-6 ) . we observed that hmgb1 levels were associated with tnf- ( r = 0.3559 , p = 0.0456 , figure 5(c ) ) and il-6 levels ( r = 0.3597 , p = 0.0432 , figure 5(d ) ) in sle patients . taken together , these data indicated that hmgb1 was pivotal for ald - dna - induced macrophage inflammatory response and correlated with the levels of tnf- and il-6 in sle . previous studies indicate that hmgb1 is an endogenous ligand of rage , tlr2 , and tlr4 [ 5052 ] . to evaluate which receptor might be involved in the hmgb1-mediated inflammatory effect in ald - dna - stimulated macrophages , we first upregulated hmgb1 expression in raw264.7 cells and then stimulated these cells with ald - dna in the presence of tlr2/4 inhibitor ( oxpapc ) or rage inhibitor ( rage - fc ) . the results showed that the production of tnf- ( figure 6(a ) ) and il-6 ( figure 6(b ) ) from raw264.7 cells exposed to tlr2/4 inhibitor was not impaired in the process of hmgb1-enhanced macrophage inflammatory response , whereas blocking the function of rage with rage - fc effectively reduced the secretion of tnf- ( figure 6(a ) ) and il-6 ( figure 6(b ) ) . to further validate the importance of rage receptor , we downregulated the expression of rage by sirna and then evaluated the effect of hmgb1 on the production of proinflammatory cytokines in ald - dna - stimulated raw264.7 cells . of importance , it was found that sirna - mediated downregulation of rage significantly inhibited hmgb1-enhanced release of tnf- ( figure 6(d ) ) and il-6 ( figure 6(e ) ) in ald - dna - stimulated macrophages . collectively , these data indicated that rage might be critical for proinflammatory signaling during the process of hmgb1-aggravated macrophages inflammatory response induced by ald - dna , whereas tlr2 and tlr4 appeared to be dispensable . recent reports show that some multifunctional proteins such as hmgb1 might be involved in autoimmune and inflammatory diseases including sle [ 31 , 5255 ] . our previous study revealed that hmgb1 was required for autoantibody induction by dna - containing immune complexes in sle patients , implying that the critical role of hmgb1 in the pathogenesis of sle . in the present investigation , we further explored the role and mechanism of hmgb1 in the pathogenesis of sle . our results demonstrated that hmgb1 levels were increased and correlated with the severity of sle in both clinical patients and murine model , consistent with previous findings [ 5760 ] . to evaluate whether hmgb1 was involved in the pathogenesis of sle , we overexpressed hmgb1 in ald - dna - immunized mice and found that hmgb1 overexpression significantly enhanced the severity of sle . to further confirm the role of hmgb1 in sle glycyrrhizin is commonly used in clinical application for its anti - inflammatory activity , and it binds to hmgb1 directly , thereby inhibiting the extracellular hmgb1 secretion . our results showed that blockade of hmgb1 function by glycyrrhizin led to dramatic downregulation of serum hmgb1 levels , and thus decreased the severity of sle . these data suggested that hmgb1 played a crucial role in the pathogenesis of sle , implicating a promising hmgb1-based therapeutic strategy against sle . the increasing number of evidence has emerged to suggest the crucial role of proinflammatory cytokines in the pathogenesis of sle . considering the importance of hmgb1 in the regulation of inflammatory response , we analyzed the correlation between hmgb1 and proinflammatory cytokines expression in sle . our studies found that the concentrations of tnf- and il-6 in sle patients were significantly higher than those in hc , consistent with previous study . further studies showed that hmgb1 levels were correlated with the levels of tnf- and il-6 in sle patients . moreover , our data found that hmgb1 promoted inflammatory response of renal macrophages in sle mice . these results suggested that hmgb1 might be involved in the pathogenesis of sle via regulating macrophage inflammatory response , however , the definite relationship between hmgb1 and macrophage inflammatory response needs further investigation . accumulating data demonstrate that activated macrophages that infiltrate kidneys mediate the onset of an aggressive adaptive immune response leading to the pathogenesis of sle in mice [ 6570 ] . these reports give a clue that macrophages play a crucial pathogenic role in the development of sle . our previous study has also indicated that ald - dna immunization lead to macrophage infiltration and aberrant activation , which mediate the onset and aggravation of sle , indicating that aberrant activation of macrophage plays a crucial pathogenic role in ald - dna - induced sle [ 3339 ] . here , we found that hmgb1 enhanced ald - dna - induced macrophage inflammatory response both in vivo and in vitro . our results suggested that rage might be critical for proinflammatory signaling during the process of hmgb1-aggravated macrophage inflammatory response induced by ald - dna , whereas tlr2 and tlr4 seemed to be dispensable . in summary , our research reported that hmgb1 levels were significantly increased and correlated with sle disease activity in both clinical patients and murine model . further study suggested that hmgb1 aggravated the severity of sle via facilitating macrophage inflammatory response . moreover , rage might be critical for proinflammatory signaling during the process of hmgb1-aggravated macrophages inflammatory response .

tissue isolation tissues were collected for research purposes with approval from the human research ethics committees at the royal women 's hospital ( 03/51 ) , mercy hospital for women ( r07/15 ) , and monash medical centre ( 07084c ) . for human placental tissue sampling , a core of full - thickness tissue was isolated from two randomly chosen sites . tissue sections from other species were washed briefly in saline before processing by maceration with a scalpel blade prior to immediate dna isolation . 39 , and purity of preparations was determined using antibodies to the trophoblast - specific cytokeratin-7 ( 1:100 dilution , clone ov - tl 12/30 ; dakocytomation , glostrup , denmark ) using immunocytochemistry . cells were fixed in ethanol , rehydrated , and treated with 0.3% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase activity . cells were then washed with tris - buffered saline and incubated with nonimmune block for 30 min . primary antibody was applied and incubated at 48 c for 16 h followed by biotinylated horse anti - mouse igg ( 1:200 ) for 30 min and then streptavidinbiotin - peroxidase complex abc ( dakocytomation ) according to the manufacturer 's instructions . peroxidase activity was visualized by application of diaminobenzidine substrate ( dakocytomation ) for 35 min . cells were counterstained with harris hematoxylin ( sigma ) , air - dried , and mounted . only cell preparations in which 95% of the cells were positive for cytokeratin-7 were used for subsequent experiments . for cvs samples ( 810 weeks of gestation ) , contaminating maternal blood and decidua were removed , and 510 mg of cleaned villi was separated from the diagnostic sample , washed in isotonic saline , pelleted by centrifugation , and stored at 20 c until dna extraction and bisulfite modification . a cell culture from each villi sample was established by finely macerating the villi and resuspending in 1.0 ml of 0.25% trypsin with incubation of the suspension at 37 c for 45 min . cells were then resuspended in 7 ml of rpmi 1640 medium ( safc biosciences ) , pelleted at 560 g , and resuspended in a small volume of amniomax medium ( invitrogen ) containing 0.5 g / ml amphotericin b. the cell suspension was then inoculated onto glass coverslips in 35-mm petri dishes with subsequent incubation at 37 c in 5% co2 in humidified air . wells were then flooded with 2 ml of amniomax medium the following day and were grown to around 95% confluence , trypsinized , pelleted , and stored at 20 c until dna extraction and bisulfite modification . cultured cells were isolated following 1418 days in culture with a maximum of three passages . jeg-3 cells ( htb-36 ( 40 ) ) , bewo ( ccl-98 ( 41 ) ) , and jar cells ( htb-144 ( 42 ) ) , were maintained in rpmi plus 10% fetal calf serum at 37 c in 5% co2 . the sv40-transformed trophoblast hipec-65 cell line was maintained as previously described ( 43 ) . cells were grown for a minimal number of passages ( < 8 ) and were split ( 1:51:8 ) prior to confluence and harvesting of genomic dna or transfection analysis . figure 1.methylation status of cyp24a1 , cyp27b1 , and vdr in human term placenta . schematic representation of methylation data generated for regulatory regions of genes coding for 24-hydroxylase ( a ) , 1-hydroxylase ( b ) , and the vitamin d receptor ( c ) . gray box , cpg island ; black boxes , exon regions ; arrow , translational start site . ii , mean methylation levels seen at each cpg site for each type of sample tested . numbers of each type of tissue are listed in parentheses . between 8 and 12 individual clones schematic representation of methylation data generated for regulatory regions of genes coding for 24-hydroxylase ( a ) , 1-hydroxylase ( b ) , and the vitamin d receptor ( c ) . gray box , cpg island ; black boxes , exon regions ; arrow , translational start site . ii , mean methylation levels seen at each cpg site for each type of sample tested . numbers of each type of tissue are listed in parentheses . between 8 and 12 individual clones , a total of 8 10 jar or hipec65 cells were plated 24 h prior to transfection in each well of a 96-well plate ( 96f nunclon delta white microwell ) . luciferase and renilla activity was measured 24 h post - transfection using the dualglo assay system ( promega , madison , wi ) on a fluorstar optima microplate reader ( bmg labtech , offenburg , germany ) . for vitamin d induction experiments , cells were treated with 10 or 100 nm 1,25-(oh)2d ( sigma ) 12 h post - transfection , with luciferase activity monitored after overnight incubation . genomic dna isolation and bisulfite dna sequencing was performed as previously described ( 37 , 38 ) . dna samples were processed using the methyl easy bisulfite modification kit ( human genetic signatures , sydney , australia ) kit according to the manufacturer 's instructions . amplification was performed on converted genomic dna using primers directed to bisulfite modified dna : vdrbisf1 , 5-aatattttttgttctttaagtgttaagta ; vdrbisf2 , 5-taaattttagtgttttttagtgttttagtt ; vdrbisr1 , 5-ctaacctaatcaacccaaataaaaata ; cyp24bisf1 , 5-ggaaagggggttttaagaaatagta ; cyp24bisr1 , 5-acttccaaccccaaaaaactctaac ; cyp24bisf2 , 5-tggttttagttagattttagaggttagttt ; cyp24bisr2 , 5-tactatttcttaaaaccccctttcc ; cyp24bisf3 , 5-gagtatgttttgggtggttaatgag ; cyp24bisr3 , 5-tccaaactaaaaatatctaactcccc ; cyp27bisf1 , 5-ttttggttgttttatatttgttttg ; cyp27bisr1 , 5-aaccataacccaaaccctcaaata . amplification conditions were as follows : 95 c for 5 min , 56 c for 1 min 30 s , and 72 c for 1 min 30 s 40 cycles , 72 c for 7 min . resulting amplicons were cloned into topo ta cloning vector ( invitrogen ) for automated fluorescent sequencing as described in ref . data were analyzed using biq analyser software ( 46 ) , and clones showing less than 80% conversion or that were identified as clonal in origin were not included in subsequent analysis . mean methylation levels for each amplicon were determined using the formula ( number of methylated cpg sites / total cpg sites examined 100 ) . cyp24a1 gene reporter assays a 758-bp region of the promoter / exon 1 of the human cyp24a1 gene previously shown to confer vitamin d responsiveness ( 47 ) was amplified using primers ; sal_cyp24_548f ( 5-ctctctgtcgacttggagctccgcagaaagccaaacttcct ) and sali_cyp24_+209r ( 5-ctctctgtcgacgtactcgagacagcctcagagcattggtg ) with phusion hi - fidelity dna polymerase ( finnzymes ) . the sali - digested amplicon was cloned into a promoterless luciferase expression vector pgl3:basic ( promega , madison , wi ) to produce the plasmid pgl3:594/+209 . reverse orientation cloning was also carried out to produce pgl:+209/594 . following sequence verification , dna was in vitro methylated using either sss1 , hpaii , or hhai methylases ( new england biolabs , ipswich , ma ) according to the manufacturer 's instructions . 100 ng of pgl3:control vector ( sv40 constitutive promoter ) , pgl3:basic ( promoterless ) , or unmethylated or methylated cyp24a1 promoter constructs were then transfected into jar and hipec65 cells . co - transfection with 10 ng of prn ( hsv - tk promoter linked to the renilla luciferase gene ) was carried out in all cases to normalize for transfection efficiency . gene induction experiments24 h prior to induction , cvs - derived cells , skin , and placental - derived fibroblasts were seeded in a 6-well plate at a density of 250,000 cells / well . half of the wells were then treated with 100 nm calcitriol ( sigma ) , and the other half were left untreated . cells were then incubated for 7 h prior to total rna isolation using trizol ( invitrogen ) according to the manufacturer 's instructions . cdna synthesis was performed with the superscript iii kit ( invitrogen ) , from 1 g of total rna . quantitative reverse transcription - pcr was performed in a 96-well optical plate ( applied biosystems , foster city , ca ) on a 7300 abi thermal cycler ( applied biosystems ) . the master mix consisted of 15 l of sybr green master mix ( sigma ) , 1 l of forward primer , 1 l of reverse primer ( 50 pm final concentration ) , 8 l of water , 5 l of cdna template in a total volume of 30 l . nondiluted cdna was used for cyp24a1 , cyp27b1 , and vdr assays , and a 1:10 dilution was used for the gapdh ( glyceraldehyde-3-phosphate dehydrogenase ) assay . primer sequences were as follows : cyp24a1_f , 5-cccaaaggaacagtgctcatg ; cyp24a1_r , 5-tctcctgaagccaacgttcag ; cyp27b1_f , 5-atgaggaacggaggacagcc ; cyp27b1_r , 5-gagcgtgttggacaccgtgt ; vdr_f , 5-ttgcgctccaatgagtccttc ; vdr_r , 5-ctgtgtccggctttggtcac ; gapdh_f , 5-ttcgacagtcagccgcatctt ; gapdh_r , 5-cccaatacgaccaaatccgtt . the cyp24a1 but not the vdr or cyp27b1 gene promoter is methylated in full term human placental tissue as a first step in the identification of potential epigenetic regulation of vitamin d levels in the placenta , we designed specific methylation amplification assays for regulatory regions of the vdr , cyp24a1 , and cyp27b1 genes . in the case of the vdr and cyp24a1 genes , assays were designed to span the transcription start site and exon 1 within the associated cpg island , whereas for cyp27b1 , we analyzed a region of exon 1 previously shown to be methylated in some cancers ( 48 , 49 ) . initial experiments on human term placenta revealed a complete lack of methylation at the vdr and cyp27b1 gene regulatory regions , with clear ( but variable ) methylation of the cyp24a1 promoter region ( fig . 1 and table 1 ) . this was not due to problems with specific assays , since methylation of both vdr and cyp27b1 were detected in choriocarcinoma cells ( see below ) . three independent assays spanning the cyp24a1-associated cpg island were then examined with each showing evidence of methylation in full term placenta ( supplemental fig . a total of 10 term placentas were then examined using the cyp24a1 - 2 assay spanning a region of differential methylation recently identified in the placenta by whole genome methylation data ( 50 ) . these results confirmed methylation of the cyp24a1 cpg island in all full term placental samples , with a mean methylation level of 56.5% ( table 1 ) . whereas some cyp24a1 alleles showed nearly complete methylation , others were almost completely unmethylated . this is reminiscent of monoallelic methylation , previously described for the apc tumor suppressor gene in the placenta ( 37 ) , but may equally reflect differing methylation profiles in a mixed population of cells . table 1mean promoter methylation levels of cyp24a1 , cyp27b1 , and vdr genes in human placental samples and cell linesa summary of cpg island methylation status for genes regulating vitamin d homeostasis and activity was determined by bisulfite sequencing . placenta , full term placental tissue ; 1st trimester ct , purified cytotrophoblasts showing > 95% ck-7 positive staining ; terminated pregnancy , placental tissue at 912 weeks gestation ; pre - eclampsia , diseased placental tissue at 912 weeks gestation ; choriocarcinoma , cell lines ( bewo , jeg-3 , and/or jar ) . placental fibroblasts were isolated following serial passaging of percoll gradient - separated cell fractions . normal tissue data consist of pooled data across a variety of different human tissues isolated from different subjects . the number of each type of cell / tissue is shown in parentheses.genetissuemean methylations . d.%cyp24a1 placenta ( n = 8) 56.53 10.49 uncultured cvs ( n = 4 ) 33.07 9.31 cultured cvs ( n = 4 ) 33.79 19.02 first trimester ct ( n = 4 ) 48.17 24.49 terminated pregnancy ( n = 3 ) 48.62 19.18 pre - eclampsia ( n = 3 ) 39.85 6.32 choriocarcinoma ( n = 3 ) 80.40 10.47 placenta fibroblasts ( n = 3 ) 2.95 2.28 decidual cells ( n = 4 ) 1.57 0.57 hipec65 ( n = 2 ) 2.03 0.96 nonplacental tissue ( n = 10 ) 1.56 1.12 cyp27b1 placenta ( n = 4 ) 5.23 4.88 choriocarcinoma ( n = 2 ) 86.63 1.51 vdr placenta ( n = 2 ) 0.65 0.14 choriocarcinoma ( n = 3 ) 85.84 11.61 mean promoter methylation levels of cyp24a1 , cyp27b1 , and vdr genes in human placental samples and cell lines a summary of cpg island methylation status for genes regulating vitamin d homeostasis and activity was determined by bisulfite sequencing . placenta , full term placental tissue ; 1st trimester ct , purified cytotrophoblasts showing > 95% ck-7 positive staining ; terminated pregnancy , placental tissue at 912 weeks gestation ; pre - eclampsia , diseased placental tissue at 912 weeks gestation ; choriocarcinoma , cell lines ( bewo , jeg-3 , and/or jar ) . placental fibroblasts were isolated following serial passaging of percoll gradient - separated cell fractions . normal tissue data consist of pooled data across a variety of different human tissues isolated from different subjects . cyp24a1 placental methylation in multiple first trimester placental cell types in order to ascertain the cell types and timing of methylation of the cyp24a1 promoter within the placenta , we examined the methylation status of chorionic villous tissue ( pre- and post - cell culturing ) , first trimester termination placental tissue , purified first trimester cytotrophoblasts ( > 90% cytokeratin-7-positive ) , and full term placental fibroblasts . promoter methylation comparable with that seen in full term placental tissue was observed both in primary and cultured cvs tissue samples ( fig . 2 and table 1 ) and in first trimester termination tissue ( table 1 ) . since the cvs culturing conditions do not favor trophoblast growth or division , the methylation of cyp24a1 seen in the cultured samples is probably mesodermal in origin . in addition , purified first trimester cytotrophoblasts also show methylation of the cyp24a1 gene ( fig . 2 ) , demonstrating that this methylation is present in multiple cell types in first trimester placenta . no methylation was detected in purified full term placental fibroblasts or an sv40-transformed first trimester trophoblast cell line ( fig . a summary of mean methylation levels obtained in this study is presented in table 1 . representative methylation data generated for the cyp24a1 - 2 assay in cytokeratin-7 positive cytotrophoblasts ( a ) , uncultured cvs ( b ) , cultured cvs ( c ) , full term placental fibroblasts ( d ) , and whole blood ( e ) . gray boxes , to gpg island locations ; arrows , start site of translation within exon 1 ( black line ) . ii , mean methylation levels seen at each cpg site for each type of sample tested . numbers of each type of tissue are listed in parentheses . representative methylation data generated for the cyp24a1 - 2 assay in cytokeratin-7 positive cytotrophoblasts ( a ) , uncultured cvs ( b ) , cultured cvs ( c ) , full term placental fibroblasts ( d ) , and whole blood ( e ) . circles , cpg sites denoted by vertical dashes . closed circles , methylation ; open circles , gray boxes , to gpg island locations ; arrows , start site of translation within exon 1 ( black line ) . ii , mean methylation levels seen at each cpg site for each type of sample tested . numbers of each type of tissue are listed in parentheses . cyp24a1 methylation is placenta - specific given the importance of the 24-hydroxylase enzyme in regulating levels of active vitamin d , it is surprising how little is known about the epigenetic regulation of this gene in human tissues . in order to address this , we assessed the methylation status of the cyp24a1 gene in a panel of human tissues consisting of kidney , skeletal muscle , skin fibroblasts , brain ( prefrontal cortex ) , sperm , whole blood , buccal mucosa , endometrial stroma , decidualized stroma , bone marrow , and umbilical cord tissue . the results , ( summarized in fig . 2 and table 1 ; detailed in supplemental fig . 2 ) , demonstrate the specificity of placental cyp24a1 methylation in humans , with no evidence for methylation in any human somatic tissue tested . the cyp24a1 gene is differentially methylated in full term placentas from different mammals in previous studies , we have demonstrated that some of the specific promoter methylation recently documented in human term placental tissue is present in primates but absent in e18 ( day 18 of pregnancy ) mouse placenta . we therefore examined methylation of the cyp24a1 gene promoter regions in full term placenta from marmoset and mouse e18 and e11.5 ( fig . assays were designed to span the region corresponding to the human cyp24a1 - 2 assay . this revealed varying levels of cyp24a1 methylation in both marmoset ( mean 27 18.7% ) and mouse ( mean 35.1 4.5% ) placental tissue with generally lower levels relative to that seen in human full term placentas ( mean 56.5 10.5% ; fig . 3 ) interestingly , less methylation was seen in e18 mouse placental tissue compared with e11.5 tissue ( data not shown ) . representative methylation data were generated for homologous cyp24a1 promoter region in marmoset ( a ) and mouse ( b ) . i , gray boxes , gpg island locations ; arrows , orientation of transcription . circles , cpg sites denoted by black dashes . closed circles , methylation ; open circles , lack of methylation . shaded bars , mean methylation levels seen at each cpg site for each type of sample tested . representative methylation data were generated for homologous cyp24a1 promoter region in marmoset ( a ) and mouse ( b ) . i , gray boxes , gpg island locations ; arrows , orientation of transcription . circles , cpg sites denoted by black dashes . closed circles , methylation ; open circles , lack of methylation . shaded bars , mean methylation levels seen at each cpg site for each type of sample tested . methylation of the core cyp24a1 gene promoter directly attenuates basal promoter activity in human trophoblast - derived cells despite circumstantial evidence of an inverse correlation of cyp24a1 mrna levels with promoter gene methylation ( above ) , no direct evidence for dna methylation - mediated silencing of this gene promoter has been reported . to investigate this further , we pcr - amplified the previously described vitamin d - responsive region of the human cyp24a1 gene promoter ( 47 ) and generated a luciferase reporter construct to directly test the effects of promoter methylation on both basal and vitamin d - induced promoter activity . the sv40-transformed human trophoblast cell line , hipec65 , and choriocarcinoma - derived jar cells were transfected with two different reporter constructs ( plus and minus orientation ) with or without in vitro methylation with sssi or hpaii methylases . sssi methylase methylates all cpgs ; hpaii methylase methylates only the cpg within the sequence ccgg . 4 , reveal a low level of promoter activity of this promoter region in the plus orientation in both cell lines , 6.5-fold ( hipec65 ; p = 0.028 ) and 12-fold ( jar ; p < 0.001 ) higher than that of the promoterless luciferase vector . promoter activity in the reverse ( minus ) orientation was negligible ( 1.3-fold ) , demonstrating a directionality of the basal promoter element ( data not shown ) . basal activity was completely attenuated by prior methylation with any of sssi ( p = 0.011 ) , hpaii ( p = 0.01 ) , or hhai ( p = 0.014 ) methylases in hipec65 cells and inhibited in the jar cell line ( p < 0.001 for each of sssi and hpaii ; supplemental table 1 ) . the 768-bp region of this gene used for reporter activity assays contains a total of 5 hhai and 15 hpaii methylase target sites , suggesting that even low level methylation of this promoter is sufficient to abrogate gene expression activity . cpg island methylation abrogates 1,25-(oh)2d - mediated up - regulation of cyp24a1 transcription in addition to examining the effects of promoter methylation on the basal cyp24a1 promoter activity , we also set out to investigate the consequences of this methylation on the previously demonstrated inducibility of this gene promoter by 1,25-(oh)2d ( 47 ) . jar and hipec65 cells were transfected with the 768-bp core promoter with or without previous methylation with sss1 methylase as above . 10 h post - transfection , one - half of cells were supplemented with 100 nm 1,25-(oh)2d , and reporter ( luciferase ) activity was then monitored . although 1,25-(oh)2d treatment resulted in a clear induction ( 3.5-fold ) of basal promoter activity in hipec65 cells in the absence of previous promoter methylation ( p = 0.008 ) , this response was abolished with in vitro methylation of the reporter construct prior to transfection ( fig . 4 ) . 1,25-(oh)2d treatment had no effect on promoter activity in jar cells ( p = 0.663 ) , most likely reflecting the methylation of the vdr gene in this cell line ( see below ) that silences the gene and inhibits exogenous 1,25-(oh)2d signaling through the vdr . sv40-transformed cytotrophoblast ( hipec-65 ) or choriocarcinoma ( jar ) cells were transfected ( n = 8 for each cell line ) with a promoterless luciferase reporter ( vector ) with or without the cloned cyp24a1 gene promoter ( 548/+209 relative to the start site of transcription ) . this region has previously been demonstrated to contain vitamin d - responsive elements ( 47 ) . basal promoter activity was detected in both cell lines and was abolished with in vitro methylation of the promoter prior to transfection ( sss1 , hpaii , and hhai methylases ) . 1,25-(oh)2d ( active vitamin d ) induction of the promoter was observed only in hipec-65 cells ( jar cells are predicted to lack the vitamin d receptor , as evidenced by complete methylation of the associated cpg island ) , but this induction was attenuated with prior methylation of the promoter region . luminescence values were normalized to renilla luciferase to correct for transfection efficiency , and all data are displayed relative to the mean promoterless control vector in hipec-65 cells . error bars denote 95% confidence intervals . statistical analysis for comparing between transfected groups was carried out using a two - tailed student 's t test with unequal variance . sv40-transformed cytotrophoblast ( hipec-65 ) or choriocarcinoma ( jar ) cells were transfected ( n = 8 for each cell line ) with a promoterless luciferase reporter ( vector ) with or without the cloned cyp24a1 gene promoter ( 548/+209 relative to the start site of transcription ) . this region has previously been demonstrated to contain vitamin d - responsive elements ( 47 ) . basal promoter activity was detected in both cell lines and was abolished with in vitro methylation of the promoter prior to transfection ( sss1 , hpaii , and hhai methylases ) . 1,25-(oh)2d ( active vitamin d ) induction of the promoter was observed only in hipec-65 cells ( jar cells are predicted to lack the vitamin d receptor , as evidenced by complete methylation of the associated cpg island ) , but this induction was attenuated with prior methylation of the promoter region . luminescence values were normalized to renilla luciferase to correct for transfection efficiency , and all data are displayed relative to the mean promoterless control vector in hipec-65 cells . error bars denote 95% confidence intervals . statistical analysis for comparing between transfected groups was carried out using a two - tailed student 's t test with unequal variance . we also examined the effect of promoter methylation on the induction of endogenous cyp24a1 , cyp27b1 , and vdr gene expression in primary cells derived from cvs tissue ( average methylation level of 33.4% ) and placental and skin fibroblasts ( both lacking cyp24a1 methylation ) . cultured skin fibroblasts showed a mean cyp24a1 induction level approaching 2000-fold ( n = 2 ; fig . 5 ) , consistent with previous data ( 47 , 51 , 52 ) . placental fibroblasts also showed a significant level of induction ( mean 172-fold ; n = 2 ) . in contrast , cultured cvs cells showed a reduced capacity for gene induction ( mean 10 fold ; n = 4 ) . in contrast to the major effects of vitamin d induction on cyp24a1 expression in skin and placental fibroblasts , vdr expression levels were slightly reduced ( mean reduction of 3-fold ) in skin fibroblasts , with little change apparent in placental fibroblasts ( mean 1.18-fold increase ) and cvs - derived cells ( mean 0.76-fold change ; fig . similarly , no major effects on cyp27b1 expression level were seen in skin fibroblasts ( mean 1.19-fold increase ) or cvs - derived cells ( mean 1.55-fold increase ) following vitamin d induction ( fig . 5 ) . altered cyp24a1 methylation in placental tissue from pre - eclampsia pregnancies ? we carried out a preliminary analysis of methylation levels of the cyp24a1 - 2 assay region in a small set of available preeclampsia tissue ( table 1 ) . these samples showed a mean methylation level of only 39.5 6% ( n = 3 ) , compared with the 56.53 10% level seen in nonpre - eclampsia placental tissue ( n = 8) . the potential significance ( p = 0.017 ) of these data will require further investigation in a larger sample size in the future . trophoblast hyperplasia is a common feature of complete and partial hydatidiform mole with the potential for malignant transformation to choriocarcinoma ( cca ) ( 53 ) . trophoblasts that comprise cca resemble the primitive trophoblast of the previllous stage during placental development , arrested in specific stages of differentiation ( 54 ) . we analyzed the methylation status of cyp24a1 in three widely studied cca cell lines , jeg-3 , bewo , and jar . in all three lines , methylation at the cyp24a1 promoter was increased to nearly complete methylation ( fig . these results correlate with the very low levels of cyp24a1 mrna seen in each of these cell types ( data not shown ) . we also assessed methylation at the vdr and cyp27b1 cpg islands , which also revealed nearly complete methylation of each of these genes in all cell lines tested . this contrasts with the complete lack of methylation seen in full term placental tissue and first trimester cytotrophoblast ( described above ) . placental insufficiency , due to inadequate remodeling of the maternal vasculature in early pregnancy , is thought to play a major role in several adverse pregnancy outcomes , including the early stages of pre - eclampsia ( 55 ) . lowered 1,25-(oh)2d and altered calcium metabolism for example , a recent cochrane review of randomized control trials demonstrated a role for calcium supplementation in reducing pre - eclampsia risk.5 altered expression levels of 1-hydroxylase , vdr , and 24-hydroxylase genes have all been reported in different placental cells and tissue from preeclamptic pregnancies , possibly in response to decreasing maternal 1,25-(oh)2d ( 19 , 57 , 58 ) . in addition , levels of cyp27b1 mrna are reduced in cultured syncytial trophoblasts derived from pre - eclamptic patients compared with controls ( 57 ) . these data highlight the likely functional importance of 1,25-(oh)2d in placental function and implicate disruption of vitamin d homeostasis in adverse pregnancy outcomes . cells were cultured in multiple wells , and 100 nm 1,25-(oh)2d3 ( active vitamin d ) was added to half of the cultures for 7 h , followed by total rna isolation and quantitative real time reverse transcription - pcr . mean levels of induction ( n = 24 ) are listed in the corresponding table . whereas cultured skin fibroblasts show a nearly 2000-fold induction of cyp24a1 expression , consistent with previous data ( 47 , 51 , 52 ) , and placental fibroblasts show a mean 172-fold induction , cultured cvs biopsies ( containing cells with cyp24a1 methylation ) show a greatly reduced level of gene induction ( 10-fold ) . no major effects on vdr or cyp27b1 expression were observed . cells were cultured in multiple wells , and 100 nm 1,25-(oh)2d3 ( active vitamin d ) was added to half of the cultures for 7 h , followed by total rna isolation and quantitative real time reverse transcription - pcr . mean levels of induction ( n = 24 ) are listed in the corresponding table . whereas cultured skin fibroblasts show a nearly 2000-fold induction of cyp24a1 expression , consistent with previous data ( 47 , 51 , 52 ) , and placental fibroblasts show a mean 172-fold induction , cultured cvs biopsies ( containing cells with cyp24a1 methylation ) show a greatly reduced level of gene induction ( 10-fold ) . no major effects on vdr or cyp27b1 expression were observed . epigenetic regulation of steroid signaling is an emerging field of research and hints at a complex interplay between environmental or dietary influences with underlying genetic variation , epigenetic variation , steroid metabolism , and subsequent downstream effects ( 59 ) . however , at present , very little is known about the consequences of altered environmental , genetic , or epigenetic regulation of vitamin d homeostasis at the fetomaternal interface . in this study , we found no evidence for methylation of the vdr or cyp27b1 genes in human full term or first trimester placental tissue . since methylation is generally associated with gene silencing , this supports previous data demonstrating high levels of expression of 1-hydroxylase and vdr in the placenta ( 18 , 19 , 60 ) . in contrast , we have identified methylation of the cyp24a1 gene , encoding 24-hydroxylase , the major catabolic enzyme of 1,25-(oh)2d and 25-(oh)2d in placental tissue and cell subtypes . we have demonstrated that this methylation is present in both the trophoblast compartment and cells of mesodermal origin and is present ( albeit at variable levels ) in primate and rodent placentas . this contrasts with the recently described placenta - specific methylation of the apc tumor suppressor gene that is primate - specific and localizes solely to the trophoblast compartment ( 37 ) and the sfrp2 tumor suppressor methylation that is primate - specific but localizes to multiple placental cell subtypes ( 38 ) . importantly , we have demonstrated ( by in vitro reporter studies ) that methylation of the cyp24a1 gene promoter directly down - regulates basal gene activity and abolishes vitamin d induction of the proximal promoter region . this is further supported by data demonstrating a reduced inducibility ( by 23 orders of magnitude ) of the methylated endogenous cyp24a1 gene in cultured primary human cvs - derived cells relative to unmethylated fibroblast cells . we observed much higher levels of induction of the endogenous gene in cultured primary fibroblast cells in comparison with that seen with a transfected reporter construct ( containing only the proximal promoter region ) in transformed trophoblast cells . this is consistent with previous data demonstrating a requirement for distal upstream sequence elements , in addition to the two previously described vitamin d - responsive elements , contained within the proximal promoter region , for maximal induction of the cyp24a1 gene ( 47 ) . interestingly , we did not find any major effects of exogenous 1,25-(oh)2d on the levels of vdr or cyp27b1 expression in skin or placental fibroblast cells . this contrasts with previous studies showing down - regulation of each of these genes in some cancer cell lines ( 61 ) in response to 1,25-(oh)2d treatment . in addition , the cyp27b1 gene was recently reported to be down - regulated in response to 1,25-(oh)2d in cultured trophoblasts undergoing syncitialization ( 62 ) . the proximal human cyp27b1 gene promoter contains putative vitamin d - responsive elements and has been shown to mediate 1,25-(oh)2d - mediated down - regulation of this gene in in vitro reporter experiments in mouse proximal tubule cells ( 63 ) . however , in primary human keratinocytes , 1,25-(oh)2d does not regulate 1-hydroxylase mrna or protein expression ( 64 ) , supporting our findings in this study . methylation of the proximal promoter region of the mouse cyp24a1 gene has been previously reported in tumor - derived endothelial cells , where it contributes to selective growth inhibition by 1,25-(oh)2d ( 30 ) . conversely , a lack of methylation in corresponding non - tumor - derived mouse endothelial cells is associated with a lack of 1,25-(oh)2d - mediated growth inhibition ( 30 ) . it is well documented that maternal circulating 1,25-(oh)2d levels show a dramatic increase during pregnancy ( reviewed in ref . 12 ) , with 50% of maternal circulating 1,25-(oh)2d derived from maternal decidua and placenta ( 13 ) . this observation demonstrates that the tight regulation of 1,25-(oh)2d levels is uncoupled during pregnancy . first , this can be achieved through an increase in expression of the cyp27b1 gene , which results in increasing 1-hydroxylase and elevated production of 1,25-(oh)2d from less active precursors . the second potential mechanism involves a decrease in the catabolism of 1,25-(oh)2d by the 24-hydroxylase enzyme encoded by cyp24a1 . increasing levels of maternal circulating 1,25-(oh)2d during pregnancy have been linked to increased synthesis rather than decreasing catabolic activity in animal studies ( 6567 ) . in addition , cyp27b1 expression is increased in early pregnancy decidua in comparison with nonpregnant endometrium ( 19 , 68 ) . in this study , we have demonstrated that the increasing synthesis of 1,25-(oh)2d occurs in concert with a placenta - specific epigenetic down - regulation of the cyp24a1 ( 24-hydroxylase ) gene . figure 6.methylation analysis of vitamin d - associated genes in cca - derived trophoblast cell lines . high levels of methylation were detected for both vdr ( a ) and cyp27b1 ( c ) in choriocarcinoma cell lines ( n = 2 ) . all choriocarcinoma cell lines ( n = 3 ; bewo not shown ) examined showed hypermethylation of the cyp24a1 gene relative to full term placental tissue or purified first trimester trophoblasts . numbers of each type of tissue are listed in parentheses . between 8 and 12 individual clones circles , cpg sites within the assayed region . closed circles , methylation ; open circles , lack of methylation . gray boxes correspond to gpg island locations , and arrows denote start site of translation within exon 1 ( black line ) . iii , mean methylation levels seen at each cpg site for each type of sample tested . methylation analysis of vitamin d - associated genes in cca - derived trophoblast cell lines . high levels of methylation were detected for both vdr ( a ) and cyp27b1 ( c ) in choriocarcinoma cell lines ( n = 2 ) . all choriocarcinoma cell lines ( n = 3 ; bewo not shown ) examined showed hypermethylation of the cyp24a1 gene relative to full term placental tissue or purified first trimester trophoblasts . numbers of each type of tissue are listed in parentheses . between 8 and 12 individual clones circles , cpg sites within the assayed region . closed circles , methylation ; open circles , lack of methylation . gray boxes correspond to gpg island locations , and arrows denote start site of translation within exon 1 ( black line ) . iii , mean methylation levels seen at each cpg site for each type of sample tested . we are currently investigating the specific role of 1,25-(oh)2d in mediating various aspects of placental functioning . 1,25-(oh)2d has widely diverse roles in regulating cell division , apoptosis , and immune modulation . it also shows antiproliferative effects in many different cell types , is proapoptotic , and is inhibitory to cell migration and invasion ( 5 , 6 , 6971 ) . each of these processes plays an important role in the development and function of the mammalian placenta . the interesting finding of simultaneous methylation of the vdr , cyp24a1 , and cyp27b1 genes in choriocarcinoma - derived trophoblast cell lines is unprecedented in cancer . methylation of the vdr gene is consistent with recent data demonstrating a lack of expression in bewo and jeg-3 choriocarcinoma cell lines ( 72 ) , and dysregulation of all three genes has been reported in cancers of different origin ( 73 ) . although abolition of vdr and cyp27b1 expression is anticipated to attenuate vitamin d - mediated growth inhibition in tumor cells , silencing of cyp24a1 would have the effect of maximizing active vitamin d bioavailability . it is possible that the precursor cells from which cca developed already had the cyp24a1 methylation described in this study and that acquisition of epigenetic silencing of the vdr and cyp27b1 genes was associated with the development of this tumor type . it is interesting to speculate that a sequential series of methylation events may occur in tumor development and progression . future studies examining the epigenetic regulation of this and other pathways in hydatidiform mole , recurring mole , and choriocarcinoma , may shed valuable light on the acquisition of methylation changes in gestational tumors . confirmation of epigenetic regulation of active vitamin d levels at the fetomaternal interface provides compelling circumstantial evidence for the intersection of two metabolic pathways in regulating pregnancy outcome , namely vitamin d and 1-carbon ( folate or methyl donor ) metabolism . one - carbon metabolism is a process by which methyl groups are passed from one donor molecule to the next ( 74 , 75 ) . this cycle produces s - adenosylmethionine , which donates its methyl group to cytosine to produce methylated cpg . each of these pathways relies on external inputs for the production of precursor molecules , and enzymes involved in each pathway ( including vdr and cyp27b1 ) have known functional polymorphic variants implicated in moderating disease risk ( 7683 ) , including adverse pregnancy outcomes ( 8489 ) . there are numerous polymorphisms throughout the cyp24a1 exonic , intronic , and regulatory gene regions ( 56 ) , but as yet there is little information regarding the functionality or clinical relevance of such variants . the convergence / intersection of 1-carbon metabolism with other metabolic and signaling pathways is potentially very important in modulating disease risk and warrants further investigation . the placenta - specific epigenetic regulation of several genes and metabolic pathways , independent of well characterized imprinted regions , is now a firmly established phenomenon . uncovering the mechanisms leading to the establishment of such markings , their timing , and sensitivity to environmental and genetic disruption has the potential to reveal valuable insights into the evolution of different placental functions and may reveal novel risk pathways associated with adverse pregnancy outcomes and tumor development .

plasma concentrations of biologically active vitamin d ( 1,25-(oh)2d ) are tightly controlled via feedback regulation of renal 1-hydroxylase ( cyp27b1 ; positive ) and 24-hydroxylase ( cyp24a1 ; catabolic ) enzymes . in pregnancy , this regulation is uncoupled , and 1,25-(oh)2d levels are significantly elevated , suggesting a role in pregnancy progression . epigenetic regulation of cyp27b1 and cyp24a1 has previously been described in cell and animal models , and despite emerging evidence for a critical role of epigenetics in placentation generally , little is known about the regulation of enzymes modulating vitamin d homeostasis at the fetomaternal interface . in this study , we investigated the methylation status of genes regulating vitamin d bioavailability and activity in the placenta . no methylation of the vdr ( vitamin d receptor ) and cyp27b1 genes was found in any placental tissues . in contrast , the cyp24a1 gene is methylated in human placenta , purified cytotrophoblasts , and primary and cultured chorionic villus sampling tissue . no methylation was detected in any somatic human tissue tested . methylation was also evident in marmoset and mouse placental tissue . all three genes were hypermethylated in choriocarcinoma cell lines , highlighting the role of vitamin d deregulation in this cancer . gene expression analysis confirmed a reduced capacity for cyp24a1 induction with promoter methylation in primary cells and in vitro reporter analysis demonstrated that promoter methylation directly down - regulates basal promoter activity and abolishes vitamin d - mediated feedback activation . this study strongly suggests that epigenetic decoupling of vitamin d feedback catabolism plays an important role in maximizing active vitamin d bioavailability at the fetomaternal interface .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION

figure 1.methylation status of cyp24a1 , cyp27b1 , and vdr in human term placenta . schematic representation of methylation data generated for regulatory regions of genes coding for 24-hydroxylase ( a ) , 1-hydroxylase ( b ) , and the vitamin d receptor ( c ) . between 8 and 12 individual clones schematic representation of methylation data generated for regulatory regions of genes coding for 24-hydroxylase ( a ) , 1-hydroxylase ( b ) , and the vitamin d receptor ( c ) . the cyp24a1 but not the vdr or cyp27b1 gene promoter is methylated in full term human placental tissue as a first step in the identification of potential epigenetic regulation of vitamin d levels in the placenta , we designed specific methylation amplification assays for regulatory regions of the vdr , cyp24a1 , and cyp27b1 genes . in the case of the vdr and cyp24a1 genes , assays were designed to span the transcription start site and exon 1 within the associated cpg island , whereas for cyp27b1 , we analyzed a region of exon 1 previously shown to be methylated in some cancers ( 48 , 49 ) . initial experiments on human term placenta revealed a complete lack of methylation at the vdr and cyp27b1 gene regulatory regions , with clear ( but variable ) methylation of the cyp24a1 promoter region ( fig . table 1mean promoter methylation levels of cyp24a1 , cyp27b1 , and vdr genes in human placental samples and cell linesa summary of cpg island methylation status for genes regulating vitamin d homeostasis and activity was determined by bisulfite sequencing . placenta , full term placental tissue ; 1st trimester ct , purified cytotrophoblasts showing > 95% ck-7 positive staining ; terminated pregnancy , placental tissue at 912 weeks gestation ; pre - eclampsia , diseased placental tissue at 912 weeks gestation ; choriocarcinoma , cell lines ( bewo , jeg-3 , and/or jar ) . d.%cyp24a1 placenta ( n = 8) 56.53 10.49 uncultured cvs ( n = 4 ) 33.07 9.31 cultured cvs ( n = 4 ) 33.79 19.02 first trimester ct ( n = 4 ) 48.17 24.49 terminated pregnancy ( n = 3 ) 48.62 19.18 pre - eclampsia ( n = 3 ) 39.85 6.32 choriocarcinoma ( n = 3 ) 80.40 10.47 placenta fibroblasts ( n = 3 ) 2.95 2.28 decidual cells ( n = 4 ) 1.57 0.57 hipec65 ( n = 2 ) 2.03 0.96 nonplacental tissue ( n = 10 ) 1.56 1.12 cyp27b1 placenta ( n = 4 ) 5.23 4.88 choriocarcinoma ( n = 2 ) 86.63 1.51 vdr placenta ( n = 2 ) 0.65 0.14 choriocarcinoma ( n = 3 ) 85.84 11.61 mean promoter methylation levels of cyp24a1 , cyp27b1 , and vdr genes in human placental samples and cell lines a summary of cpg island methylation status for genes regulating vitamin d homeostasis and activity was determined by bisulfite sequencing . placenta , full term placental tissue ; 1st trimester ct , purified cytotrophoblasts showing > 95% ck-7 positive staining ; terminated pregnancy , placental tissue at 912 weeks gestation ; pre - eclampsia , diseased placental tissue at 912 weeks gestation ; choriocarcinoma , cell lines ( bewo , jeg-3 , and/or jar ) . cyp24a1 placental methylation in multiple first trimester placental cell types in order to ascertain the cell types and timing of methylation of the cyp24a1 promoter within the placenta , we examined the methylation status of chorionic villous tissue ( pre- and post - cell culturing ) , first trimester termination placental tissue , purified first trimester cytotrophoblasts ( > 90% cytokeratin-7-positive ) , and full term placental fibroblasts . promoter methylation comparable with that seen in full term placental tissue was observed both in primary and cultured cvs tissue samples ( fig . in addition , purified first trimester cytotrophoblasts also show methylation of the cyp24a1 gene ( fig . no methylation was detected in purified full term placental fibroblasts or an sv40-transformed first trimester trophoblast cell line ( fig . cyp24a1 methylation is placenta - specific given the importance of the 24-hydroxylase enzyme in regulating levels of active vitamin d , it is surprising how little is known about the epigenetic regulation of this gene in human tissues . in order to address this , we assessed the methylation status of the cyp24a1 gene in a panel of human tissues consisting of kidney , skeletal muscle , skin fibroblasts , brain ( prefrontal cortex ) , sperm , whole blood , buccal mucosa , endometrial stroma , decidualized stroma , bone marrow , and umbilical cord tissue . 2 ) , demonstrate the specificity of placental cyp24a1 methylation in humans , with no evidence for methylation in any human somatic tissue tested . the cyp24a1 gene is differentially methylated in full term placentas from different mammals in previous studies , we have demonstrated that some of the specific promoter methylation recently documented in human term placental tissue is present in primates but absent in e18 ( day 18 of pregnancy ) mouse placenta . we therefore examined methylation of the cyp24a1 gene promoter regions in full term placenta from marmoset and mouse e18 and e11.5 ( fig . this revealed varying levels of cyp24a1 methylation in both marmoset ( mean 27 18.7% ) and mouse ( mean 35.1 4.5% ) placental tissue with generally lower levels relative to that seen in human full term placentas ( mean 56.5 10.5% ; fig . methylation of the core cyp24a1 gene promoter directly attenuates basal promoter activity in human trophoblast - derived cells despite circumstantial evidence of an inverse correlation of cyp24a1 mrna levels with promoter gene methylation ( above ) , no direct evidence for dna methylation - mediated silencing of this gene promoter has been reported . to investigate this further , we pcr - amplified the previously described vitamin d - responsive region of the human cyp24a1 gene promoter ( 47 ) and generated a luciferase reporter construct to directly test the effects of promoter methylation on both basal and vitamin d - induced promoter activity . 4 , reveal a low level of promoter activity of this promoter region in the plus orientation in both cell lines , 6.5-fold ( hipec65 ; p = 0.028 ) and 12-fold ( jar ; p < 0.001 ) higher than that of the promoterless luciferase vector . promoter activity in the reverse ( minus ) orientation was negligible ( 1.3-fold ) , demonstrating a directionality of the basal promoter element ( data not shown ) . cpg island methylation abrogates 1,25-(oh)2d - mediated up - regulation of cyp24a1 transcription in addition to examining the effects of promoter methylation on the basal cyp24a1 promoter activity , we also set out to investigate the consequences of this methylation on the previously demonstrated inducibility of this gene promoter by 1,25-(oh)2d ( 47 ) . although 1,25-(oh)2d treatment resulted in a clear induction ( 3.5-fold ) of basal promoter activity in hipec65 cells in the absence of previous promoter methylation ( p = 0.008 ) , this response was abolished with in vitro methylation of the reporter construct prior to transfection ( fig . 1,25-(oh)2d treatment had no effect on promoter activity in jar cells ( p = 0.663 ) , most likely reflecting the methylation of the vdr gene in this cell line ( see below ) that silences the gene and inhibits exogenous 1,25-(oh)2d signaling through the vdr . this region has previously been demonstrated to contain vitamin d - responsive elements ( 47 ) . basal promoter activity was detected in both cell lines and was abolished with in vitro methylation of the promoter prior to transfection ( sss1 , hpaii , and hhai methylases ) . 1,25-(oh)2d ( active vitamin d ) induction of the promoter was observed only in hipec-65 cells ( jar cells are predicted to lack the vitamin d receptor , as evidenced by complete methylation of the associated cpg island ) , but this induction was attenuated with prior methylation of the promoter region . basal promoter activity was detected in both cell lines and was abolished with in vitro methylation of the promoter prior to transfection ( sss1 , hpaii , and hhai methylases ) . 1,25-(oh)2d ( active vitamin d ) induction of the promoter was observed only in hipec-65 cells ( jar cells are predicted to lack the vitamin d receptor , as evidenced by complete methylation of the associated cpg island ) , but this induction was attenuated with prior methylation of the promoter region . we also examined the effect of promoter methylation on the induction of endogenous cyp24a1 , cyp27b1 , and vdr gene expression in primary cells derived from cvs tissue ( average methylation level of 33.4% ) and placental and skin fibroblasts ( both lacking cyp24a1 methylation ) . in contrast , cultured cvs cells showed a reduced capacity for gene induction ( mean 10 fold ; n = 4 ) . in contrast to the major effects of vitamin d induction on cyp24a1 expression in skin and placental fibroblasts , vdr expression levels were slightly reduced ( mean reduction of 3-fold ) in skin fibroblasts , with little change apparent in placental fibroblasts ( mean 1.18-fold increase ) and cvs - derived cells ( mean 0.76-fold change ; fig . we analyzed the methylation status of cyp24a1 in three widely studied cca cell lines , jeg-3 , bewo , and jar . we also assessed methylation at the vdr and cyp27b1 cpg islands , which also revealed nearly complete methylation of each of these genes in all cell lines tested . lowered 1,25-(oh)2d and altered calcium metabolism for example , a recent cochrane review of randomized control trials demonstrated a role for calcium supplementation in reducing pre - eclampsia risk.5 altered expression levels of 1-hydroxylase , vdr , and 24-hydroxylase genes have all been reported in different placental cells and tissue from preeclamptic pregnancies , possibly in response to decreasing maternal 1,25-(oh)2d ( 19 , 57 , 58 ) . cells were cultured in multiple wells , and 100 nm 1,25-(oh)2d3 ( active vitamin d ) was added to half of the cultures for 7 h , followed by total rna isolation and quantitative real time reverse transcription - pcr . cells were cultured in multiple wells , and 100 nm 1,25-(oh)2d3 ( active vitamin d ) was added to half of the cultures for 7 h , followed by total rna isolation and quantitative real time reverse transcription - pcr . however , at present , very little is known about the consequences of altered environmental , genetic , or epigenetic regulation of vitamin d homeostasis at the fetomaternal interface . in this study , we found no evidence for methylation of the vdr or cyp27b1 genes in human full term or first trimester placental tissue . in contrast , we have identified methylation of the cyp24a1 gene , encoding 24-hydroxylase , the major catabolic enzyme of 1,25-(oh)2d and 25-(oh)2d in placental tissue and cell subtypes . importantly , we have demonstrated ( by in vitro reporter studies ) that methylation of the cyp24a1 gene promoter directly down - regulates basal gene activity and abolishes vitamin d induction of the proximal promoter region . this is consistent with previous data demonstrating a requirement for distal upstream sequence elements , in addition to the two previously described vitamin d - responsive elements , contained within the proximal promoter region , for maximal induction of the cyp24a1 gene ( 47 ) . the proximal human cyp27b1 gene promoter contains putative vitamin d - responsive elements and has been shown to mediate 1,25-(oh)2d - mediated down - regulation of this gene in in vitro reporter experiments in mouse proximal tubule cells ( 63 ) . in this study , we have demonstrated that the increasing synthesis of 1,25-(oh)2d occurs in concert with a placenta - specific epigenetic down - regulation of the cyp24a1 ( 24-hydroxylase ) gene . figure 6.methylation analysis of vitamin d - associated genes in cca - derived trophoblast cell lines . high levels of methylation were detected for both vdr ( a ) and cyp27b1 ( c ) in choriocarcinoma cell lines ( n = 2 ) . all choriocarcinoma cell lines ( n = 3 ; bewo not shown ) examined showed hypermethylation of the cyp24a1 gene relative to full term placental tissue or purified first trimester trophoblasts . high levels of methylation were detected for both vdr ( a ) and cyp27b1 ( c ) in choriocarcinoma cell lines ( n = 2 ) . all choriocarcinoma cell lines ( n = 3 ; bewo not shown ) examined showed hypermethylation of the cyp24a1 gene relative to full term placental tissue or purified first trimester trophoblasts . each of these processes plays an important role in the development and function of the mammalian placenta . the interesting finding of simultaneous methylation of the vdr , cyp24a1 , and cyp27b1 genes in choriocarcinoma - derived trophoblast cell lines is unprecedented in cancer . methylation of the vdr gene is consistent with recent data demonstrating a lack of expression in bewo and jeg-3 choriocarcinoma cell lines ( 72 ) , and dysregulation of all three genes has been reported in cancers of different origin ( 73 ) . although abolition of vdr and cyp27b1 expression is anticipated to attenuate vitamin d - mediated growth inhibition in tumor cells , silencing of cyp24a1 would have the effect of maximizing active vitamin d bioavailability . it is possible that the precursor cells from which cca developed already had the cyp24a1 methylation described in this study and that acquisition of epigenetic silencing of the vdr and cyp27b1 genes was associated with the development of this tumor type . confirmation of epigenetic regulation of active vitamin d levels at the fetomaternal interface provides compelling circumstantial evidence for the intersection of two metabolic pathways in regulating pregnancy outcome , namely vitamin d and 1-carbon ( folate or methyl donor ) metabolism .

the senile plaques and neurofibrillary tangles ( nfts ) , which are composed of self - polymerized amyloid- peptide ( a ) and hyperphosphorylated tau proteins , respectively , are the two major pathological hallmarks in brains of ad . pathogenic a aggregates initiate a cascade of molecular events that foster widespread neurodegeneration , although the exact pathogenesis from a aggregation to neurodegeneration is not clear . a originates from proteolysis of the amyloid precursor protein ( app ) by the sequential enzymatic actions of beta - site amyloid precursor protein - cleaving enzyme 1 ( bace1 ) , -secretase , and -secretase , a protein complex with presenilin 1 ( ps1 ) at its catalytic core . -secretase adam10 conversely cleaves app within the eventual a sequence to preclude a generation , and it yields the n - terminal protein of app ( soluble app- [ sapp ] ) . sapp decreases a generation by directly associating with bace1 , and so it may be a potential agent to ameliorate imbalances in app processing.4 , 5 overexpression of adam10 markedly reduces a plaque load and soluble a , restoring learning deficits in a double - transgenic alzheimer mouse model . neurotrophins ( neurotrophic factors [ ntfs ] ) are important in the development , differentiation , and regeneration of brain neurons . in recent years , some neurotrophins , such as nerve growth factor ( ngf ) and brain - derived neurotrophic factor ( bdnf ) , were explored as candidate therapeutics for ad.8 , 9 human acidic fibroblast growth factor ( hafgf ) has neuroprotective functions similar to ntfs. exogenous administration of hafgf has been shown to prevent degeneration and apoptosis of neurons in epileptic seizure.10 , 11 hafgf is involved in the regulation of synaptic plasticity , processes attributed to learning and memory through improving cholinergic nerve functions . hafgf showed a neuroprotective effect against brain injury resulting from focal ischemia - reperfusion in rats,13 , 14 and it repaired human spinal cord injury in a clinical trial.15 , 16 it is reported that the concentration of acid fibroblast growth factor ( afgf ) is increased in the serum and cerebrospinal fluid of patients with ad . however , due to its large molecular size of 17 kda , the 154-amino acid protein hafgf is unable to freely pass through biological membranes and the blood - brain barrier ( bbb ) . novel strategies were used to address this bottleneck problem , such as fusing the target protein with tat - ptd and utilizing intranasal delivery.19 , 20 our previous study showed that modified protein tat - hafgf14154 could penetrate the bbb and was distributed in hippocampus and cortex via intravenous injection . following intranasal administration , the distribution of tat - hafgf14154 was observed at 15 min in the brains of rats . tat - ptd could increase the concentration of hafgf in brain , and tat - hafgf14154 improved cognition and reduced a plaques more significantly than hafgf14154 in senescence - accelerated mouse prone-8 ( samp8 ) and app / ps1 mice.22 , 23 meanwhile , liposomes have gained increasing attention as a promising strategy for brain - targeted drug delivery due to their large delivery capabilities , capacity for surface decoration , low toxicity , and biocompatibility with biodegradability . studies have shown that liposomes easily cross the bbb through absorptive mediated transcytosis . in the current study , tat - hafgf14154 was encapsulated with cationic liposomes , and it was delivered to the app / ps1 double - transgenic mouse model for ad via intranasal administration to investigate neuroprotection . the molecular mechanism related to neuroprotection of tat - hafgf14154 was also explored in primary cortical neurons of rats injured by a142 . results demonstrated that tat - hafgf14154 ameliorated the ad phenotype by increasing adam10 expression at the transcriptional level through the pi3k - creb - ire1/xbp1 pathway , thereby enhancing adam10 enzyme activity . the highest entrapment efficiency was achieved with a 1:2 ratio of tat - hafgf14154 and liposomes incubated at 25c or 30c for 0.5 hr ( 97% ; figure s1a ) . these conditions did not alter the electrophoretic mobility profile of tat - hafgf14154 on sds - page ( figure s1b ) . after a single intranasal administration , afgf was detected in the olfactory bulb ( ob ) and entorhinal cortex ( ec ) of 7-month - old c57bl/6 mice ( figure s1c ) . both tat - hafgf14154 ( 600 g / kg , t600 ) and tat - hafgf14154-loaded cationic liposomes ( 600 g / kg , l + t600 ) significantly enhanced the levels of afgf in the ob ( p < 0.05 for t600 , p < 0.01 for l + t600 versus vehicle ; figure s1d ) ; only l + t600 enhanced the levels of afgf significantly in the ec ( p < 0.01 versus vehicle ; figure s1e ) . intranasal treatment of tat - hafgf14154-loaded cationic liposomes demonstrated a higher efficiency for the delivery of afgf compared with tat - hafgf14154 alone , as evidenced by the significantly elevated levels of afgf in the ec ( p < 0.01 for l + t600 versus t600 ; figure s1e ) . tat - hafgf14154-loaded cationic liposomes were delivered intranasally to 7-month - old app / ps1 mice every 2 days for 6 weeks , followed by a morris water maze ( mwm ) test in the fifth week and nest construction ( nc ) and open field ( of ) tests in the sixth week ( figure 1a ) . representative movement tracks of mice on day 5 in the place navigation component of the mwm are shown in figure 1b . both t600 and l + t600 significantly decreased the escape latency compared with vehicle ( p < 0.001 for t600 and l + t600 ; figure 1c ) . mice treated with l + t600 exhibited a shorter escape latency compared to those treated with t600 ( p < 0.01 ; figure 1c ) during the 6 days of testing . t600 and l + t600 also decreased the path length in app / ps1 mice compared with vehicle during the 6 days of tests ( p < 0.001 for t600 and l + t600 ; figure 1d ) . l + t600 exhibited a notably shorter path length from day 3 ( p < 0.05 on day 3 , p < 0.01 on days 4 and 5 ; figure 1e ) , whereas this was only evident with t600 on day 5 ( p < 0.05 ; figure 1e ) compared with vehicle . after finishing the navigation test on day 5 , a spatial probe test was conducted 24 hr later . movement tracks , the incidence of crossing the removed - platform area , and percentage of time spent in target quadrant were recorded ( figures 1f1h ) . l + t600 significantly increased the incidence of crossing the removed - platform area ( p < 0.01 ; figure 1 g ) and time spent in the target quadrant ( p < 0.01 ; figure 1h ) compared with vehicle . there were no differences in the swimming speed of mice among all groups in the place navigation and spatial probe tests ( figure s2a ) . these results indicate that tat - hafgf14154-loaded cationic liposomes significantly attenuated cognitive behavior deficits and that the liposome tat - hafgf14154 preparation was more potent than tat - hafgf14154 alone.figure 1tat - hafgf14154-loaded cationic liposomes attenuated cognitive behavioral deficits in app / ps1 mice(a ) schematic of the protocol followed for the experiments . aliquots of 0.9% saline ( vehicle ) , tat - hafgf14154 ( 600 g / kg , t600 ) , and tat - hafgf14154-loaded cationic liposomes ( 600 g / kg , l + t600 ) were delivered intranasally to 7-month - old app / ps1 mice every 2 days for 6 weeks ( n = 8) . mwm was conducted in the fifth week , and nest construction ( nc ) and of tests were in the sixth week . representative movement tracks of mice on day 5 ( b ) , escape latencies ( c ) , and path length ( d and e ) over the 5 days are shown . ( f h ) following the completion of the navigation test , a spatial probe test was conducted 24 hr later . representative movement tracks ( f ) , percentage of time mice spent in the target quadrant ( g ) , and times of crossing the removed - platform area ( h ) were assessed . all values are presented as means sem ( n = 8 ; * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 , two - way anova with bonferroni post hoc test for c e , one - way anova with bonferroni s post hoc test for g and h ) . , intranasal administration ; mwm , morris water maze ; of , open field ; ns . tat - hafgf14154-loaded cationic liposomes attenuated cognitive behavioral deficits in app / ps1 mice ( a ) schematic of the protocol followed for the experiments . aliquots of 0.9% saline ( vehicle ) , tat - hafgf14154 ( 600 g / kg , t600 ) , and tat - hafgf14154-loaded cationic liposomes ( 600 g / kg , l + t600 ) were delivered intranasally to 7-month - old app / ps1 mice every 2 days for 6 weeks ( n = 8) . mwm was conducted in the fifth week , and nest construction ( nc ) and of tests were in the sixth week . representative movement tracks of mice on day 5 ( b ) , escape latencies ( c ) , and path length ( d and e ) over the 5 days are shown . ( f h ) following the completion of the navigation test , a spatial probe test was conducted 24 hr later . representative movement tracks ( f ) , percentage of time mice spent in the target quadrant ( g ) , and times of crossing the removed - platform area ( h ) were assessed . all values are presented as means sem ( n = 8 ; * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 , two - way anova with bonferroni post hoc test for c e , one - way anova with bonferroni s post hoc test for g and h ) . i.n . , intranasal administration ; mwm , morris water maze ; of , open field ; ns . , no significance . next , non - cognitive behaviors were examined in app / ps1 mice , including nc and of tests in the sixth week after intranasal administration . body weights of mice had no significant change in the whole experimental period ( figure s2b ) . it is reported that app / ps1 mice fail to construct nests and display an increased locomotor activity compared with wild - type mice.28 , 29 the nesting scores of the ps1/app mice improved significantly with both t600 and l + t600 treatments ( p < 0.01 for t600 , p < 0.01 for l + t600 ; figures s3a and s3b ) . in the of test , l + t600 significantly decreased time spent ( p < 0.05 ; figure s3c ) and crossing times ( p < 0.01 ; figure s3d ) in the central area , and it shortened the total distance of movement ( p < 0.01 ; figure s3e ) compared with vehicle . l + t600 treatment was significantly more effective than t600 in the of test ( p < 0.05 in figure s3c and p < 0.01 in figure s3e ) . overload of a plaque is the major pathology in the brain of app / ps1 mice . a plaques were assessed by immunohistochemisty in the cortex and hippocampus ( cornu ammonis 1 [ ca1 ] is illustrated in figure 2a ) . l + t600 treatment reduced the area ( p < 0.01 ; figure 2b ) and the number ( p < 0.001 ; figure 2c ) of a plaques compared with vehicle . although t600 decreased the a plaque number ( p < 0.05 ; figure 2c ) , it did not significantly change the percentage of the a plaque area . to further confirm these results , tissue homogenates from cortex and hippocampus were used to detect a by western blotting ( figures 2d2f ) . l + t600 reduced a levels both in cortex ( p < 0.05 ; figure 2e ) and hippocampus ( p < 0.01 ; figure 2f ) compared with vehicle . these results indicate that tat - hafgf14154-loaded cationic liposomes decreased a burden in the brain of app / ps1 mice and were more effective than tat - hafgf14154 alone.figure 2tat - hafgf14154-loaded cationic liposomes reduced a loading in the brain of app / ps1 mice(a ) representative images of a plaques in coronal section and ca1 area of brain ( scale bar , 80 m ) . ( b and c ) percentage of a plaque area ( b ) and plaque number ( c ) in coronal sections were analyzed by image - pro plus ( n = 8) . ( d f ) representative immunoblots ( d ) and quantification of a expression in cortex ( e ) and hippocampus ( f ) ( n = 6 ) . all values are presented as means sem ( * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 , one - way anova with bonferroni post hoc test ) . tat - hafgf14154-loaded cationic liposomes reduced a loading in the brain of app / ps1 mice ( a ) representative images of a plaques in coronal section and ca1 area of brain ( scale bar , 80 m ) . ( b and c ) percentage of a plaque area ( b ) and plaque number ( c ) in coronal sections were analyzed by image - pro plus ( n = 8) . ( d f ) representative immunoblots ( d ) and quantification of a expression in cortex ( e ) and hippocampus ( f ) ( n = 6 ) . all values are presented as means sem ( * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 , one - way anova with bonferroni post hoc test ) . od , relative optical density . to determine whether the reduction of a loading could be due to a change in app - processing modulation , the expression of app and its processing proteins ( adam10 , bace1 , and ps1 ) in cortex and hippocampus was measured by western blotting ( figure 3a ) . the expression of adam10 ( p < 0.01 in cortex , figure 3b ; p < 0.05 in hippocampus , figure 3f ) , but not full - length app ( fl - app , figures 3c and 3 g ) or bace1 ( figures 3d and 3h ) , was enhanced significantly by l + t600 both in cortex and hippocampus compared with vehicle . l + t600 enhanced the expression of ps1 ( p < 0.05 ; figure 3i ) in hippocampus , but not in cortex . t600 had no influence on the expression of the above proteins ( figures 3b3i ) . enzyme activities of adam10 and bace1 , two major secretases in app processing , were assayed from hippocampus homogenates of app / ps1 mice . the activity of adam10 ( p < 0.05 ; figure 3j ) , but not bace1 ( figure 3k ) , was increased by l + t600 compared with vehicle . these results revealed that the reduction of a loading in the brain of app / ps1 mice treated with tat - hafgf14154-loaded cationic liposomes could be due to the enhancement of adam10 in expression and activity.figure 3tat - hafgf14154-loaded cationic liposomes enhanced adam10 expression and activity in the brain of app / ps1 mice(a i ) representative immunoblots ( a ) and quantification expression of app - processing proteins in cortex ( b e ) and hippocampus ( f i ) of app / ps1 mice . app - processing proteins , including fl - app , adam10 , bace1 , and ps1 , were assayed by western blotting . ( j and k ) adam10 activity ( j ) and bace1 activity ( k ) in homogenate of hippocampus were measured by an enzymatic cleavage assays . all values are presented as means sem ( n = 6 ; * p < 0.05 and * * p < 0.01 , one - way anova with bonferroni post hoc test ) . tat - hafgf14154-loaded cationic liposomes enhanced adam10 expression and activity in the brain of app / ps1 mice ( a i ) representative immunoblots ( a ) and quantification expression of app - processing proteins in cortex ( b e ) and hippocampus ( f i ) of app / ps1 mice . app - processing proteins , including fl - app , adam10 , bace1 , and ps1 , were assayed by western blotting . ( j and k ) adam10 activity ( j ) and bace1 activity ( k ) in homogenate of hippocampus were measured by an enzymatic cleavage assays . all values are presented as means sem ( n = 6 ; * p < 0.05 and * * p < 0.01 , one - way anova with bonferroni post hoc test ) . primary cortical neurons at 5 days in vitro ( div ) were incubated with a142 oligomers for 48 hr at 37c . a142 ( 1.5 m ) suppressed the viability of primary cortical neurons to 69.7% 1.4% ( p < 0.001 ; figure 4a ) , and apoptotic bodies / karyopyknosis affected 34.3% 4.4% of the cells ( p < 0.001 ; figures 4b and 4c ) , compared to control cells . neurons at 5 div were pre - treated with tat - hafgf14154 ( 60 ng / ml [ t60 ] , 300 ng / ml [ t300 ] , and 1,500 ng / ml [ t1500 ] ) for 2 hr at 37c . a was added to the culture medium with continuous tat - hafgf14154 treatment for 48 hr . upon treatment with tat - hafgf14154 , cell viability was significantly increased to 84.5% 3.0% ( t60 + a ) , 90.4% 3.5% ( t300 + a ) , and 97.4% 1.9% ( t1500 + a ) , compared with untreated cells ( p < 0.01 for t60 + a , p < 0.001 for t300 + a , and p < 0.001 for t1500 + a ; figure 4a ) . nuclear staining ( hoechst 33342 ) revealed that the prevalence of karyopyknosis or apoptotic body formation induced by a142 was diminished by tat - hafgf14154 in a concentration - dependent manner ( p < 0.05 for t60 + a , p < 0.001 for t300 + a , and p < 0.001 for t1500 + a ; figures 4b and 4c ) . morphologic structure of neurons was observed by scanning electron microscopy ( figure 4d ) . as showed in micrographs , somata were flat and fractured and neurites were fragmented in neurons injured by a. this damaged appearance was reversed by the treatment with tat - hafgf14154 in a concentration - dependent manner.figure 4tat - hafgf14154 protected neurons from damage induced by a142primary cortical neurons were isolated from brains of newborn sd rats . div5 ( 5 days in vitro ) neurons were pre - treated with tat - hafgf14154 ( 60 , 300 , and 1,500 ng / ml ) for 2 hr at 37c , and 1.5 m a142 was added into the culture medium with continuous tat - hafgf14154 treatment for 48 hr . ( a ) cell viability was detected by mtt assay ( n = 6 ) . ( b ) representative immunofluorescence images of hoechst 33342 staining ( scale bar , 20 m ) . ( c ) the percentage of cells exhibiting apoptotic bodies or karyopyknosis ( blue ) was calculated ( n = 20 ) . ( d ) representative images of cell surface structure were captured by scanning electron microscope ( scale bar , 4 m ) . the yellow arrows show fractured neurites or axons , and the red arrows show desiccated or shrinking somata . values are presented as means sem ( * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 , one - way anova with bonferroni post hoc test ) . tat - hafgf14154 protected neurons from damage induced by a142 primary cortical neurons were isolated from brains of newborn sd rats . div5 ( 5 days in vitro ) neurons were pre - treated with tat - hafgf14154 ( 60 , 300 , and 1,500 ng / ml ) for 2 hr at 37c , and 1.5 m a142 was added into the culture medium with continuous tat - hafgf14154 treatment for 48 hr . ( a ) cell viability was detected by mtt assay ( n = 6 ) . ( b ) representative immunofluorescence images of hoechst 33342 staining ( scale bar , 20 m ) . ( c ) the percentage of cells exhibiting apoptotic bodies or karyopyknosis ( blue ) was calculated ( n = 20 ) . ( d ) representative images of cell surface structure were captured by scanning electron microscope ( scale bar , 4 m ) . the yellow arrows show fractured neurites or axons , and the red arrows show desiccated or shrinking somata . values are presented as means sem ( * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 , one - way anova with bonferroni post hoc test ) . to further estimate the degree of injury in neurites , two synaptic markers , synaptophysin ( syn ) and growth - associated protein 43 ( gap43 ) , were assessed by immunofluorescence and western blotting in primary cortical neurons ( figure s4 ) . both assays revealed that levels of syn and gap43 were suppressed by a ( p < 0.05 or p < 0.01 ) but that this was successfully rescued by the tat - hafgf14154 treatment ( p < 0.01 or p < 0.001 ) . levels of syn and gap43 were also assessed in the brains of app / ps1 mice by immunohistochemistry and western blotting ( figure s5 ) . the immunohistochemistry detected syn and gap43 located in different regions of hippocampus in wild - type healthy control mice ( figure s5a ) . syn was mainly located in the dentate gyrus ( dg ) area , whereas gap43 was mainly located in ca1 area . therefore , the effects of treatment on the levels of syn in the dg area ( figures s5b and s5d ) and gap43 in the ca1 area ( figures s5c and s5e ) were assessed . l + t600 increased the levels of syn in the dg area ( p < 0.01 ; figure s5d ) and gap43 in the ca1 area ( p < 0.001 ; figure s5e ) compared with vehicle . western blotting for syn and gap43 ( figures s5f s5j ) validated these conclusions . although there were no significant changes of syn and gap43 levels in the cortex ( figures s5 g and s5h ) , l + t600 enhanced the levels of syn ( p < 0.05 ; figure s5i ) and gap43 ( p < 0.05 ; figure s5j ) in the hippocampus compared with vehicle . t600 did not change the expression levels of syn and gap43 in the cortex and hippocampus ( figures s5d s5j ) . the expression of adam10 , both mrna and protein , in primary neurons was significantly enhanced by tat - hafgf14154 ( p < 0.05 for t300 + a and p < 0.01 for t1500 + a in mrna level , figure 5a ; p < 0.01 for t1500 + a in protein level , figure 5c ) . adam10 activity in primary neurons was also elevated by tat - hafgf14154 ( p < 0.01 for t300 + a and p < 0.001 for t1500 + a ; figure 5d ) . the enhancement of adam10 in expression and enzyme activity both in vivo ( figure 3 ) and in vitro indicated that adam10 might be a key target for tat - hafgf14154 to protect neurons from injury in ad models.figure 5tat - hafgf14154 enhanced expression and activity of adam10 in primary neurons injured by a142expressions of adam10 mrna and protein levels were assayed by qrt - pcr and western blotting , respectively . ( b and c ) representative immunoblots ( b ) and quantification ( c ) of adam10 expression . all values are presented as means sem ( n = 6 ; * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 , one - way anova with bonferroni post hoc test ) . tat - hafgf14154 enhanced expression and activity of adam10 in primary neurons injured by a142 expressions of adam10 mrna and protein levels were assayed by qrt - pcr and western blotting , respectively . ( b and c ) representative immunoblots ( b ) and quantification ( c ) of adam10 expression . all values are presented as means sem ( n = 6 ; * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 , one - way anova with bonferroni post hoc test ) . od , relative optical density . to validate this possibility , small interfering rna ( sirna ) targeting of adam10 was tested in rat primary cortical neurons at 5 div . knockdown was confirmed over 2472 hr by qrt - pcr and western blotting , and it was not affected by a treatment for 48 hr ( figure s6 ) . normal neurons ( t1500 + a ) or adam10-knockdown ( kd ) neurons ( kd + t1500 + a ) were incubated with tat - hafgf14154 and a142 as described above . the sirna knockdown blocked the elevation of adam10 induced by tat - hafgf14154 ( p < 0.01 for a and p < 0.001 for kd + t1500 + a ; figure 6b ) , and it significantly impaired the ability of t1500 to rescue a toxicity ( p < 0.01 ; figure 6c ) and a damage to soma and neurites ( figure 6d).figure 6knockdown of adam10 by sirna blocks tat - hafgf14154 neuroprotectionneurons were treated with or without a142/tat - hafgf14154/sirna as described above . ( a and b ) expression of adam10 in neurons was detected by western blotting . the yellow arrows show fractured neurites or axons , and the red arrows show desiccated or damaged somas . all values are presented as means sem ( * * p < 0.01 and * * * p < 0.001 , one - way anova with bonferroni post hoc test ) . knockdown of adam10 by sirna blocks tat - hafgf14154 neuroprotection neurons were treated with or without a142/tat - hafgf14154/sirna as described above . ( a and b ) expression of adam10 in neurons was detected by western blotting . the yellow arrows show fractured neurites or axons , and the red arrows show desiccated or damaged somas . all values are presented as means sem ( * * p < 0.01 and * * * p < 0.001 , one - way anova with bonferroni post hoc test ) . x - box - binding protein 1 ( xbp1 ) is one of the transcription factors of adam10 , and it is also linked with the unfolded protein response ( upr ) . spliced xbp1 protein ( xbp1s ) is the activated form of xbp1 , and the ratio of xbp1s and unspliced xbp1 ( xbp1u ) reflects the activity of xbp1 . after nuclear translocation , xbp1s binds to its binding site on the adam10 mrna promoter and promotes transcription . xbp1s / xbp1u was significantly increased by l + t600 in the cortex ( p < 0.05 ; figure s7b ) , by t600 and l + t600 in the hippocampus ( p < 0.01 for t600 and p < 0.05 for l + t600 ; figure s7c ) compared to vehicle - treated mice , indicating that xbp1 was activated by tat - hafgf14154 . primary cortical neurons in vitro were used to confirm these results and explore the signaling pathway related to the upregulation of adam10 by tat - hafgf14154 . inositol - requiring enzyme 1 ( ire1 ) , one of the three endoplasmic reticulum ( er ) stress sensors , can active xbp1 by splicing xbp1u to xbp1s . phosphoinositide 3-kinase ( pi3k ) is one of the major signaling mediators of afgf , and its activation can be blocked by ly294002 . regulatory subunits p85 of pi3k promote the nuclear translocation of xbp1s.35 , 36 , 37 camp response element - binding protein ( creb ) is activated in ad models by pi3k / akt , and its phosphorylation is neuroprotective.38 , 39 creb is also activated by er stress , and it binds to the promoter of the ire1 gene and regulates its expression . based on these findings , we hypothesized that tat - hafgf14154 promotes the expression of adam10 through this pi3k - creb - ire1/xbp1 pathway . levels of xbp1u , xbp1s , p - pi3k ( p - p85 and tyr467 ) , pi3k ( p85 ) , p - creb ( ser133 ) , and creb were assayed by western blotting , and the effects of pi3k inhibitor ( ly294002 ) were assessed ( figures 7a7e ) . after treatment with tat - hafgf14154 , xbp1s / xbp1u , phosphorylation of pi3k ( p - pi3k / p - p85 and tyr467 ) , and phosphorylation of creb ( p - creb and ser133 ) were significantly increased in neurons injured by a ( p < 0.01 ) , and they were suppressed by pi3k inhibitor ( p < 0.001 in figures 7c and 7d and p < 0.01 in figure 7e ) . these results fueled our hypothesis that the pi3k - creb - ire1/xbp1 pathway was activated by tat - hafgf14154 . pi3k inhibition abolished the rise in adam10 expression seen in neurons treated with t1500 + a ( p < 0.01 for p + t1500 + a ; figures 7f and 7 g ) as well as thwarted hafgf14154 rescue of cell viability ( p < 0.001 for p + t1500 + a ; figure 7h ) and morphological damage ( figure 7i).figure 7tat - hafgf14154 upregulated the expression of adam10 via the pi3k - creb - ire1/xbp1 pathwayprimary neurons were pre - treated for 30 min with pi3k inhibitor ( ly294002 , p ) , followed by incubation with tat - hafgf14154 and a142 as described above . ( a and b ) representative immunoblots of xbp1s , xbp1u , p - pi3k(p - p85 and tyr467 ) , pi3k(p85 ) , p - creb ( ser133 ) , and creb . ( c e ) quantitative analysis for xbp1s / xbp1u ( c ) , expression of pi3k p85 and p - p85 ( d ) , and p - creb ( ser133 ) and creb ( e ) . ( f and g ) upregulation of adam10 induced by tat - hafgf14154 was blocked by the pi3k inhibitor . ( i ) the pi3k inhibitor subverted the improvement effect of tat - hafgf14154 on cell morphology . the yellow arrows show fractured neurites or axons , and the red arrows show desiccated or damaged somas . all values are presented as means sem ( n = 6 ; * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 , one - way anova with bonferroni post hoc test for c , g , and h , two - way anova with bonferroni s post hoc test for d and e ) . p , pi3k inhibitor ly294002 ; xbp1s , spliced xbp1 protein ; xbp1u , unspliced xbp1protein ; od , relative optical density . tat - hafgf14154 upregulated the expression of adam10 via the pi3k - creb - ire1/xbp1 pathway primary neurons were pre - treated for 30 min with pi3k inhibitor ( ly294002 , p ) , followed by incubation with tat - hafgf14154 and a142 as described above . ( a and b ) representative immunoblots of xbp1s , xbp1u , p - pi3k(p - p85 and tyr467 ) , pi3k(p85 ) , p - creb ( ser133 ) , and creb . ( c e ) quantitative analysis for xbp1s / xbp1u ( c ) , expression of pi3k p85 and p - p85 ( d ) , and p - creb ( ser133 ) and creb ( e ) . ( f and g ) upregulation of adam10 induced by tat - hafgf14154 was blocked by the pi3k inhibitor . ( i ) the pi3k inhibitor subverted the improvement effect of tat - hafgf14154 on cell morphology . the yellow arrows show fractured neurites or axons , and the red arrows show desiccated or damaged somas . all values are presented as means sem ( n = 6 ; * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 , one - way anova with bonferroni post hoc test for c , g , and h , two - way anova with bonferroni s post hoc test for d and e ) . p , pi3k inhibitor ly294002 ; xbp1s , spliced xbp1 protein ; xbp1u , unspliced xbp1protein ; od , relative optical density . ad has become a major public health problem owing to the increasing prevalence , long course of disease , burden on caregivers , and high financial cost of care . new effective treatments that will prevent , delay , or treat the symptoms of ad are urgently needed . neuroprotection of afgf has been widely reported over the recent years,15 , 42 , 43 yet its mechanism is still uncertain . in our study , tat - hafgf14154-loaded cationic liposomes were delivered to app / ps1 mice via intranasal administration to investigate neuroprotection of tat - hafgf14154 , and the related mechanism was explored in primary cortical neurons of rats injured by a142 . one of the great challenges for drugs targeting neurodegenerative diseases is that the bbb restricts the access of drugs to the brain by oral and intravenous administration . intranasal delivery has proven to be a new and noninvasive strategy , which can circumvent the bbb and facilitate direct transport of the drugs to the brain through neuronal and extracellular pathways.44 , 45 our previous studies showed that intranasal administration of tat - hafgf could improve cognition and reduce a deposits more significantly in app / ps1 mice compared with intravenous injection.18 , 21 , 22 , 23 in an animal model of parkinson s disease induced by 6-ohda , liposomes markedly assisted the delivery of basic fibroblast growth factor ( bfgf ) to the striatum and substantia nigra ( sn ) , and they enhanced the neuroprotective effects of bfgf on dopaminergic neurons . liposomes also enhanced the levels of afgf significantly in the ec , accompanied by amelioration in cognitive and non - cognitive behavioral deficits . consistent with the behavioral improvement , a burden was reduced in both the cortex and hippocampus of brain by intranasal treatment with tat - hafgf14154-loaded cationic liposomes . a originates from proteolysis of app by the sequential enzymatic actions of bace1 and ps1 , called app amyloidogenic processing . conversely , adam10 precludes a generation by cleaving app within the eventual a sequence , called non - amyloidogenic processing . there was a marked increase of adam10 in both expression and activity in the brains of mice with treatment of tat - hafgf14154-loaded cationic liposomes . adam10-mediated non--amyloidosis processing via enhancing expression and activity of adam10 may be involved in the amelioration of behavioral deficits and the reduction of a burden in app / ps1 mice treated with tat - hafgf14154 . recent reports have provided several candidate drugs or therapeutic methods to attenuate pathology in ad ; most of them show its mechanism of modulating app processing so as to alter app toward non - amyloidogenic processing.30 , 47 , 48 , 49 , 50 overexpression of adam10 impressively reduced a plaque load and soluble a , and it additionally restored learning deficits in double - transgenic alzheimer model mice . adam10 is a metalloprotease with multiple functions in the brain . besides the -secretase in app processing , adam10 also plays a critical role in regulating functional membrane proteins at the synapse . postnatal disruption of the adam10 in mouse brain caused epileptic seizures , learning deficits , altered spine morphology , and defective synaptic functions . with the upregulation of adam10 , tat - hafgf14154 dose - dependently enhanced cell viability , and it reversed damage in soma and neurites of neurons damaged by a. tat - hafgf14154 increased synaptic proteins syn and gap43 significantly both in vitro and in vivo , indicating that tat - hafgf14154 promoted nerve growth and possessed the potential of modulating synaptic plasticity . after sirna knockdown of adam10 , damage in soma and neurites induced by a could not be reversed by the tat - hafgf14154 treatment with a decrease in cell viability , which suggested that adam10 is a key target for tat - hafgf14154 to protect neurons from injury in the ad model . pischedda and piccoli also reported that pharmacological inhibition of adam10 results in impairment of neurite outgrowth , and this modulation is involved in the fgfr2-signaling pathway . recent progress in understanding the substrates and function as well as the regulation and cell biology of adam10 in the cns highlights the value of adam10 as a drug target in brain diseases . according to our results that tat - hafgf14154 promoted mrna expression of adam10 xbp1 efficiently upregulated adam10 expression ; a reduced activity or presence of xbp1 could potentially be associated with an increased plaque burden and progression of ad . pc12 cells overexpressing mxbp1s were fully protected at lethal doses of a oligomers ( 18 mg / ml ) and partially protected at higher concentrations of a. endogenous xbp1 plays a critical role in preventing a neurotoxicity , as shown by the exacerbated cell death induced by a in ad models when xbp1 was eliminated . ire1 cleaves xbp1 pre - mrna encoding for xbp1u in the cytoplasm , and this cytoplasmic splicing results in a new protein with transcriptional activity called xbp1s . mammalian ire1 initiates diverse downstream signaling of the upr either through splicing of xbp1 pre - mrna to maintain cell survival or through posttranscriptional modifications via regulated ire1-dependent decay ( ridd ) of multiple substrates , which triggers cell apoptosis.55 , 56 tat - hafgf14154 enhanced the ratio of xbp1s / xbp1u in the brain of app / ps1 mice and primary neurons injured by a , indicating that splicing xbp1 was activated by tat - hafgf14154 . nuclear translocation of active xbp1s could be improved by the overexpression of pi3k regulatory subunits p85 , and deletion of p85 showed a reduced accumulation of xbp1s in the nucleus , revealing a link between pi3k and the ire1/xbp1 pathway . the pi3k pathway is one of the most important intracellular signaling cascades induced by afgf too . phosphorylation of pi3k was upregulated by tat - hafgf14154 in primary neurons damaged by a , but no change of pi3k expression was found . it meant that tat - hafgf14154 activated the pi3k pathway but did nt influence the translocation of xbp1s . creb is critically involved in the regulation of synaptic plasticity , intrinsic excitability , and long - term memory formation . its activation exhibits neuroprotection via the pi3k / akt / creb pathway in ad models.38 , 39 creb is also activated by er stress : a chromatin immunoprecipitation assay indicated that creb binds to the promoter region of ire1 genes and regulates its expression . expression of ire1 was increased concomitantly with creb phosphorylation in human glioma cells treated with nitric oxide ( no ) . phosphorylation of creb was upregulated by tat - hafgf14154 , and no change of creb expression was found in primary neurons injured by a. the activation of the creb pathway and the splicing of xbp1 induced by tat - hafgf14154 were blocked by pi3k inhibitor , resulting in a disappearing rescue of cell viability and morphological damage . these results suggest that the activation of the pi3k pathway is related to the activation of creb and the downstream splicing of xbp1 induced by ire1. in conclusion , tat - hafgf14154 treatment significantly ameliorated the behavioral deficits of app / ps1 mice , relieved a burden , and increased the expression and activity of adam10 in the brain . tat - hafgf14154 antagonized a142-induced cell death and structure damage in primary neurons in an adam10-dependent manner following the activation of xbp1 splicing and the pi3k - creb pathway . as shown in figure 8 , tat - hafgf14154 binds to its receptor , activating the pi3k pathway , leading to creb activation followed by the splicing of xbp1u mrna to xbp1s mrna in the er . the xbp1s mrna generated is translated to active xbp1s protein , a transcription factor for adam10 . these results highlight the important role of adam10 as a key target in neuroprotection of tat - hafgf14154 , suggesting that tat - hafgf14154 has the potential to be an alternative therapy to attenuate the ad pathological process.figure 8signaling pathway for tat - hafgf14154 neuroprotectiontat - hafgf14154 binds to its receptor , activating the pi3k pathway , leading to creb activation followed by the splicing of xbp1u mrna to xbp1s mrna in the er . the xbp1s mrna generated is translated to active xbp1s protein , a transcription factor for adam10 . the nuclear translocation of active xbp1s can be promoted by the pi3k regulatory subunit p85,35 , 36 , 37 but tat - hafgf14154 does not alter the level of p85. ly294002 inhibits the phosphorylation of pi3k and the splicing of xbp1 , inhibiting the expression of adam10 . signaling pathway for tat - hafgf14154 neuroprotection tat - hafgf14154 binds to its receptor , activating the pi3k pathway , leading to creb activation followed by the splicing of xbp1u mrna to xbp1s mrna in the er . the xbp1s mrna generated is translated to active xbp1s protein , a transcription factor for adam10 . the nuclear translocation of active xbp1s can be promoted by the pi3k regulatory subunit p85,35 , 36 , 37 but tat - hafgf14154 does not alter the level of p85. ly294002 inhibits the phosphorylation of pi3k and the splicing of xbp1 , inhibiting the expression of adam10 . recombinant fusion protein tat - hafgf14154 was expressed and purified from our lab as reported previously.14 , 18 liposomes were produced by the method of ph gradient . tat - hafgf14154 was incubated with liposomes in different ratios ( 1:100 , 1:50 , 1:10 , 1:5 , 1:2 , and 4:5 ) for 30 min at 25c or 30c . encapsulation efficiency of cationic liposomes was calculated by a formula : encapsulation efficiency ( q ) % = ( ctotal cfree)/ctotal 100 . free tat - hafgf14154 ( cfree ) and total tat - hafgf14154 ( ctotal ) were separated by microcolumn centrifugation method . the content of tat - hafgf14154-loaded cationic liposomes was assayed by human fgf acidic elisa kit ( r&d systems ) , according to the manufacturer s instructions . coomassie blue staining was applied after electrophoresis with 12% sds - page to detect sds - resistant polymerization of tat - hafgf14154 after 0 , 0.5 , or 1 hr incubation with cationic liposomes . appswe / ps1d9 double - transgenic mice ( app / ps1 ) and wild - type background - matched mice ( 7 months old ) were purchased from beijing huafukang biotechnology . mice were kept under conditions of controlled temperature ( 24c 1c ) , humidity ( 50%60% ) , and a light / dark cycle of 12/12 hr ( light on at 8:00 a.m. ) . aliquots of 0.9% saline ( vehicle ) , 600 g / kg tat - hafgf14154 ( t600 ) , and 600 g / kg tat - hafgf14154-loaded cationic liposomes ( l + t600 ) were intranasally administered as a single dose to wild - type mice , which were then sacrificed in after 30 min . app / ps1 mice were treated with the same doses of the same agents by intranasal delivery every 2 days for 6 weeks according to the modified method of capsoni . all experiments were conducted according to the guidelines for animal care and use of china , and they were approved by the animal ethics committee of the chinese academy of medical science . after treatment for 5 weeks , learning and memory abilities of mice were assayed by mwm . the maze consisted of a steel pool ( 120-cm diameter ) filled with opaque water at 22c 1c and a steel platform ( 10 cm ) . an overhead video camera connected to ethovsion xt ( noldus information technology ) was used to track the movement of mice and record all trials . in the place navigation test , the hidden platform ( 1 cm under water level ) was kept constant in the middle of one quadrant throughout training . the training consisted of 6-day trials ( one trial per quadrant and four quadrants per day , with a 20-min interval between each trial ) . each trial lasted until the mouse climbed onto the hidden platform target within 60 s , and the escape latency onto the platform and the path length were recorded . the spatial probe test without the platform was conducted 24 hr after completing the last training trial ( day 5 ) . each mouse was put into the pool for 60 s from the most distant to the target quadrant , and the percentage of time spent on each quadrant and times of crossing the removed - platform area were measured . mice were transferred to individual cages with bedding but no environmental enrichment items 1 hr before the dark phase commenced . the mouse was given access to a piece of cotton gauze ( 5-cm squares , weighing 3 g ) in each cage . the next morning the nesting score of each mouse was calculated according to a standard five - point nest - rating scale . the chamber was a brightly and evenly illuminated square area ( 50 50 25 cm ) made of white glacial polyvinyl chloride and illuminated with four 60-w lamps ( mounted 1.5 m above ) . mice were placed individually in the center of the of , left to explore for 5 min , and videotaped under white illumination . time spent in the center , number of crossings through the central area , and total distance movement were recorded and analyzed by ethovision xt ( noldus information technology ) . the right hemisphere of app / ps1 mice was fixed in 4% paraformaldehyde ( guoyao ) and embedded with paraffin . coronal paraffin sections ( 6-m thick ) were dewaxed , rehydrated , and treated with 3% h2o2/methanol solution for 10 min . then , sections were boiled for 15 min in 0.01 m citrate buffer ( ph 6.0 ) . after the citrate buffer returned to room temperature , sections were washed with pbs and incubated in blocking solution ( 5% bsa ) for 1 hr at 37c , followed by being incubated overnight with primary antibodies at 4c in a humidified chamber . primary antibodies included anti - synaptophysin ( 1:250 , abcam ) and anti - gap43 ( 1:250 , abcam ) rabbit antibodies and mouse monoclonal anti - a antibody ( 6e10 , 1:500 , covance ) . after being washed with pbs , the sections were incubated with anti - mouse or rabbit igg - horseradish peroxidase ( hrp ) secondary antibody for 1 hr at 37c , followed by diaminobenzidine ( dab ) ( boster biotech ) and hematoxylin staining . slides were assessed by light microscope ( ix71 , olympus ) and micrographs were analyzed by image - pro plus 6.0 software . tissue homogenates and cultured cells were lysed in radioimmunoprecipitation assay ( ripa ) lysis buffer ( beyotime ) with pmsf ( sigma - aldrich ) and protease inhibitor cocktail ( roche ) on ice . lysates in supernatant were separated by centrifuging at 13,000 rpm at 4c for 15 min . the concentration of total protein was measured by bca protein assay kit ( thermo fisher scientific ) . samples were electrophoresed in nupage 4%12% bis - tris gels ( thermo fisher scientific ) with nupage mes sds running buffer ( thermo fisher scientific ) , and they were electrophoretically transferred to polyvinylidene difluoride ( pvdf ) membranes ( millipore ) . after being dipped in 0.2% glutaraldehyde ( sigma - aldrich ) for 30 min , membranes were blocked for 12 hr at room temperature and then incubated at 4c overnight with the following primary antibodies : mouse afgf monoclonal antibody ( 1:1,000 , sigma ) , rabbit anti - synaptopysin antibody ( 1:1,000 , abcam ) , rabbit anti - gap43 antibody ( 1:1,000 , abcam ) , 6e10 mouse monoclonal antibody ( 1:2,000 , covance ) , adam10 rabbit monoclonal antibody , bace1 rabbit monoclonal antibody , ps1 rabbit monoclonal antibody ( 1:1,000 , abcam ) , anti - app(c - terminal ) rabbit monoclonal antibody ( 1:4,000 , sigma ) , phospho - pi3k p85 / p55 ( tyr 467/tyr 199 ) rabbit antibody , pi3 kinase p85 / p55 rabbit monoclonal antibody ( 1:1,000 , bioworld ) , -actin rabbit monoclonal antibody ( 1:1,000 , cell signaling technology ) , xbp-1 rabbit polyclonal antibody ( 1:1,000 , abcam ) , creb rabbit monoclonal antibody ( 1:1,000 , cell signaling technology ) , phospho - creb ( ser 133 ) rabbit monoclonal antibody ( 1:1,000 , cell signaling technology ) , and gapdh rabbit monoclonal antibody ( 1:1,000 , cell signaling technology ) . after incubation with secondary antibody ( peroxidase - conjugated affinipure goat anti - rabbit / mouse igg , 1:2,000 , proteintech ) for 2 hr at room temperature , the blots were detected with supersignal west pico chemiluminescent substrate ( pierce biotechnology ) . activities of adam10 and bace1 were measured by sensolyte 520 adam10 activity assay kit ( anaspec ) and -secretase ( bace1 ) activity detection kit ( sigma - aldrich ) , according to the manufacturers instructions . primary cortical neurons were separated from brains of newborn sprague - dawley ( sd ) rats . tissues were cut rapidly into small pieces in d - hank s buffer , then digested with 0.25% trypsin for 10 min at 37c . dmem ( high glucose , thermo fisher scientific ) containing 10% fetal bovine serum ( fbs ) ( thermo fisher scientific ) was then added . the cell suspension was filtered , then centrifuged at 250 g for 5 min . cells were cultured in neurobasal - a medium ( thermo fisher scientific ) supplemented with 2% b-27 supplement ( 50 , serum free , thermo fisher scientific ) at 37c . small tissue mass and dead cells were removed by changing culture medium 46 hr after seeding . neurons ( 5 div ) were pre - treated with tat - hafgf14154 ( 60 , 300 , and 1,500 ng / ml ) for 2 hr at 37c , and 1.5 m a142 ( millipore ) was added into the culture medium with continuous tat - hafgf14154 treatment for 48 hr . a142 was dissolved in 0.1% dmso and incubated in 37c for 24 hr . in some cases , cells were treated with 50 nm pi3k inhibitor ( ly294002 , cell signaling technology ) 30 min before tat - hafgf14154 treatment . for viability analysis , cells were seeded in a 96-well culture plate at a density of 1 10 cells / well . after treatments , cells were incubated with 10 ml 5 mg / ml methyl thiazolyl tetrazolium ( mtt ) ( sigma ) for 4 hr at 37c . the supernatants were aspirated , and the formazan precipitates were solubilized by the addition of 100 l dmso per well . absorbance at 570 nm was monitored by a multiskan go spectrophotometer ( thermo scientific ) . for apoptosis analysis , cells were seeded in a six - well culture plate at a density of 1.5 10 cells / well . after treatments , cells were washed with pbs and fixed with 4% paraformaldehyde for 10 min at room temperature , then exposed to hoechst 33342 ( 5 g / ml ) images were recorded by interactive laser cytometer ( lsm 710 meta , carl zeiss ) . cells were seeded on 15-mm circular glass coverslips ( nest ) in a 24-well culture plate . after treatments , samples were fixed with glutaraldehyde ( sigma - aldrich ) overnight at 4c , followed by being rinsed and dehydrated in an alcohol gradient ( 30% , 50% , 70% , and 90% for 3 min each and 100% for 15 min ) . cells on the coverslips were incubated with isoamyl acetate ( dongzheng ) for 5 min twice , dried by a critical point dryer ( emitech k850x , quorum ) , followed by gold - palladium sputtering . the coverslips were fixed on specimen mount with conductive adhesive , and the cell surfaces were examined by scanning electron microscope ( carl zeiss ) . to detect the location and expression of synaptic proteins , primary cortical neurons were seeded in 35-mm confocal dishes ( corning ) at a density of 1.5 10 cells / well . after treatments , cells were fixed with 4% paraformaldehyde ( pfa ) for 15 min and permeabilized with 0.1% triton x-100 ( sigma ) for 20 min at room temperature , followed by incubation with blocking buffer ( beyotime ) for 1 hr at room temperature . then , samples were incubated with primary antibodies overnight at 4c , including rabbit anti - gap43 antibody ( 1:250 , abcam ) and rabbit anti - synaptopysin antibody ( 1:250 , abcam ) . after being washed with pbs , cells were incubated with anti - rabbit igg - h&l secondary antibody ( dylight 488 , 1:200 , abcam ) for 1 hr at 37c and kept in the dark , followed by dapi nuclear staining ( 0.5 g / ml , beyotime ) for 15 min . images of cells were recorded by interactive laser cytometer ( lsm 710 meta , carl zeiss ) , and micrographs were analyzed using image - pro plus 6.0 software . total rna of cultured cells was extracted with hipure total rna mini kit ( magen ) and quantified by nanodrop 2000 ( thermo fisher scientific ) . the iscript cdna synthesis kit ( bio - rad ) was used to synthesize the first - strand cdna . the qpcr was performed with ssoadvanced universal supermixes ( bio - rad ) by cfx96 connect real - time pcr detection system ( bio - rad ) . -actin was used as an internal control and its primers were cccgcgagtacaaccttcttg ( forward ) and tcatccatggcgaactggtgg ( reverse ) . the sirna duplexes against adam10 were designed by us and synthesized by genepharma with the following sequences : ccagcagagagauauauuatt ( sense ) and uaauauaucucucugcuggtt ( anti - sense ) . scrambled - sirna sequences were uucuccgaacgugucacgutt ( sense ) and acgugacacguucggagaatt ( anti - sense ) . primary cortical neurons were seeded on six - well plates at a density of 1.5 10 cells / well . cells ( 5 div ) were transfected with 20 nm scrambled - sirna or targeted - sirna using lipofectamine rnaimax reagent ( invitrogen ) . the knockdown efficiency was validated by qrt - pcr and western blot after sirnas were transfected for 48 or 72 hr . one - way anova followed by bonferroni post hoc test or two - way anova followed by bonferroni multiple comparison tests was used for data analyses with statistics software ( spss 19.0 ) . all experimental data represent means sem , and p < 0.05 was considered statistically significant .

acid fibroblast growth factor ( afgf ) has shown neuroprotection in alzheimer s disease ( ad ) models in previous studies , yet its mechanism is still uncertain . here we report that the efficacy of tat - hafgf14154 is markedly increased when loaded cationic liposomes for intranasal delivery are intranasally administered to app / ps1 mice . our results demonstrated that liposomal tat - hafgf14154 treatment significantly ameliorated behavioral deficits , relieved brain a burden , and increased the expression and activity of disintegrin and metalloproteinase domain - containing protein 10 ( adam10 ) in the brain . tat - hafgf14154 antagonized a142-induced cell death and structural damage in rat primary neurons in an adam10-dependent manner , which , in turn , was promoted by the activation of xbp1 splicing and modulated by the pi3k - creb pathway . both knockdown of adam10 and inhibition of pi3k ( ly294002 ) negated tat - hafgf14154 rescue . thus , tat - hafgf14154 activates the ire1/xbp1 pathway of the unfolded protein response ( upr ) against the endoplasmic reticulum ( er ) stress induced by a , and , subsequently , the nuclear translocation of spliced xbp1 ( xbp1s ) promotes transcription of adam10 . these results highlight the important role of adam10 and its activation through the pi3k - creb - ire1/xbp1 pathway as a key factor in the mechanism of neuroprotection for tat - hafgf14154 .

Introduction Results Discussion Materials and Methods Author Contributions Conflicts of Interest

hafgf showed a neuroprotective effect against brain injury resulting from focal ischemia - reperfusion in rats,13 , 14 and it repaired human spinal cord injury in a clinical trial.15 , 16 it is reported that the concentration of acid fibroblast growth factor ( afgf ) is increased in the serum and cerebrospinal fluid of patients with ad . tat - ptd could increase the concentration of hafgf in brain , and tat - hafgf14154 improved cognition and reduced a plaques more significantly than hafgf14154 in senescence - accelerated mouse prone-8 ( samp8 ) and app / ps1 mice.22 , 23 meanwhile , liposomes have gained increasing attention as a promising strategy for brain - targeted drug delivery due to their large delivery capabilities , capacity for surface decoration , low toxicity , and biocompatibility with biodegradability . in the current study , tat - hafgf14154 was encapsulated with cationic liposomes , and it was delivered to the app / ps1 double - transgenic mouse model for ad via intranasal administration to investigate neuroprotection . results demonstrated that tat - hafgf14154 ameliorated the ad phenotype by increasing adam10 expression at the transcriptional level through the pi3k - creb - ire1/xbp1 pathway , thereby enhancing adam10 enzyme activity . intranasal treatment of tat - hafgf14154-loaded cationic liposomes demonstrated a higher efficiency for the delivery of afgf compared with tat - hafgf14154 alone , as evidenced by the significantly elevated levels of afgf in the ec ( p < 0.01 for l + t600 versus t600 ; figure s1e ) . tat - hafgf14154-loaded cationic liposomes were delivered intranasally to 7-month - old app / ps1 mice every 2 days for 6 weeks , followed by a morris water maze ( mwm ) test in the fifth week and nest construction ( nc ) and open field ( of ) tests in the sixth week ( figure 1a ) . these results indicate that tat - hafgf14154-loaded cationic liposomes significantly attenuated cognitive behavior deficits and that the liposome tat - hafgf14154 preparation was more potent than tat - hafgf14154 alone.figure 1tat - hafgf14154-loaded cationic liposomes attenuated cognitive behavioral deficits in app / ps1 mice(a ) schematic of the protocol followed for the experiments . aliquots of 0.9% saline ( vehicle ) , tat - hafgf14154 ( 600 g / kg , t600 ) , and tat - hafgf14154-loaded cationic liposomes ( 600 g / kg , l + t600 ) were delivered intranasally to 7-month - old app / ps1 mice every 2 days for 6 weeks ( n = 8) . tat - hafgf14154-loaded cationic liposomes attenuated cognitive behavioral deficits in app / ps1 mice ( a ) schematic of the protocol followed for the experiments . aliquots of 0.9% saline ( vehicle ) , tat - hafgf14154 ( 600 g / kg , t600 ) , and tat - hafgf14154-loaded cationic liposomes ( 600 g / kg , l + t600 ) were delivered intranasally to 7-month - old app / ps1 mice every 2 days for 6 weeks ( n = 8) . these results indicate that tat - hafgf14154-loaded cationic liposomes decreased a burden in the brain of app / ps1 mice and were more effective than tat - hafgf14154 alone.figure 2tat - hafgf14154-loaded cationic liposomes reduced a loading in the brain of app / ps1 mice(a ) representative images of a plaques in coronal section and ca1 area of brain ( scale bar , 80 m ) . tat - hafgf14154-loaded cationic liposomes reduced a loading in the brain of app / ps1 mice ( a ) representative images of a plaques in coronal section and ca1 area of brain ( scale bar , 80 m ) . to determine whether the reduction of a loading could be due to a change in app - processing modulation , the expression of app and its processing proteins ( adam10 , bace1 , and ps1 ) in cortex and hippocampus was measured by western blotting ( figure 3a ) . these results revealed that the reduction of a loading in the brain of app / ps1 mice treated with tat - hafgf14154-loaded cationic liposomes could be due to the enhancement of adam10 in expression and activity.figure 3tat - hafgf14154-loaded cationic liposomes enhanced adam10 expression and activity in the brain of app / ps1 mice(a i ) representative immunoblots ( a ) and quantification expression of app - processing proteins in cortex ( b e ) and hippocampus ( f i ) of app / ps1 mice . tat - hafgf14154-loaded cationic liposomes enhanced adam10 expression and activity in the brain of app / ps1 mice ( a i ) representative immunoblots ( a ) and quantification expression of app - processing proteins in cortex ( b e ) and hippocampus ( f i ) of app / ps1 mice . nuclear staining ( hoechst 33342 ) revealed that the prevalence of karyopyknosis or apoptotic body formation induced by a142 was diminished by tat - hafgf14154 in a concentration - dependent manner ( p < 0.05 for t60 + a , p < 0.001 for t300 + a , and p < 0.001 for t1500 + a ; figures 4b and 4c ) . as showed in micrographs , somata were flat and fractured and neurites were fragmented in neurons injured by a. this damaged appearance was reversed by the treatment with tat - hafgf14154 in a concentration - dependent manner.figure 4tat - hafgf14154 protected neurons from damage induced by a142primary cortical neurons were isolated from brains of newborn sd rats . both assays revealed that levels of syn and gap43 were suppressed by a ( p < 0.05 or p < 0.01 ) but that this was successfully rescued by the tat - hafgf14154 treatment ( p < 0.01 or p < 0.001 ) . the expression of adam10 , both mrna and protein , in primary neurons was significantly enhanced by tat - hafgf14154 ( p < 0.05 for t300 + a and p < 0.01 for t1500 + a in mrna level , figure 5a ; p < 0.01 for t1500 + a in protein level , figure 5c ) . the enhancement of adam10 in expression and enzyme activity both in vivo ( figure 3 ) and in vitro indicated that adam10 might be a key target for tat - hafgf14154 to protect neurons from injury in ad models.figure 5tat - hafgf14154 enhanced expression and activity of adam10 in primary neurons injured by a142expressions of adam10 mrna and protein levels were assayed by qrt - pcr and western blotting , respectively . tat - hafgf14154 enhanced expression and activity of adam10 in primary neurons injured by a142 expressions of adam10 mrna and protein levels were assayed by qrt - pcr and western blotting , respectively . the sirna knockdown blocked the elevation of adam10 induced by tat - hafgf14154 ( p < 0.01 for a and p < 0.001 for kd + t1500 + a ; figure 6b ) , and it significantly impaired the ability of t1500 to rescue a toxicity ( p < 0.01 ; figure 6c ) and a damage to soma and neurites ( figure 6d).figure 6knockdown of adam10 by sirna blocks tat - hafgf14154 neuroprotectionneurons were treated with or without a142/tat - hafgf14154/sirna as described above . x - box - binding protein 1 ( xbp1 ) is one of the transcription factors of adam10 , and it is also linked with the unfolded protein response ( upr ) . spliced xbp1 protein ( xbp1s ) is the activated form of xbp1 , and the ratio of xbp1s and unspliced xbp1 ( xbp1u ) reflects the activity of xbp1 . inositol - requiring enzyme 1 ( ire1 ) , one of the three endoplasmic reticulum ( er ) stress sensors , can active xbp1 by splicing xbp1u to xbp1s . regulatory subunits p85 of pi3k promote the nuclear translocation of xbp1s.35 , 36 , 37 camp response element - binding protein ( creb ) is activated in ad models by pi3k / akt , and its phosphorylation is neuroprotective.38 , 39 creb is also activated by er stress , and it binds to the promoter of the ire1 gene and regulates its expression . based on these findings , we hypothesized that tat - hafgf14154 promotes the expression of adam10 through this pi3k - creb - ire1/xbp1 pathway . levels of xbp1u , xbp1s , p - pi3k ( p - p85 and tyr467 ) , pi3k ( p85 ) , p - creb ( ser133 ) , and creb were assayed by western blotting , and the effects of pi3k inhibitor ( ly294002 ) were assessed ( figures 7a7e ) . after treatment with tat - hafgf14154 , xbp1s / xbp1u , phosphorylation of pi3k ( p - pi3k / p - p85 and tyr467 ) , and phosphorylation of creb ( p - creb and ser133 ) were significantly increased in neurons injured by a ( p < 0.01 ) , and they were suppressed by pi3k inhibitor ( p < 0.001 in figures 7c and 7d and p < 0.01 in figure 7e ) . these results fueled our hypothesis that the pi3k - creb - ire1/xbp1 pathway was activated by tat - hafgf14154 . pi3k inhibition abolished the rise in adam10 expression seen in neurons treated with t1500 + a ( p < 0.01 for p + t1500 + a ; figures 7f and 7 g ) as well as thwarted hafgf14154 rescue of cell viability ( p < 0.001 for p + t1500 + a ; figure 7h ) and morphological damage ( figure 7i).figure 7tat - hafgf14154 upregulated the expression of adam10 via the pi3k - creb - ire1/xbp1 pathwayprimary neurons were pre - treated for 30 min with pi3k inhibitor ( ly294002 , p ) , followed by incubation with tat - hafgf14154 and a142 as described above . ( f and g ) upregulation of adam10 induced by tat - hafgf14154 was blocked by the pi3k inhibitor . tat - hafgf14154 upregulated the expression of adam10 via the pi3k - creb - ire1/xbp1 pathway primary neurons were pre - treated for 30 min with pi3k inhibitor ( ly294002 , p ) , followed by incubation with tat - hafgf14154 and a142 as described above . ( f and g ) upregulation of adam10 induced by tat - hafgf14154 was blocked by the pi3k inhibitor . in our study , tat - hafgf14154-loaded cationic liposomes were delivered to app / ps1 mice via intranasal administration to investigate neuroprotection of tat - hafgf14154 , and the related mechanism was explored in primary cortical neurons of rats injured by a142 . intranasal delivery has proven to be a new and noninvasive strategy , which can circumvent the bbb and facilitate direct transport of the drugs to the brain through neuronal and extracellular pathways.44 , 45 our previous studies showed that intranasal administration of tat - hafgf could improve cognition and reduce a deposits more significantly in app / ps1 mice compared with intravenous injection.18 , 21 , 22 , 23 in an animal model of parkinson s disease induced by 6-ohda , liposomes markedly assisted the delivery of basic fibroblast growth factor ( bfgf ) to the striatum and substantia nigra ( sn ) , and they enhanced the neuroprotective effects of bfgf on dopaminergic neurons . there was a marked increase of adam10 in both expression and activity in the brains of mice with treatment of tat - hafgf14154-loaded cationic liposomes . adam10-mediated non--amyloidosis processing via enhancing expression and activity of adam10 may be involved in the amelioration of behavioral deficits and the reduction of a burden in app / ps1 mice treated with tat - hafgf14154 . recent reports have provided several candidate drugs or therapeutic methods to attenuate pathology in ad ; most of them show its mechanism of modulating app processing so as to alter app toward non - amyloidogenic processing.30 , 47 , 48 , 49 , 50 overexpression of adam10 impressively reduced a plaque load and soluble a , and it additionally restored learning deficits in double - transgenic alzheimer model mice . with the upregulation of adam10 , tat - hafgf14154 dose - dependently enhanced cell viability , and it reversed damage in soma and neurites of neurons damaged by a. tat - hafgf14154 increased synaptic proteins syn and gap43 significantly both in vitro and in vivo , indicating that tat - hafgf14154 promoted nerve growth and possessed the potential of modulating synaptic plasticity . after sirna knockdown of adam10 , damage in soma and neurites induced by a could not be reversed by the tat - hafgf14154 treatment with a decrease in cell viability , which suggested that adam10 is a key target for tat - hafgf14154 to protect neurons from injury in the ad model . according to our results that tat - hafgf14154 promoted mrna expression of adam10 xbp1 efficiently upregulated adam10 expression ; a reduced activity or presence of xbp1 could potentially be associated with an increased plaque burden and progression of ad . mammalian ire1 initiates diverse downstream signaling of the upr either through splicing of xbp1 pre - mrna to maintain cell survival or through posttranscriptional modifications via regulated ire1-dependent decay ( ridd ) of multiple substrates , which triggers cell apoptosis.55 , 56 tat - hafgf14154 enhanced the ratio of xbp1s / xbp1u in the brain of app / ps1 mice and primary neurons injured by a , indicating that splicing xbp1 was activated by tat - hafgf14154 . nuclear translocation of active xbp1s could be improved by the overexpression of pi3k regulatory subunits p85 , and deletion of p85 showed a reduced accumulation of xbp1s in the nucleus , revealing a link between pi3k and the ire1/xbp1 pathway . phosphorylation of pi3k was upregulated by tat - hafgf14154 in primary neurons damaged by a , but no change of pi3k expression was found . phosphorylation of creb was upregulated by tat - hafgf14154 , and no change of creb expression was found in primary neurons injured by a. the activation of the creb pathway and the splicing of xbp1 induced by tat - hafgf14154 were blocked by pi3k inhibitor , resulting in a disappearing rescue of cell viability and morphological damage . these results suggest that the activation of the pi3k pathway is related to the activation of creb and the downstream splicing of xbp1 induced by ire1. in conclusion , tat - hafgf14154 treatment significantly ameliorated the behavioral deficits of app / ps1 mice , relieved a burden , and increased the expression and activity of adam10 in the brain . tat - hafgf14154 antagonized a142-induced cell death and structure damage in primary neurons in an adam10-dependent manner following the activation of xbp1 splicing and the pi3k - creb pathway . as shown in figure 8 , tat - hafgf14154 binds to its receptor , activating the pi3k pathway , leading to creb activation followed by the splicing of xbp1u mrna to xbp1s mrna in the er . these results highlight the important role of adam10 as a key target in neuroprotection of tat - hafgf14154 , suggesting that tat - hafgf14154 has the potential to be an alternative therapy to attenuate the ad pathological process.figure 8signaling pathway for tat - hafgf14154 neuroprotectiontat - hafgf14154 binds to its receptor , activating the pi3k pathway , leading to creb activation followed by the splicing of xbp1u mrna to xbp1s mrna in the er . the nuclear translocation of active xbp1s can be promoted by the pi3k regulatory subunit p85,35 , 36 , 37 but tat - hafgf14154 does not alter the level of p85. signaling pathway for tat - hafgf14154 neuroprotection tat - hafgf14154 binds to its receptor , activating the pi3k pathway , leading to creb activation followed by the splicing of xbp1u mrna to xbp1s mrna in the er . the nuclear translocation of active xbp1s can be promoted by the pi3k regulatory subunit p85,35 , 36 , 37 but tat - hafgf14154 does not alter the level of p85. aliquots of 0.9% saline ( vehicle ) , 600 g / kg tat - hafgf14154 ( t600 ) , and 600 g / kg tat - hafgf14154-loaded cationic liposomes ( l + t600 ) were intranasally administered as a single dose to wild - type mice , which were then sacrificed in after 30 min . app / ps1 mice were treated with the same doses of the same agents by intranasal delivery every 2 days for 6 weeks according to the modified method of capsoni .

teeth can be moved through the alveolar bone when appropriate orthodontic force is applied to them . this is mainly due to the fact that mechanical loading of a biological system results in strain , which subsequently leads to biological responses at the cellular and molecular level , aiming at adaptation of the system to the changed conditions . as a result of this principle , remodeling of the periodontal ligament ( pdl ) and recent research in the biological basis of tooth movement has provided detailed insight into cellular , molecular , and tissue - level reactions to orthodontic forces . the pressure - tension theory proposed by schwartz in 1932 is the simplest theory describing tooth movement on mechanical loading . on the pressure side , the biological events are as follows : disturbance of blood flow in the compressed pdl , cell death in the compressed area of the pdl ( hyalinization ) , resorption of the hyalinized tissue by macrophages , and undermining bone resorption by osteoclasts beside the hyalinized tissue , which ultimately results in tooth movement . on the tension side , blood flow is activated where the pdl is stretched , which promote osteoblastic activity and osteoid deposition , which later mineralizes . the fluid flow hypothesis , describing a mechanism by which osteocytes respond to mechanical forces , states that locally evoked strain derived from the displacement of fluid in the canaliculi is very important . when loading occurs , interstitial fluid is squeezed through the thin layer of the non - mineralized matrix surrounding the cell bodies and cell processes , resulting in local strain at the cell membrane and activation of the affected osteocytes . according to piezoelectric theory , there is production of piezoelectric signal on application of orthodontic force , which quickly reduces to zero . on removal of the mechanical force the possible sources of this electric current could be collagen , hydroxyapatite , or the mucopolysaccharide fraction of the ground substance . on application of orthodontic force , the alveolar bone adjacent to the tooth bends and the area of concavity accumulates negative charges , resulting in bone deposition . the small voltages thus generated are called streaming potentials . recently , it has been proposed that the pressure - tension theory is not simple and might involve more complicated biological tissue response , suggesting that bone apposition could be induced by ( 1 ) the load exerted by stretched fibers of the pdl , which may also induce a slight bending of the alveolar wall ; ( 2 ) direct resorption by unloading of the alveolar wall in the case of low forces ; and ( 3 ) indirect resorption as repair due to ischemia following the application of high forces . the force leading to a change in tissue pressure that approximated the capillary vessels blood pressure , thus preventing their occlusion in the compressed periodontal ligament . according to schwarz , forces well below the optimal level cause no reaction in the pdl . forces exceeding the optimal level would lead to areas of tissue necrosis , thereby preventing frontal bone resorption . tooth movement would thus be delayed until undermining resorption eliminates the necrotic tissue obstacle . prostaglandins ( pgs ) are a group of chemical messengers and are derivatives of arachidonic acid . prostaglandin e2 ( pge2 ) is a potent vasodilator and can increase the vascular permeability . it also has chemotactic properties and can stimulate the formation of osteoclasts , resulting in a subsequent increase in bone resorption . pgs are produced through two different pathways by the action of the enzyme cyclooxygenase on arachidonic acid : the constitutive isoform or cyclooxygenase-1 ( cox-1 ) and the inducible isoform or cyclooxygenase-2 ( cox-2 ) . the cox family of enzymes consists of two proteins that convert arachidonic acid , a 20-carbon polyunsaturated fatty acid comprising a portion of the plasma membrane phospholipids of most cells , to pgs . the constitutive isoform ( cox-1 ) is found in almost all tissues and is tissue protective . in contrast , cox-2 , the inducible isoform of cox , appears to be limited in basal conditions within most tissues , and de novo synthesis is activated by cytokines , bacterial lipopolysaccharides , or growth factors to produce pgs in large amounts during inflammatory processes occurring due to cell injury . it has been found that pgs have an important role in promoting bone resorption . although the exact role of pgs in bone resorption is not clear , it is thought to do so by stimulating cells to produce cyclic adenosine monophosphate , which is an important chemical messenger for bone resorption . research proved that the application of orthodontic force increased the synthesis of pgs , which in turn stimulated osteoclastic bone resorption . a similar study on cats by davidovitch et al , also showed increased levels of pge2 in the alveolar bone , as a result of application of orthodontic force . the histological data were supported by the finding of chumbley et al , that indomethacin , an inhibitor of pg synthesis , also inhibited orthodontic tooth movement . leiker et al studied the long - term effects of varying concentrations and frequencies of injectable , exogenous pge2 on the rate of tooth movement in rats and reported that injections of exogenous pge2 over an extended period of time in rats did enhance the amount of tooth movement . however , there was an increase in the amount of root resorption with increasing numbers and concentrations of the pge2 injections , which could be a potential concern . sekhavat et al , reported that misoprostol was effective in enhancing tooth movement in doses as low as 1025 g / kg , twice daily . it was also noticed that misoprostol did not significantly increase the amount of root resorption . they suggested that oral misoprostol could be used to enhance orthodontic tooth with minimal root resorption . however , long - term studies are needed to justify the use of pgs to accelerate orthodontic tooth movement in clinical practice . orthodontic forces result in capillary vasodilatation in the pdl , resulting in migration of inflammatory cells as well as cytokine production by these cells . cytokines are proteins acting as signals between the cells of the immune system , produced during the activation of immune cells and usually act locally although some act systemically with overlapping functions . cytokines like interleukin-1 ( il-1 ) , interleukin-6 ( il-6 ) , interleukin-8 ( il-8 ) , and tumor necrosis factor- ( tnf- ) have been proved to be associated with bone remodeling . il-1 predominantly exists in two forms , and , of which il-1 is the form mainly involved in bone metabolism , stimulation of bone resorption , and inhibition of bone formation . il-1 also plays a central role in the inflammatory process , and large amounts of il-1 are present in inflamed gingival tissues . they are released within 12 to 24 h after orthodontic force application and play an important role in initiating bone resorption and tooth movement . it was found that both macrophages and neutrophils predominate in il-1 production in inflamed gingival tissues . the staining of feline pdl cells for il-1 showed the presence of bound signal complexes in the plasma membrane , which was expected , as it is known that receptors for il-1 are present on fibroblasts . il-6 is produced by both lymphoid and non - lymphoid cells and can induce osteoclastic bone resorption through an effect on osteo - clastogenesis . il-6 has been identified in human gingival tissues and cells ; and participates in the molecular events associated with inflammatory periodontal diseases and tissue destruction in periodontitis . the levels of il-6 increase significantly 24 h after mechanical loading and play an important role in initiating tooth movement . after the application of force , the induction of both il-1 and il-6 was observed to reach a maximum on day 3 and to decline thereafter . tnf- is a pro - inflammatory cytokine that is often over - expressed in periodontitis and is responsible for alveolar bone resorption during periodontal breakdown . the possibility that tnf- is involved in normal physiological processes is supported by its function in osteoclastogenesis . receptor activator of nuclear factor kappa - b ligand ( rankl ) and its receptor receptor activator of nuclear factor k b ( rank ) , which are present on osteoblasts and precursor osteoclasts , respectively , have been identified as the key factors that stimulate osteoclast formation . recent studies analyzing the cytokine expression pattern in compression and tension sides of the pdl during orthodontic tooth movement in humans , by means of real - time polymerase chain reaction , found that both the pressure and tension sides showed higher expression of all the cytokines when compared to the pdl of normal teeth which served as control . the compression side exhibited higher expression of tnf- , rankl , and matrix metalloproteinase i ( mmp-1 ) , whereas the tension side presented higher expression of il-10 , tissue inhibitor of mmp - i(timp-1 ) , type i collagen , osteoprotegerin ( opg ) , and osteocalcin . these findings strongly suggest that tnf- plays a pivotal role in the bone resorption process , thus helping in orthodontic tooth movement . it is now clear that rankl , together with macrophage - colony stimulating factor , is required for osteoclast formation from precursor monocytes and macrophages . the natural inhibitor of rank - rankl interactions is the soluble tnf receptor - like molecule opg , which binds to rankl and prevents its ligation , thereby preventing osteoclast differentiation and activation . the rankl protein was found to be predominant in inflammatory cells around inflamed tissues adjacent to areas of pathological bone loss in periodontal disease and is associated with the progress of periodontal disease . prostaglandins ( pgs ) are a group of chemical messengers and are derivatives of arachidonic acid . prostaglandin e2 ( pge2 ) is a potent vasodilator and can increase the vascular permeability . it also has chemotactic properties and can stimulate the formation of osteoclasts , resulting in a subsequent increase in bone resorption . pgs are produced through two different pathways by the action of the enzyme cyclooxygenase on arachidonic acid : the constitutive isoform or cyclooxygenase-1 ( cox-1 ) and the inducible isoform or cyclooxygenase-2 ( cox-2 ) . the cox family of enzymes consists of two proteins that convert arachidonic acid , a 20-carbon polyunsaturated fatty acid comprising a portion of the plasma membrane phospholipids of most cells , to pgs . the constitutive isoform ( cox-1 ) is found in almost all tissues and is tissue protective . in contrast , cox-2 , the inducible isoform of cox , appears to be limited in basal conditions within most tissues , and de novo synthesis is activated by cytokines , bacterial lipopolysaccharides , or growth factors to produce pgs in large amounts during inflammatory processes occurring due to cell injury . it has been found that pgs have an important role in promoting bone resorption . although the exact role of pgs in bone resorption is not clear , it is thought to do so by stimulating cells to produce cyclic adenosine monophosphate , which is an important chemical messenger for bone resorption . research proved that the application of orthodontic force increased the synthesis of pgs , which in turn stimulated osteoclastic bone resorption . a similar study on cats by davidovitch et al , also showed increased levels of pge2 in the alveolar bone , as a result of application of orthodontic force . the histological data were supported by the finding of chumbley et al , that indomethacin , an inhibitor of pg synthesis , also inhibited orthodontic tooth movement . leiker et al studied the long - term effects of varying concentrations and frequencies of injectable , exogenous pge2 on the rate of tooth movement in rats and reported that injections of exogenous pge2 over an extended period of time in rats did enhance the amount of tooth movement . however , there was an increase in the amount of root resorption with increasing numbers and concentrations of the pge2 injections , which could be a potential concern . sekhavat et al , reported that misoprostol was effective in enhancing tooth movement in doses as low as 1025 g / kg , twice daily . it was also noticed that misoprostol did not significantly increase the amount of root resorption . they suggested that oral misoprostol could be used to enhance orthodontic tooth with minimal root resorption . however , long - term studies are needed to justify the use of pgs to accelerate orthodontic tooth movement in clinical practice . orthodontic forces result in capillary vasodilatation in the pdl , resulting in migration of inflammatory cells as well as cytokine production by these cells . cytokines are proteins acting as signals between the cells of the immune system , produced during the activation of immune cells and usually act locally although some act systemically with overlapping functions . cytokines like interleukin-1 ( il-1 ) , interleukin-6 ( il-6 ) , interleukin-8 ( il-8 ) , and tumor necrosis factor- ( tnf- ) have been proved to be associated with bone remodeling . il-1 predominantly exists in two forms , and , of which il-1 is the form mainly involved in bone metabolism , stimulation of bone resorption , and inhibition of bone formation . il-1 also plays a central role in the inflammatory process , and large amounts of il-1 are present in inflamed gingival tissues . they are released within 12 to 24 h after orthodontic force application and play an important role in initiating bone resorption and tooth movement . it was found that both macrophages and neutrophils predominate in il-1 production in inflamed gingival tissues . the staining of feline pdl cells for il-1 showed the presence of bound signal complexes in the plasma membrane , which was expected , as it is known that receptors for il-1 are present on fibroblasts . il-6 is produced by both lymphoid and non - lymphoid cells and can induce osteoclastic bone resorption through an effect on osteo - clastogenesis . il-6 has been identified in human gingival tissues and cells ; and participates in the molecular events associated with inflammatory periodontal diseases and tissue destruction in periodontitis . the levels of il-6 increase significantly 24 h after mechanical loading and play an important role in initiating tooth movement . after the application of force , the induction of both il-1 and il-6 was observed to reach a maximum on day 3 and to decline thereafter . tnf- is a pro - inflammatory cytokine that is often over - expressed in periodontitis and is responsible for alveolar bone resorption during periodontal breakdown . the possibility that tnf- is involved in normal physiological processes is supported by its function in osteoclastogenesis . receptor activator of nuclear factor kappa - b ligand ( rankl ) and its receptor receptor activator of nuclear factor k b ( rank ) , which are present on osteoblasts and precursor osteoclasts , respectively , have been identified as the key factors that stimulate osteoclast formation . recent studies analyzing the cytokine expression pattern in compression and tension sides of the pdl during orthodontic tooth movement in humans , by means of real - time polymerase chain reaction , found that both the pressure and tension sides showed higher expression of all the cytokines when compared to the pdl of normal teeth which served as control . the compression side exhibited higher expression of tnf- , rankl , and matrix metalloproteinase i ( mmp-1 ) , whereas the tension side presented higher expression of il-10 , tissue inhibitor of mmp - i(timp-1 ) , type i collagen , osteoprotegerin ( opg ) , and osteocalcin . these findings strongly suggest that tnf- plays a pivotal role in the bone resorption process , thus helping in orthodontic tooth movement . it is now clear that rankl , together with macrophage - colony stimulating factor , is required for osteoclast formation from precursor monocytes and macrophages . the natural inhibitor of rank - rankl interactions is the soluble tnf receptor - like molecule opg , which binds to rankl and prevents its ligation , thereby preventing osteoclast differentiation and activation . the rankl protein was found to be predominant in inflammatory cells around inflamed tissues adjacent to areas of pathological bone loss in periodontal disease and is associated with the progress of periodontal disease . the pdl lies between the cementum and alveolar bone , acting as a cushion to withstand mechanical forces applied to teeth . it is likely that pdl cells stimulated by forces of mastication , occlusal contact , and orthodontic treatment produce local factors that participate not only in the maintenance and remodeling of the ligament itself , but also in the metabolism of adjacent alveolar bone . application of a continuous force on the crown of the tooth leads to tooth movement within the alveolus that is marked initially by narrowing of the periodontal membrane , particularly in the marginal area . if the duration of movement is divided into an initial and a secondary period , direct bone resorption is found notably in the secondary period , when the hyalinized tissue has disappeared after undermining bone resorption . during the crucial stage of initial application of force it represents a sterile necrotic area , generally limited to 1 or 2 mm in diameter . the process displays three main stages : degeneration , elimination of destroyed tissue , and establishment of a new tooth attachment . a clear relationship between force level , timing , and extent of hyalinization even with a force as low as 5 cn , hyalinization occurred and the timing of the event seemed to be independent of the force level . an interesting finding from a recent study , however , was that an initially light and gradually increasing force resulted in less hyalinization than a heavier initial force that increased to the same end force level . it is prudent to use lighter forces for initiating orthodontic tooth movement and then gradually increasing the force levels , instead of using heavy force right at the start . in the secondary period of tooth movement , the osteoclasts attack the bone surface over a much wider area and , provided the force is kept within certain limits , further bone resorption will be predominantly of the direct type . the fibrous attachment apparatus is somewhat reorganized by the production of new periodontal fibrils . these are attached to the root surface and parts of the alveolar bone wall where direct resorption is not occurring by the deposition of new tissue , in which the fibrils become embedded . the main feature is the deposition of new bone on the alveolar surface from which the tooth is moving away . shortly after cell proliferation has started , osteoid tissue is deposited on the tension side . the original periodontal fibres become embedded in the new layers of osteoid , which mineralizes in the deeper parts . new bone is deposited until the width of the membrane has returned to normal limits , and simultaneously fibrous system is remodelled . concomitantly with bone apposition on the periodontal surface on the tension side , an accompanying resorption process occurs on the spongiosa surface of the alveolar bone . have proved that the expression and production of some inflammatory mediators ( pge2 , il-1 ) are promoted by mechanical stimulation of the pdl . pgs of the e series play an important role in the pathogenesis of chronic periodontitis by regulating production of osteoclast activating factor in activated lymphocytes . pge2 have an important role in orthodontic tooth movement , which has been conclusively proven in the literature . the induction of both il-1 and il-6 was observed to reach a maximum on day 3 after mechanical loading and declines thereafter . this shows that they have an important role in initiating bone resorption and subsequent tooth movement during orthodontic treatment . it must be acknowledged that any interference in the signaling pathway resulting in reduced production of pge2 , il-1 , and il-6 would significantly delay tooth movement , which might be the reason for differences in the rate of initial tooth movement in different patients . the same could be the reason for the initial delay in orthodontic tooth movement seen in adults when compared to adolescent patients . pdl cells , under mechanical stress , may induce secretion of osteoclasts through up - regulation of rankl expression via pge2 synthesis during orthodontic tooth movement . it has been shown that compressive force up - regulated rankl expression and induction of cox-2 in human pdl cells in vitro . it is a well - known fact that there is a local increase in pgs in the pdl and alveolar bone during orthodontic treatment . several studies have shown an arrest in tooth movement in experimental animals when non - steroidal anti - inflammatory drugs were administered . macrophages have the ability to produce cytokines , such as il-1 and il-6 , the levels of which are known to increase during orthodontic tooth movement . non - steroidal anti - inflammatory drugs must be avoided in orthodontic patients , as they would delay tooth movement and prolong the treatment duration . the number and distribution patterns of rankl- and rank - expressing osteoclasts change when excessive orthodontic force is applied to periodontal tissue , and il-1 and tnf- are expressed in osteoclasts in inflamed rat periodontal tissues . the presence of rankl in periodontal tissues , during experimental tooth movement of rat molars , shows that rankl is regulated by inflammatory cytokines in the pdl in response to mechanical stress . the rankl levels increase 24 h after mechanical loading and play an important role in initiating orthodontic tooth movement . compressive forces are more important for an increase in rankl levels compared to tensile forces . this indicates that the biomechanics used to initiate tooth movement could play a role in the increase in rankl levels and subsequent rate of tooth movement . showed that the local rankl gene transfer into the periodontal tissue significantly enhanced rankl expression and secretion of osteoclast in periodontal tissue without any systemic effects . it was conclusively proved that the transfer of rankl gene to the periodontal - tissue , activated osteoclast secretion and accelerated the amount of experimental tooth movement . they proposed that local rankl gene transfer might be a useful tool not only for shortening the duration of orthodontic treatment , but also for moving ankylosed teeth . long - term studies are needed to validate the effectiveness of the local rankl gene transfer as an useful tool to reduce the treatment time , by accelerating the rate of tooth movement in orthodontic patients undergoing fixed appliance treatment . the potential use of the local rankl gene transfer in moving ankylosed teeth has to be further evaluated to justify its use . it consist of the odontoblastic zone , cell free zone of weil , cell - rich zone , and the pulp core , which can be seen in a histological slide preparation . the principal cells of the pulp are odontoblast , fibroblast , undifferentiated ectomesenchymal cells , macrophages , and dendritic cells . the innervation is from the sensory afferent from trigeminal nerve and sympathetic branches from superior cervical ganglion . an extensive plexus of nerves in the cell - free zone is seen called the subodontoid plexus of rushkov . orthodontic forces not only tend to produce mechanical damage and inflammatory reactions in the periodontium but also cause cell damage , inflammatory changes , and circulatory disturbances in dental pulp . calcitonin gene - related peptide ( cgrp ) and substance p present in the dental pulp have been associated with the mediation of pulpal inflammation . cgrp is a major sensory neuropeptide which has been found to evoke the release of il-6 and il-8 from synovial fibroblasts in patients with rheumatoid arthritis . substance p is another sensory neuropeptide released from the peripheral endings of sensory nerves during inflammation , capable of modifying the secretion of pro - inflammation cytokines from immunocompetent cells , and has also been reported to induce the secretion of il-1 , il-6 , and tnf- from monocytes . the fact that the expression of cgrp and substance p is increased in dental pulp in response to bucally directed orthodontic tooth movement of the upper first molar in rats positively reinforces the fact that these neuropeptides might be involved in inflammation of the dental pulp at the time of orthodontic tooth movement . gingival crevicular fluid ( gcf ) is an inflammatory exudate present in the gingival sulcus . the aqueous component of gcf is primarily derived from the serum ; however , the gingival tissue through which the fluid passes , along with bacteria present in the tissue and gingival crevice , can modify its composition . therefore , its constituents , which are derived from a variety of sources , including microbial dental plaque , host inflammatory cells , host tissue , and serum , vary according to the condition of the periodontal tissues and the predominant bacterial flora present . in general , cells , immunoglobulins , lysosomal enzymes , microorganisms , and toxins can be detected in gcf , while the mechanism of bone resorption might also be related to the release of inflammatory mediators present in gcf . recently , a number of gcf constituents have been shown to be diagnostic markers of active tissue destruction in periodontal diseases although only a few studies have focused on those involved in bone remodeling during orthodontic tooth movement . during the course of orthodontic treatment , the forces exerted produce a distortion of the pdl extracellular matrix , resulting in alterations in cellular shape and cytoskeletal configuration . such events lead to the synthesis and presence in the deeper periodontal tissues of extracellular matrix components , tissue - degrading enzymes , acids , and inflammatory mediators ; induce cellular proliferation and differentiation ; and promote wound healing and tissue remodeling . glycosaminoglycans ( gag ) have been detected in gcf samples from sites around teeth affected by such conditions as chronic gingivitis , chronic periodontitis , and juvenile periodontitis . gcf collected from around the canine tooth subjected to orthodontic force showed an increase in the gag components particularly hyaluronic acid and chondroitin sulfate . the gcf concentrations of il-1 and il-6 were found to be significantly higher in a group with severe periodontal disease compared with controls . it was demonstrated that the gcf il-1 and tnf- levels had a positive correlation to mean pocket depths , which led to the suggestion that those cytokines may be involved in the pathogenesis of periodontal diseases . the gcf isolated from tooth , 24 h after being subjected to orthodontic forces , showed an increase in the levels of il-1 , il-6 , tnf- , epidermal growth factor and 2-microglobulin , when compared to controls . the level of pro - inflammatory cytokines such as il-1 , il-6 , and tnf- reached a significant level at 24 h after application of orthodontic forces . it was also found that force induced il-8 secretion from the pdl cells required the presence of il-1 in sufficient quantity . the lysosomal cysteine protease cathepsin b is known to play an important role in the resolution of organic matrix , a final step in bone resorption . during human orthodontic tooth movement , mechanically stimulated cathepsin b levels were analyzed with the help of fluorospectrometry and western blot analysis . the cathepsin b levels were found to be significantly increased when compared to the control teeth , which may be involved in extracellular matrix degradation in response to mechanical stress . immunocytochemical studies demonstrated that cathepsin b and cathepsin l were localized in the pdl of the rat molar and were expressed in compressed sites during experimental tooth movement . there was a three - fold increase in cathepsin b and four - fold increase in cathepsin this proves that cathepsin b and cathepsin l play an important role in initial orthodontic tooth movement by initiating bone resorption . the expression of cathepsin k , a novel collagenolytic enzyme specifically expressed in osteoclasts , was investigated in the rat maxillary teeth during experimental tooth movement by in situ hybridization histochemistry with a non - radioisotopic complementary ribonucleic acid ( crna ) probe for rat cathepsin k. cathepsin k messenger ribonucleic acid ( mrna ) expression was detected in the mono- and multi - nuclear osteoclasts on the pressure side of the alveolar bone at 12 h after force application , and the distribution and number of cathepsin k mrna - positive osteoclasts increased time - dependently on the pressure side . at 3 - 4 days , there was a marked increase in cathepsin k mrna - positive osteoclasts on both pressure and tension side of the alveolar bone in response to tooth movement . at 7 - 12 days , the cathepsin k mrna - positive osteoclasts on both sides had disappeared . these suggest that the recruitment of osteoclasts on the pressure side begins during the initial stage of orthodontic tooth movement and the site - specific early induction of cathepsin k mrna may cause an imbalance in the relative resorption activities on the pressure and tension side incident to such movement . the role of cathepsin k in orthodontic tooth movement and its mechanism of action could be made further clear by controlled experimental studies . mmps are enzymes that play a central role in pdl remodeling , both in physiological and in pathological conditions . collagenase-1 ( mmp-1 ) and collagenase-2 ( mmp-8 ) , because they share a unique ability to cleave native triple - helical interstitial collagens , can initiate this tissue remodeling . mmp-8 degranulation by polymorphonuclear leukocytes is among the pivotal factors in pathological collagen destruction during periodontal diseases . study done by apajalahti showed that mmp-2 levels significantly increased in the gcf 4 - 8 h after force application , while mmp-1 level failed to show any significant increase . ingman too found similar results when conducting the study by the immunofluorometric assay over a period of 1 month . there was a 12-fold increase in the orthodontic gcf when compared to the control gcf . the mmp-8 levels in orthodontic gcf were lower than those detected in gingivitis and periodontitis gcf , but significantly higher than in control gcf . this proves that mmps , particularly mmp-2 , play an important role in orthodontic tooth movement and tissue remodeling . cantarellaa found that compression force induced a significant increase of mmp-1 protein after 1 h ; the increase lasted until the third hour of force application and disappeared thereafter . the tension force induced significantly increased levels of the mmp-1 protein after just 1 h of force application . mmp-2 protein was induced by compression and increased significantly in a time - dependent fashion , reaching a peak after 8 h of force application . on the tension side , mmp-2 was significantly increased after 1 h , but gradually returned to basal levels within 8 h. immunolocalization of collagenase-3 ( mmp-13 ) done by leonardi showed that there was an increase in mmp-13 very early following the application of an orthodontic force in both pdl and alveolar bone . analysis of the gcf in patients with periodontal disease revealed that there was an increase in rankl and a decrease in the opg levels ; and subsequently the ratio of rankl concentration to that of opg in gcf samples was significantly higher for periodontal disease patients than for healthy subjects . this shows that both rankl and opg contribute to osteoclastic bone destruction in periodontal disease . experimental compressive forces on the pdl resulted in a 16.7-fold increase in rankl secretion and a 2.9-fold decrease in opg secretion when compared to the control . this confirms that the rankl / rank / opg system plays an important role in orthodontic tooth movement . this decrease was found to be significant during the first month of active tooth movement and started to stabilize later . batra found that the alkaline phosphatase activity in the gcf showed an increase during canine retraction which peaked during the 14th day of retraction followed by a significant fall in activity in the 21st day . it is one of the important enzymes required in initiating orthodontic tooth movement and sustaining it for a 2 week period . any defect in the production of this enzyme could interfere and delay orthodontic tooth movement . aspartate aminotransferase ( ast ) is a soluble enzyme that is normally confined to the cytoplasm of cells , but is released to the extra - cellular environment upon cell death . the activity levels of ast in the gcf are considered to be important in regulating alveolar bone resorption during orthodontic tooth movement . ever since the presence of ast enzyme in gcf has been demonstrated , several studies have observed that the levels of ast activity in gcf may reflect the magnitude of periodontal tissue destruction in periodontitis . however , there are only few studies which have investigated a possible role of ast activity levels in tissue remodeling incidental to orthodontic forces . ast activity in the gcf was found to be highest in the first week of orthodontic force application and there was a gradual reduction in the activity during the next 3 weeks . thus , it has the potential to serve as a biological marker to monitor orthodontic tooth movement . similarly , lactate dehydrogenase activity in the gcf can be used as a diagnostic tool for monitoring orthodontic tooth movement in clinical practice . leptin , a polypeptide hormone , has been classified as a cytokine and is mainly secreted from the adipose tissue in humans . leptin and its receptor share structural and functional similarities with members of the long - chain helical cytokines : il-6 , il-11 , il-12 , leukemia inhibitory factor , granulocyte - colony - stimulating factor , and oncostatin m. it has been suggested that leptin orchestrates the host response to inflammatory and infectious stimuli ; as it stimulates the immune system by enhancing cytokine production and phagocytosis by macrophages . thus , the overall increase in leptin during inflammation and infection indicates that leptin is part of the immune response and host defense mechanisms . since the presence of leptin within healthy and marginally inflamed gingiva has been demonstrated , several studies have observed that the levels of gcf leptin activity may play an important role in the development of periodontal disease . recently , it has been suggested that leptin plays a significant role in bone formation by virtue of its direct effect on osteoblast proliferation and differentiation , and in prolonging the life span of human primary osteoblasts by inhibiting apoptosis . leptin is also involved in anti - osteogenic effects by acting centrally on the hypothalamus . it was recently found that the concentration of leptin in gcf is decreased by orthodontic tooth movement and this conclusively proved that leptin may have been one of the mediators responsible for orthodontic tooth movement . il-17 has been found to be increased in patients with periodontitis , while it was barely detectable in sera from periodontally healthy individuals . the role of il-17 in orthodontic tooth movement and its potential to serve as a marker for validating the tooth movement remains a potential area for future research . the orthodontic displacement of a tooth is the result of a mechanical stimulus , generated by a force applied to the crown of a tooth , which results in an acute inflammatory response in periodontal tissues , which in turn may trigger the cascade of biological events associated with bone remodeling . this leads to the synthesis and release of various neurotransmitters , arachidonic acid , growth factors , metabolites , cytokines , colony - stimulating factors , and enzymes like cathepsin k , mmps , and ast , which are ultimately responsible for initiating bone remodeling and subsequent tooth movement . any interference with the release of these neurotransmitters and enzymes could delay or hamper orthodontic tooth movement . with the increased scope of gene therapy , the possibility that the rate of tooth movement could be increased by local gene transfer is very much real and could go a long way in reducing the treatment duration of orthodontic patients . however , well - designed experimental studies are needed for the same in order to evaluate their clinical efficiency and validate their use , as this is an era of evidence - based dentistry .

orthodontic force elicits a biological response in the tissues surrounding the teeth , resulting in remodeling of the periodontal ligament and the alveolar bone . the force - induced tissue strain result in reorganization of both cellular and extracellular matrix , besides producing changes in the local vascularity . this in turn leads to the synthesis and release of various neurotransmitters , arachidonic acid , growth factors , metabolites , cytokines , colony - stimulating factors , and enzymes like cathepsin k , matrix metalloproteinases , and aspartate aminotransferase . despite the availability of many studies in the orthodontic and related scientific literature , a concise integration of all data is still lacking . such a consolidation of the rapidly accumulating scientific information should help in understanding the biological processes that underlie the phenomenon of tooth movement in response to mechanical loading . therefore , the aim of this review was to describe the biological processes taking place at the molecular level on application of orthodontic force and to provide an update of the current literature .

INTRODUCTION INFLAMMATORY MEDIATORS IN THE PERIODONTAL LIGAMENT Prostaglandins Cytokines TISSUE RESPONSE TO MECHANICAL FORCES PULP TISSUE RESPONSE TO MECHANICAL FORCES GINGIVAL CREVICULAR FLUID DURING ORTHODONTIC TOOTH MOVEMENT CONCLUSION

teeth can be moved through the alveolar bone when appropriate orthodontic force is applied to them . this is mainly due to the fact that mechanical loading of a biological system results in strain , which subsequently leads to biological responses at the cellular and molecular level , aiming at adaptation of the system to the changed conditions . as a result of this principle , remodeling of the periodontal ligament ( pdl ) and recent research in the biological basis of tooth movement has provided detailed insight into cellular , molecular , and tissue - level reactions to orthodontic forces . the pressure - tension theory proposed by schwartz in 1932 is the simplest theory describing tooth movement on mechanical loading . on the pressure side , the biological events are as follows : disturbance of blood flow in the compressed pdl , cell death in the compressed area of the pdl ( hyalinization ) , resorption of the hyalinized tissue by macrophages , and undermining bone resorption by osteoclasts beside the hyalinized tissue , which ultimately results in tooth movement . the fluid flow hypothesis , describing a mechanism by which osteocytes respond to mechanical forces , states that locally evoked strain derived from the displacement of fluid in the canaliculi is very important . when loading occurs , interstitial fluid is squeezed through the thin layer of the non - mineralized matrix surrounding the cell bodies and cell processes , resulting in local strain at the cell membrane and activation of the affected osteocytes . according to piezoelectric theory , there is production of piezoelectric signal on application of orthodontic force , which quickly reduces to zero . on removal of the mechanical force the possible sources of this electric current could be collagen , hydroxyapatite , or the mucopolysaccharide fraction of the ground substance . on application of orthodontic force , the alveolar bone adjacent to the tooth bends and the area of concavity accumulates negative charges , resulting in bone deposition . recently , it has been proposed that the pressure - tension theory is not simple and might involve more complicated biological tissue response , suggesting that bone apposition could be induced by ( 1 ) the load exerted by stretched fibers of the pdl , which may also induce a slight bending of the alveolar wall ; ( 2 ) direct resorption by unloading of the alveolar wall in the case of low forces ; and ( 3 ) indirect resorption as repair due to ischemia following the application of high forces . the force leading to a change in tissue pressure that approximated the capillary vessels blood pressure , thus preventing their occlusion in the compressed periodontal ligament . it also has chemotactic properties and can stimulate the formation of osteoclasts , resulting in a subsequent increase in bone resorption . pgs are produced through two different pathways by the action of the enzyme cyclooxygenase on arachidonic acid : the constitutive isoform or cyclooxygenase-1 ( cox-1 ) and the inducible isoform or cyclooxygenase-2 ( cox-2 ) . the cox family of enzymes consists of two proteins that convert arachidonic acid , a 20-carbon polyunsaturated fatty acid comprising a portion of the plasma membrane phospholipids of most cells , to pgs . in contrast , cox-2 , the inducible isoform of cox , appears to be limited in basal conditions within most tissues , and de novo synthesis is activated by cytokines , bacterial lipopolysaccharides , or growth factors to produce pgs in large amounts during inflammatory processes occurring due to cell injury . research proved that the application of orthodontic force increased the synthesis of pgs , which in turn stimulated osteoclastic bone resorption . a similar study on cats by davidovitch et al , also showed increased levels of pge2 in the alveolar bone , as a result of application of orthodontic force . leiker et al studied the long - term effects of varying concentrations and frequencies of injectable , exogenous pge2 on the rate of tooth movement in rats and reported that injections of exogenous pge2 over an extended period of time in rats did enhance the amount of tooth movement . however , there was an increase in the amount of root resorption with increasing numbers and concentrations of the pge2 injections , which could be a potential concern . sekhavat et al , reported that misoprostol was effective in enhancing tooth movement in doses as low as 1025 g / kg , twice daily . however , long - term studies are needed to justify the use of pgs to accelerate orthodontic tooth movement in clinical practice . orthodontic forces result in capillary vasodilatation in the pdl , resulting in migration of inflammatory cells as well as cytokine production by these cells . they are released within 12 to 24 h after orthodontic force application and play an important role in initiating bone resorption and tooth movement . il-6 has been identified in human gingival tissues and cells ; and participates in the molecular events associated with inflammatory periodontal diseases and tissue destruction in periodontitis . the levels of il-6 increase significantly 24 h after mechanical loading and play an important role in initiating tooth movement . after the application of force , the induction of both il-1 and il-6 was observed to reach a maximum on day 3 and to decline thereafter . recent studies analyzing the cytokine expression pattern in compression and tension sides of the pdl during orthodontic tooth movement in humans , by means of real - time polymerase chain reaction , found that both the pressure and tension sides showed higher expression of all the cytokines when compared to the pdl of normal teeth which served as control . it also has chemotactic properties and can stimulate the formation of osteoclasts , resulting in a subsequent increase in bone resorption . pgs are produced through two different pathways by the action of the enzyme cyclooxygenase on arachidonic acid : the constitutive isoform or cyclooxygenase-1 ( cox-1 ) and the inducible isoform or cyclooxygenase-2 ( cox-2 ) . the cox family of enzymes consists of two proteins that convert arachidonic acid , a 20-carbon polyunsaturated fatty acid comprising a portion of the plasma membrane phospholipids of most cells , to pgs . in contrast , cox-2 , the inducible isoform of cox , appears to be limited in basal conditions within most tissues , and de novo synthesis is activated by cytokines , bacterial lipopolysaccharides , or growth factors to produce pgs in large amounts during inflammatory processes occurring due to cell injury . research proved that the application of orthodontic force increased the synthesis of pgs , which in turn stimulated osteoclastic bone resorption . a similar study on cats by davidovitch et al , also showed increased levels of pge2 in the alveolar bone , as a result of application of orthodontic force . leiker et al studied the long - term effects of varying concentrations and frequencies of injectable , exogenous pge2 on the rate of tooth movement in rats and reported that injections of exogenous pge2 over an extended period of time in rats did enhance the amount of tooth movement . sekhavat et al , reported that misoprostol was effective in enhancing tooth movement in doses as low as 1025 g / kg , twice daily . however , long - term studies are needed to justify the use of pgs to accelerate orthodontic tooth movement in clinical practice . orthodontic forces result in capillary vasodilatation in the pdl , resulting in migration of inflammatory cells as well as cytokine production by these cells . il-1 also plays a central role in the inflammatory process , and large amounts of il-1 are present in inflamed gingival tissues . they are released within 12 to 24 h after orthodontic force application and play an important role in initiating bone resorption and tooth movement . the levels of il-6 increase significantly 24 h after mechanical loading and play an important role in initiating tooth movement . after the application of force , the induction of both il-1 and il-6 was observed to reach a maximum on day 3 and to decline thereafter . recent studies analyzing the cytokine expression pattern in compression and tension sides of the pdl during orthodontic tooth movement in humans , by means of real - time polymerase chain reaction , found that both the pressure and tension sides showed higher expression of all the cytokines when compared to the pdl of normal teeth which served as control . it is likely that pdl cells stimulated by forces of mastication , occlusal contact , and orthodontic treatment produce local factors that participate not only in the maintenance and remodeling of the ligament itself , but also in the metabolism of adjacent alveolar bone . application of a continuous force on the crown of the tooth leads to tooth movement within the alveolus that is marked initially by narrowing of the periodontal membrane , particularly in the marginal area . a clear relationship between force level , timing , and extent of hyalinization even with a force as low as 5 cn , hyalinization occurred and the timing of the event seemed to be independent of the force level . it is prudent to use lighter forces for initiating orthodontic tooth movement and then gradually increasing the force levels , instead of using heavy force right at the start . in the secondary period of tooth movement , the osteoclasts attack the bone surface over a much wider area and , provided the force is kept within certain limits , further bone resorption will be predominantly of the direct type . these are attached to the root surface and parts of the alveolar bone wall where direct resorption is not occurring by the deposition of new tissue , in which the fibrils become embedded . concomitantly with bone apposition on the periodontal surface on the tension side , an accompanying resorption process occurs on the spongiosa surface of the alveolar bone . pgs of the e series play an important role in the pathogenesis of chronic periodontitis by regulating production of osteoclast activating factor in activated lymphocytes . pge2 have an important role in orthodontic tooth movement , which has been conclusively proven in the literature . the induction of both il-1 and il-6 was observed to reach a maximum on day 3 after mechanical loading and declines thereafter . it must be acknowledged that any interference in the signaling pathway resulting in reduced production of pge2 , il-1 , and il-6 would significantly delay tooth movement , which might be the reason for differences in the rate of initial tooth movement in different patients . it is a well - known fact that there is a local increase in pgs in the pdl and alveolar bone during orthodontic treatment . several studies have shown an arrest in tooth movement in experimental animals when non - steroidal anti - inflammatory drugs were administered . macrophages have the ability to produce cytokines , such as il-1 and il-6 , the levels of which are known to increase during orthodontic tooth movement . the presence of rankl in periodontal tissues , during experimental tooth movement of rat molars , shows that rankl is regulated by inflammatory cytokines in the pdl in response to mechanical stress . this indicates that the biomechanics used to initiate tooth movement could play a role in the increase in rankl levels and subsequent rate of tooth movement . showed that the local rankl gene transfer into the periodontal tissue significantly enhanced rankl expression and secretion of osteoclast in periodontal tissue without any systemic effects . it was conclusively proved that the transfer of rankl gene to the periodontal - tissue , activated osteoclast secretion and accelerated the amount of experimental tooth movement . long - term studies are needed to validate the effectiveness of the local rankl gene transfer as an useful tool to reduce the treatment time , by accelerating the rate of tooth movement in orthodontic patients undergoing fixed appliance treatment . the potential use of the local rankl gene transfer in moving ankylosed teeth has to be further evaluated to justify its use . it consist of the odontoblastic zone , cell free zone of weil , cell - rich zone , and the pulp core , which can be seen in a histological slide preparation . the principal cells of the pulp are odontoblast , fibroblast , undifferentiated ectomesenchymal cells , macrophages , and dendritic cells . orthodontic forces not only tend to produce mechanical damage and inflammatory reactions in the periodontium but also cause cell damage , inflammatory changes , and circulatory disturbances in dental pulp . the fact that the expression of cgrp and substance p is increased in dental pulp in response to bucally directed orthodontic tooth movement of the upper first molar in rats positively reinforces the fact that these neuropeptides might be involved in inflammation of the dental pulp at the time of orthodontic tooth movement . therefore , its constituents , which are derived from a variety of sources , including microbial dental plaque , host inflammatory cells , host tissue , and serum , vary according to the condition of the periodontal tissues and the predominant bacterial flora present . in general , cells , immunoglobulins , lysosomal enzymes , microorganisms , and toxins can be detected in gcf , while the mechanism of bone resorption might also be related to the release of inflammatory mediators present in gcf . recently , a number of gcf constituents have been shown to be diagnostic markers of active tissue destruction in periodontal diseases although only a few studies have focused on those involved in bone remodeling during orthodontic tooth movement . during the course of orthodontic treatment , the forces exerted produce a distortion of the pdl extracellular matrix , resulting in alterations in cellular shape and cytoskeletal configuration . such events lead to the synthesis and presence in the deeper periodontal tissues of extracellular matrix components , tissue - degrading enzymes , acids , and inflammatory mediators ; induce cellular proliferation and differentiation ; and promote wound healing and tissue remodeling . it was demonstrated that the gcf il-1 and tnf- levels had a positive correlation to mean pocket depths , which led to the suggestion that those cytokines may be involved in the pathogenesis of periodontal diseases . the level of pro - inflammatory cytokines such as il-1 , il-6 , and tnf- reached a significant level at 24 h after application of orthodontic forces . the lysosomal cysteine protease cathepsin b is known to play an important role in the resolution of organic matrix , a final step in bone resorption . the cathepsin b levels were found to be significantly increased when compared to the control teeth , which may be involved in extracellular matrix degradation in response to mechanical stress . immunocytochemical studies demonstrated that cathepsin b and cathepsin l were localized in the pdl of the rat molar and were expressed in compressed sites during experimental tooth movement . the expression of cathepsin k , a novel collagenolytic enzyme specifically expressed in osteoclasts , was investigated in the rat maxillary teeth during experimental tooth movement by in situ hybridization histochemistry with a non - radioisotopic complementary ribonucleic acid ( crna ) probe for rat cathepsin k. cathepsin k messenger ribonucleic acid ( mrna ) expression was detected in the mono- and multi - nuclear osteoclasts on the pressure side of the alveolar bone at 12 h after force application , and the distribution and number of cathepsin k mrna - positive osteoclasts increased time - dependently on the pressure side . at 3 - 4 days , there was a marked increase in cathepsin k mrna - positive osteoclasts on both pressure and tension side of the alveolar bone in response to tooth movement . at 7 - 12 days , the cathepsin k mrna - positive osteoclasts on both sides had disappeared . these suggest that the recruitment of osteoclasts on the pressure side begins during the initial stage of orthodontic tooth movement and the site - specific early induction of cathepsin k mrna may cause an imbalance in the relative resorption activities on the pressure and tension side incident to such movement . there was a 12-fold increase in the orthodontic gcf when compared to the control gcf . on the tension side , mmp-2 was significantly increased after 1 h , but gradually returned to basal levels within 8 h. immunolocalization of collagenase-3 ( mmp-13 ) done by leonardi showed that there was an increase in mmp-13 very early following the application of an orthodontic force in both pdl and alveolar bone . analysis of the gcf in patients with periodontal disease revealed that there was an increase in rankl and a decrease in the opg levels ; and subsequently the ratio of rankl concentration to that of opg in gcf samples was significantly higher for periodontal disease patients than for healthy subjects . it is one of the important enzymes required in initiating orthodontic tooth movement and sustaining it for a 2 week period . any defect in the production of this enzyme could interfere and delay orthodontic tooth movement . the activity levels of ast in the gcf are considered to be important in regulating alveolar bone resorption during orthodontic tooth movement . ast activity in the gcf was found to be highest in the first week of orthodontic force application and there was a gradual reduction in the activity during the next 3 weeks . similarly , lactate dehydrogenase activity in the gcf can be used as a diagnostic tool for monitoring orthodontic tooth movement in clinical practice . leptin and its receptor share structural and functional similarities with members of the long - chain helical cytokines : il-6 , il-11 , il-12 , leukemia inhibitory factor , granulocyte - colony - stimulating factor , and oncostatin m. it has been suggested that leptin orchestrates the host response to inflammatory and infectious stimuli ; as it stimulates the immune system by enhancing cytokine production and phagocytosis by macrophages . thus , the overall increase in leptin during inflammation and infection indicates that leptin is part of the immune response and host defense mechanisms . it was recently found that the concentration of leptin in gcf is decreased by orthodontic tooth movement and this conclusively proved that leptin may have been one of the mediators responsible for orthodontic tooth movement . the orthodontic displacement of a tooth is the result of a mechanical stimulus , generated by a force applied to the crown of a tooth , which results in an acute inflammatory response in periodontal tissues , which in turn may trigger the cascade of biological events associated with bone remodeling . this leads to the synthesis and release of various neurotransmitters , arachidonic acid , growth factors , metabolites , cytokines , colony - stimulating factors , and enzymes like cathepsin k , mmps , and ast , which are ultimately responsible for initiating bone remodeling and subsequent tooth movement . any interference with the release of these neurotransmitters and enzymes could delay or hamper orthodontic tooth movement . with the increased scope of gene therapy , the possibility that the rate of tooth movement could be increased by local gene transfer is very much real and could go a long way in reducing the treatment duration of orthodontic patients .

superficial fungal skin infections ( dermatophytosis ) caused by dermatophytes are known as tinea and will have a specific name depending on body location , as in tinea capitis , tinea corporis , or tinea pedis . most studies in this field dealt with adults or with a specific form of dermatophytosis . the few surveys performed in children reported a prevalence that varies from 2.5% to 15.2% [ 57 ] and differ from country to country . tinea capitis is predominantly a disease of children , especially under 12 years of age , and rare in adults [ 24 , 712 ] . the frequency of tinea capitis is declining in many developed nations ; however in the united kingdom and the united states it is becoming a major public health problem , and afro - caribbean children are particularly affected [ 5 , 913 ] . initial analysis by otters et al showed that the incidence rate of dermatophytosis in children in dutch general practice has increased between 1987 and 2001 . it is unclear whether this increase reflected an increase of the incidence in the population and is related to socio - demographic characteristics . knowledge about the factors contributing to the increasing incidence might improve the care for patients with dermatophytosis in general practice and its prevention . both topical and oral treatments have been proven to be effective , though not for all dermatophytoses [ 1517 ] . however , in a british study only 7% of children had received appropriate treatment for tinea capitis before referral to dermatology practice . the evaluation of management of dermatophytosis in children in general practice is therefore important . in 1997 , the dutch college of general practitioners issued a clinical guideline for the diagnosis and treatment of dermatophytosis for all ages . we do not have a clear insight in the degree of application of this guideline in children . in the present study we compared the results of two consecutive surveys in general practice performed in 1987 and 2001 respectively and aimed to answer the following research questions : what was the reported incidence rate of dermatophytosis in children aged 017 in general practice in 1987 and in 2001?were these incidence rates related to sociodemographic characteristics?how did the gp manage dermatophytosis ( prescription , referral ) and did this change between 1987 and 2001 ? what was the reported incidence rate of dermatophytosis in children aged 017 in general practice in 1987 and in 2001 ? how did the gp manage dermatophytosis ( prescription , referral ) and did this change between 1987 and 2001 ? we used data from the first and second dutch national surveys of general practice , which were performed by the netherlands institute for health services research ( nivel ) in 1987 and 2001 . general practices have a fixed list size , all inhabitants are listed in a general practice , and gps have a gate - keeping role , for adults as well as for children . usually , the first contact with health care , in a broad sense , is the contact with the general practitioner . in this respect the dutch health care structure did not change between 1987 and 2001 . in 1987 a sample , non - proportionally stratified by region , degree of urbanisation and distance to nearest hospital , of 161 gps in 103 practices was selected randomly to participate in the first national survey . with respect to age and gender the participating gps and practices were representative of dutch gps and practices in 1987 . the gps were divided into four groups , and each group used registration forms to register data ( e.g. diagnosis , prescription and referrals ) on all contacts between patient and practice during one of four consecutive 3-month periods . other socio - demographic characteristics such as socio - economic status ( ses ) and ethnic origin were obtained by a questionnaire and filled out by parents , or by the children themselves if they were older than 12 years ( response rate 91.2% ) . ses was based on the father s occupation , which was categorized into five classes non - manual work high ( class i ) , non - manual work middle ( class ii ) , non - manual low and farmers ( class iii ) , manual work high / middle ( class iv ) and manual work low ( class v ) . if either parent was born in turkey , africa , asia ( except japan and indonesia ) and central or south america , their children were considered to be children of non - western origin ( in accordance with the classification of statistics netherlands ) . the degree of urbanization was derived from the general practice s postal code and categorized into four classes under 30,000 inhabitants , 30,00050,000 inhabitants , over 50,000 inhabitants and the three large dutch cities amsterdam , rotterdam and the hague. the netherlands were divided into a northern , central and southern region . season was divided into four categories : spring was defined as months april june , summer as july the diagnoses made by the gps were coded afterwards by clerks using the international classification of primary care ( icpc ) , a classification commonly used in primary care . in 2001 , data about all physician - patient contacts over 12 months were derived from the electronic medical records of all listed patients in 104 practices ( 195 gps ) . the gps registered data on diagnosis , prescriptions and referrals , and coded the diagnosis themselves using the icpc . patient demographic characteristics such as age and gender were derived from the gps computerized patient files . as in 1987 , degree of urbanization , region and season were defined as in 1987 . in both surveys were episode orientated , meaning that a consultation on a new health problem marked the beginning of a new episode . if there were multiple consultations in a single episode , the diagnosis made during the last consultation was regarded as the episode - diagnosis . in both surveys all prescriptions were coded according to the anatomical - therapeutical - chemical ( atc ) classification , as developed by who ( www.whocc.no/atcddd ) . we excluded data from nine practices from the analyses , mainly because of technical problems with registration . the dutch guideline allows the gp to diagnose dermatophytes without a koh preparation or culture in most cases and we do not know how certain the gp was of this diagnosis . this study analyzed data from both surveys for children aged 017 years presenting with dermatophytosis which was coded as s74 ( icpc ) . because of an underrepresentation of deprived areas , the 1987 survey population was weighted to the dutch population of 1987 . incidence rates were calculated by dividing the weighted number of reported new episodes ( numerator ) by the study population at risk ( denominator ) . for 2001 we calculated the incidence rate of dermatophytosis by dividing the total number of reported new episodes ( numerator ) by the average study population at risk , the mid - time population ( denominator ) . the mid - time population was calculated as the mean of all listed patients of all participating gps , aged 017 years , at the beginning and at the end of the registration period . data were stratified by age , gender , urbanization level , region , season , ses and ethnic origin . incidence rates per 1,000 person - years and 95% confidence intervals were calculated assuming a poisson distribution . in both surveys this study analyzed data from both surveys for children aged 017 years presenting with dermatophytosis which was coded as s74 ( icpc ) . because of an underrepresentation of deprived areas , the 1987 survey population was weighted to the dutch population of 1987 . incidence rates were calculated by dividing the weighted number of reported new episodes ( numerator ) by the study population at risk ( denominator ) . for 2001 we calculated the incidence rate of dermatophytosis by dividing the total number of reported new episodes ( numerator ) by the average study population at risk , the mid - time population ( denominator ) . the mid - time population was calculated as the mean of all listed patients of all participating gps , aged 017 years , at the beginning and at the end of the registration period . data were stratified by age , gender , urbanization level , region , season , ses and ethnic origin . incidence rates per 1,000 person - years and 95% confidence intervals were calculated assuming a poisson distribution . in both surveys the study population in 1987 consisted of 86,577 children aged 017 years yielding 21,644 person years . these children had 559 contacts concerning dermatophytosis which contributed to 450 episodes ; 85.3% of these episodes included a single contact with the gp . in 2001 these children had 2,318 contacts concerning dermatophytosis which contributed to 2,007 episodes ; 88.2% of these episodes consisted of only one contact with the gp . table 1 shows the distribution of reported incidence rates of dermatophytosis in 1987 and 2001 in general practice stratified for several background characteristics . compared to 1987 , in 2001 the total incidence rate of dermatophytosis in general practice table 1incidence rates per 1,000 person years of all new episodes of dermatophytosis in general practice in 1987 and 200119872001p - valueincidence rates95% confidence intervalsincidence rates95% confidence intervalsage categories<1 year12.87.520.535.129.841.1<0.0114 years17.413.721.724.822.627.2<0.0159 years19.716.323.718.917.220.80.691014 years24.820.929.224.022.126.10.731517 years22.718.527.727.624.930.60.07total20.818.922.824.623.525.7<0.01genderboys20.017.422.822.621.224.10.08girls21.718.924.726.024.427.6<0.01urbanization<30,00016.613.919.823.021.524.7<0.0130,00050,00023.320.026.918.216.120.50.01>50,00020.416.425.119.818.121.60.80big cities27.720.836.224.120.128.70.41regionnorthern14.410.020.024.822.027.9<0.01central22.319.924.923.922.525.30.28southern19.816.224.025.723.727.9<0.01seasonwinter19.315.623.722.720.724.90.14spring23.119.627.226.424.228.70.13summer19.916.024.525.423.327.70.02autumn20.316.824.222.220.324.30.36sesnon - manual high21.017.125.621.920.023.90.70non - manual middle20.015.126.024.021.726.50.18non - manual low and farmers23.614.835.825.621.530.30.71manual high / middle21.717.127.023.419.128.40.62manual low26.721.233.325.121.429.30.66ethnic originnatives and western immigrants21.719.424.122.321.023.60.65non - western immigrants29.920.941.433.629.038.70.50 amsterdam , rotterdam , the hague according to father s occupation incidence rates per 1,000 person years of all new episodes of dermatophytosis in general practice in 1987 and 2001 amsterdam , rotterdam , the hague according to father s occupation in 2001 versus 1987 the incidence rate of dermatophytosis in general practice increased in young children ( 04 years ) ; infants ( < 1 year ) showed in 2001 a threefold higher incidence rate ( p < 0.01 ) . the gp more often diagnosed dermatophytosis in girls than boys 2 ( not significant for 1987 ; p < 0.01 for 2001 ) . in 1987 we found the highest incidence rate in the three big cities and the lowest in the rural areas ( < 30,000 ) whereas in 2001 it was distributed more equally over all urbanization levels . compared to 1987 , in 2001 the incidence rate increased in the rural areas ( < 30,000 ) and decreased in small cities ( 30,00050,000 ) , whereas it remained stable in larger cities ( > 50,000 ) and the three big cities . in 1987 we found the highest incidence rate in the central part of the netherlands compared to the northern part ( p < 0.01 ) whereas in 2001 the incidence rates were not different between regions . in 2001 , the incidence rates increased only in the northern and southern part of the netherlands compared to 1987 . in both surveys the incidence rates were distributed equally over all seasons and ses classes . in 2001 versus 1987 the incidence rate of dermatophytosis in general practice increased in summer ( p = 0.02 ) . in 2001 , the gp more often diagnosed dermatophytosis in children of non - western immigrants than in children of natives and western immigrants ( p < 0.01 ) . in 1987 the gps made 388 prescriptions in the first contact of the episode ; in 92.5% of these episodes only one drug was prescribed . in 10% of the episodes the gps did not prescribe any medication in the first contact . during all episodes the gps made 464 prescriptions resulting in an average prescription rate of 1.16 per episode ; in 7.5% of the episodes the gps did not prescribe any medication . in 2001 the gps made 1,715 prescriptions in the first contact of the episode ; in 90.4% of these episodes only one drug was prescribed . in 22.5% of the episodes the gps did not prescribe any medication in the first contact . during all episodes the gps made 2,333 prescriptions resulting in an average prescription rate of 1.16 per episode ; in 18.3% of the episodes the gps did not prescribe any medication . in table 2 we present the drugs prescribed in the first contact of the episode . in both surveys about three quarters of the dermatophytosis cases were treated with topical drugs ; gps prescribed in about 50% of the children with dermatophytosis topical antifungals and in about 20% topical antifungals combined with topical steroids . oral antifungals were applied in only a very small proportion of the cases . however , compared to 1987 , in 2001 the proportion of oral antifungal prescriptions almost doubled from 3.3% to 5.6% ( p = 0.05 ) . for oral treatment in 1987 only azoles were prescribed whereas in 2001 both azoles and allylamines were prescribed in almost equal proportions . table 2prescriptions in the first contact of episodenumber ( % ) 19872001total number of episodes400 ( 100)2,007 ( 100)oral treatmentsantifungals13 ( 3.3)113 ( 5.6)azoles13 ( 3.3)48 ( 2.4)allylamines0 ( 0)60 ( 3.0)others0 ( 0)5 ( 0.2)topical treatmentsantifungals231 ( 57.8)1,098 ( 54.7)azoles209 ( 52.3)1,002 ( 49.9)allylamines0 ( 0)43 ( 2.1)undecenoic acid20 ( 5.0)41 ( 2.0)others2 ( 0.5)12 ( 0.6)combinations ( antifungals and corticosteroids)94 ( 23.5)375 ( 18.7)corticosteroids10 ( 2.5)20 ( 1.0)emollients3 ( 0.8)31 ( 1.5)others37 ( 9.3)78 ( 3.9)no prescription in first contact40 ( 10)451 ( 22.5 ) unweighted number of new episodes number of new episodes in the mid - time population prescriptions in the first contact of episode unweighted number of new episodes number of new episodes in the mid - time population in 1987 twelve ( 2.6% ) and in 2001 thirty - two ( 1.6% ) of the children with dermatophytosis were referred to the dermatologist . in 1987 the boys to girls ratio of referred children was 3:1 , whereas in 2001 this was 1:2 . the study population in 1987 consisted of 86,577 children aged 017 years yielding 21,644 person years . these children had 559 contacts concerning dermatophytosis which contributed to 450 episodes ; 85.3% of these episodes included a single contact with the gp . in 2001 these children had 2,318 contacts concerning dermatophytosis which contributed to 2,007 episodes ; 88.2% of these episodes consisted of only one contact with the gp . table 1 shows the distribution of reported incidence rates of dermatophytosis in 1987 and 2001 in general practice stratified for several background characteristics . compared to 1987 , in 2001 the total incidence rate of dermatophytosis in general practice table 1incidence rates per 1,000 person years of all new episodes of dermatophytosis in general practice in 1987 and 200119872001p - valueincidence rates95% confidence intervalsincidence rates95% confidence intervalsage categories<1 year12.87.520.535.129.841.1<0.0114 years17.413.721.724.822.627.2<0.0159 years19.716.323.718.917.220.80.691014 years24.820.929.224.022.126.10.731517 years22.718.527.727.624.930.60.07total20.818.922.824.623.525.7<0.01genderboys20.017.422.822.621.224.10.08girls21.718.924.726.024.427.6<0.01urbanization<30,00016.613.919.823.021.524.7<0.0130,00050,00023.320.026.918.216.120.50.01>50,00020.416.425.119.818.121.60.80big cities27.720.836.224.120.128.70.41regionnorthern14.410.020.024.822.027.9<0.01central22.319.924.923.922.525.30.28southern19.816.224.025.723.727.9<0.01seasonwinter19.315.623.722.720.724.90.14spring23.119.627.226.424.228.70.13summer19.916.024.525.423.327.70.02autumn20.316.824.222.220.324.30.36sesnon - manual high21.017.125.621.920.023.90.70non - manual middle20.015.126.024.021.726.50.18non - manual low and farmers23.614.835.825.621.530.30.71manual high / middle21.717.127.023.419.128.40.62manual low26.721.233.325.121.429.30.66ethnic originnatives and western immigrants21.719.424.122.321.023.60.65non - western immigrants29.920.941.433.629.038.70.50 amsterdam , rotterdam , the hague according to father s occupation incidence rates per 1,000 person years of all new episodes of dermatophytosis in general practice in 1987 and 2001 amsterdam , rotterdam , the hague according to father s occupation in 2001 versus 1987 the incidence rate of dermatophytosis in general practice increased in young children ( 04 years ) ; infants ( < 1 year ) showed in 2001 a threefold higher incidence rate ( p < 0.01 ) . the gp more often diagnosed dermatophytosis in girls than boys 2 ( not significant for 1987 ; p < 0.01 for 2001 ) . in 1987 we found the highest incidence rate in the three big cities and the lowest in the rural areas ( < 30,000 ) whereas in 2001 it was distributed more equally over all urbanization levels . compared to 1987 , in 2001 the incidence rate increased in the rural areas ( < 30,000 ) and decreased in small cities ( 30,00050,000 ) , whereas it remained stable in larger cities ( > 50,000 ) and the three big cities . in 1987 we found the highest incidence rate in the central part of the netherlands compared to the northern part ( p < 0.01 ) whereas in 2001 the incidence rates were not different between regions . in 2001 , the incidence rates increased only in the northern and southern part of the netherlands compared to 1987 . in both surveys the incidence rates were distributed equally over all seasons and ses classes . in 2001 versus 1987 the incidence rate of dermatophytosis in general practice increased in summer ( p = 0.02 ) . in 2001 , the gp more often diagnosed dermatophytosis in children of non - western immigrants than in children of natives and western immigrants ( p < 0.01 ) . in 1987 the gps made 388 prescriptions in the first contact of the episode ; in 92.5% of these episodes only one drug was prescribed . in 10% of the episodes the gps did not prescribe any medication in the first contact . during all episodes the gps made 464 prescriptions resulting in an average prescription rate of 1.16 per episode ; in 7.5% of the episodes the gps did not prescribe any medication . in 2001 the gps made 1,715 prescriptions in the first contact of the episode ; in 90.4% of these episodes only one drug was prescribed . in 22.5% of the episodes the gps did not prescribe any medication in the first contact . during all episodes the gps made 2,333 prescriptions resulting in an average prescription rate of 1.16 per episode ; in 18.3% of the episodes the gps did not prescribe any medication . in table 2 we present the drugs prescribed in the first contact of the episode . in both surveys about three quarters of the dermatophytosis cases were treated with topical drugs ; gps prescribed in about 50% of the children with dermatophytosis topical antifungals and in about 20% topical antifungals combined with topical steroids . oral antifungals were applied in only a very small proportion of the cases . however , compared to 1987 , in 2001 the proportion of oral antifungal prescriptions almost doubled from 3.3% to 5.6% ( p = 0.05 ) . for oral treatment in 1987 only azoles were prescribed whereas in 2001 both azoles and allylamines were prescribed in almost equal proportions . table 2prescriptions in the first contact of episodenumber ( % ) 19872001total number of episodes400 ( 100)2,007 ( 100)oral treatmentsantifungals13 ( 3.3)113 ( 5.6)azoles13 ( 3.3)48 ( 2.4)allylamines0 ( 0)60 ( 3.0)others0 ( 0)5 ( 0.2)topical treatmentsantifungals231 ( 57.8)1,098 ( 54.7)azoles209 ( 52.3)1,002 ( 49.9)allylamines0 ( 0)43 ( 2.1)undecenoic acid20 ( 5.0)41 ( 2.0)others2 ( 0.5)12 ( 0.6)combinations ( antifungals and corticosteroids)94 ( 23.5)375 ( 18.7)corticosteroids10 ( 2.5)20 ( 1.0)emollients3 ( 0.8)31 ( 1.5)others37 ( 9.3)78 ( 3.9)no prescription in first contact40 ( 10)451 ( 22.5 ) unweighted number of new episodes number of new episodes in the mid - time population prescriptions in the first contact of episode unweighted number of new episodes number of new episodes in the mid - time population in 1987 twelve ( 2.6% ) and in 2001 thirty - two ( 1.6% ) of the children with dermatophytosis were referred to the dermatologist . in 1987 the boys to girls ratio of referred children was 3:1 , whereas in 2001 this was 1:2 . the incidence rate of dermatophytosis in general practice increased over the past 14 years which is consistent with a previous study performed in the netherlands . considering the decrease of the overall consultation rate of children in dutch general practice ( second dutch national survey ) probably our finding is a consequence of an increased prevalence in the population as reported by sladden et al . who showed that dermatophytosis is becoming a major health problem in the uk and the usa . a previous study reported that the consultation rate for onychomycosis in dutch general practice increased from 5.9 ( 1999 ) to 8.2 ( 20002001 ) and fell to 4.9 ( 2002 ) following a nationwide information campaign performed by the manufacturer of terbinafine in the netherlands , advising people with onychomycosis to visit their gp . alternatively the increased incidence rate does not reflect an increase in incidence of dermatophytosis in the population but a higher inclination to present this disease to the gp . the gps more often diagnosed dermatophytosis in girls which is different from a previous dutch study . however , the prevalence of dermatophytosis in the population was found to be higher in boys [ 3 , 4 , 6 ] in three studies in mediterranean countries . probably girls are more sensitive for the esthetic aspects of the disease and therefore present this problem more easily to their gp . the incidence rate increased in rural areas ( < 30,000 ) and remained stable in urban areas . a previous study performed in children in rural areas in turkey reported that the prevalence of dermatophytosis is higher under poor hygienical conditions . however , in the netherlands , the difference in hygiene conditions between rural and urban areas is negligible . considering the decrease of the overall consultation rate of children in dutch general practice ( second dutch national survey ) it might reflect an increase in incidence rate of dermatophytosis in children in general practice in urban areas . children of non - western immigrants consulted their gp more often with dermatophytosis ; in 2001 this difference became more apparent . the significantly higher overall consultation rate in non - western children might explain this difference . the increasing racial and ethnic heterogeneity of the dutch childhood population might contribute to an increased incidence of dermatophytosis in the population . previous studies reported that afro - caribbean children are particularly affected by tinea capitis in the uk and that the prevalence of tinea capitis in stockholm increased corresponding with the increased immigration from africa . between 1987 and 2001 the prescription pattern of the gp changed . in 2001 probably , this was influenced by the nationwide information campaign , as mentioned earlier . in 2001 probably this has to do with the fact that nowadays more over - the - counter drugs are available for dermatophytosis ; patients who initially use these medications may consult their gp in a later phase of the disease which should have consequences for the gps management . the majority of the dermatophytosis patients were treated with topical azoles and a very small proportion with allylamines or other antifungals indicating that dermatophytoses were primarily treated with azoles . this is in accordance with the results of the cochrane reviews [ 15 , 16 ] and the clinical guideline for the treatment of dermatophytosis issued by the dutch college of general practitioners in 1997 . in 2001 there were more episodes that included only one contact with the gp and more oral medication especially allylamines were prescribed . in both surveys the prescription rate per episode is the same , but in 2001 there were more episodes in which the gp did not prescribe any medication . these changes in disease management could be the consequence of the introduction of the clinical guideline for diagnosis and treatment of dermatophytosis issued by the dutch college of general practitioners in 1997 which may have improved the care for patients with dermatophytosis in general practice . the boys to girls ratio of referred children concerning dermatophytosis strikingly changed from 3:1 ( 1987 ) to 1:2 ( 2001 ) . as suggested earlier , girls may be more sensitive for the esthetic reasons of the disease and therefore consulted the gp more often than boys in 2001 . this is supported by our previous analysis showing that girls more often consulted the gp than boys concerning all skin diseases combined . possibly , for cosmetic reasons girls or their parents put more pressure on the gps for being referred to a dermatologist . these two large representative and comprehensive surveys enabled us to assess accurately epidemiological data on dermatophytosis in children . for this study data of only two points in time were available . to identify a sustained trend of the incidence of dermatophytosis in general practice data of multiple points in time there were small differences in the design of the two national surveys , which might disturb the comparability of data . for example icpc coding of the diagnoses was not performed equally in both surveys : in 1987 clerks coded diagnoses afterwards , whereas in 2001 the gps coded the diagnoses themselves during the consultation . we assume that coding by clerks more often led to a specific diagnostic icpc code . in the present study the accuracy of diagnoses ( s74 ) made by the gps could be subject of debate . the dutch guideline allows the gp to make the diagnosis of dermatophyte infection in most cases without koh preparation or culture . in our analysis the participating gps were trained in coding the diagnoses correctly using icpc codes . overall these trained gps classified diagnoses correctly in about 81% of the test cases . however , initial analysis showed that the incidence rate of diaper rash ( s89 ) decreased by about 50% in 2001 . possibly gps have coded diaper rash as dermatophytosis which may have lead to an overestimation of the incidence rate of dermatophytosis in 2001 . another possibility is that gps have coded dermatophytosis as diaper rash in 1987 , which may have led to an underestimation of the incidence rate of dermatophytosis in 1987 . the incidence rate of dermatophytosis in general practice increased over the past 14 years which is consistent with a previous study performed in the netherlands . considering the decrease of the overall consultation rate of children in dutch general practice ( second dutch national survey ) probably our finding is a consequence of an increased prevalence in the population as reported by sladden et al . who showed that dermatophytosis is becoming a major health problem in the uk and the usa . a previous study reported that the consultation rate for onychomycosis in dutch general practice increased from 5.9 ( 1999 ) to 8.2 ( 20002001 ) and fell to 4.9 ( 2002 ) following a nationwide information campaign performed by the manufacturer of terbinafine in the netherlands , advising people with onychomycosis to visit their gp . alternatively the increased incidence rate does not reflect an increase in incidence of dermatophytosis in the population but a higher inclination to present this disease to the gp . the gps more often diagnosed dermatophytosis in girls which is different from a previous dutch study . however , the prevalence of dermatophytosis in the population was found to be higher in boys [ 3 , 4 , 6 ] in three studies in mediterranean countries . probably girls are more sensitive for the esthetic aspects of the disease and therefore present this problem more easily to their gp . the incidence rate increased in rural areas ( < 30,000 ) and remained stable in urban areas . a previous study performed in children in rural areas in turkey reported that the prevalence of dermatophytosis is higher under poor hygienical conditions . however , in the netherlands , the difference in hygiene conditions between rural and urban areas is negligible . considering the decrease of the overall consultation rate of children in dutch general practice ( second dutch national survey ) it might reflect an increase in incidence rate of dermatophytosis in children in general practice in urban areas . children of non - western immigrants consulted their gp more often with dermatophytosis ; in 2001 this difference became more apparent . the significantly higher overall consultation rate in non - western children might explain this difference . the increasing racial and ethnic heterogeneity of the dutch childhood population might contribute to an increased incidence of dermatophytosis in the population . previous studies reported that afro - caribbean children are particularly affected by tinea capitis in the uk and that the prevalence of tinea capitis in stockholm increased corresponding with the increased immigration from africa . between 1987 and 2001 the prescription pattern of the gp changed . in 2001 more oral antifungals especially terbinafine ( not available in 1987 ) were prescribed . probably , this was influenced by the nationwide information campaign , as mentioned earlier . in 2001 probably this has to do with the fact that nowadays more over - the - counter drugs are available for dermatophytosis ; patients who initially use these medications may consult their gp in a later phase of the disease which should have consequences for the gps management . the majority of the dermatophytosis patients were treated with topical azoles and a very small proportion with allylamines or other antifungals indicating that dermatophytoses were primarily treated with azoles . this is in accordance with the results of the cochrane reviews [ 15 , 16 ] and the clinical guideline for the treatment of dermatophytosis issued by the dutch college of general practitioners in 1997 . in 2001 there were more episodes that included only one contact with the gp and more oral medication especially allylamines were prescribed . in both surveys the prescription rate per episode is the same , but in 2001 there were more episodes in which the gp did not prescribe any medication . these changes in disease management could be the consequence of the introduction of the clinical guideline for diagnosis and treatment of dermatophytosis issued by the dutch college of general practitioners in 1997 which may have improved the care for patients with dermatophytosis in general practice . the boys to girls ratio of referred children concerning dermatophytosis strikingly changed from 3:1 ( 1987 ) to 1:2 ( 2001 ) . as suggested earlier , girls may be more sensitive for the esthetic reasons of the disease and therefore consulted the gp more often than boys in 2001 . this is supported by our previous analysis showing that girls more often consulted the gp than boys concerning all skin diseases combined . possibly , for cosmetic reasons girls or their parents put more pressure on the gps for being referred to a dermatologist . these two large representative and comprehensive surveys enabled us to assess accurately epidemiological data on dermatophytosis in children . for this study data of only two points in time were available . to identify a sustained trend of the incidence of dermatophytosis in general practice data of multiple points in time there were small differences in the design of the two national surveys , which might disturb the comparability of data . for example icpc coding of the diagnoses was not performed equally in both surveys : in 1987 clerks coded diagnoses afterwards , whereas in 2001 the gps coded the diagnoses themselves during the consultation . we assume that coding by clerks more often led to a specific diagnostic icpc code . in the present study the accuracy of diagnoses ( s74 ) made by the gps could be subject of debate . the dutch guideline allows the gp to make the diagnosis of dermatophyte infection in most cases without koh preparation or culture . in our analysis the participating gps were trained in coding the diagnoses correctly using icpc codes . overall these trained gps classified diagnoses correctly in about 81% of the test cases . however , initial analysis showed that the incidence rate of diaper rash ( s89 ) decreased by about 50% in 2001 . possibly gps have coded diaper rash as dermatophytosis which may have lead to an overestimation of the incidence rate of dermatophytosis in 2001 . another possibility is that gps have coded dermatophytosis as diaper rash in 1987 , which may have led to an underestimation of the incidence rate of dermatophytosis in 1987 . in general practice the reported incidence rate of dermatophytosis in children increased , especially in girls , children of non - western immigrants , rural areas and in the northern and southern part of the netherlands . this increase in incidence rate could be a consequence of an increasing prevalence in the population . more studies on the population prevalence of dermatophytosis and help seeking behaviour are needed , especially in children of non - western immigrants and rural areas to test this hypothesis . gps generally follow the clinical guideline for diagnosis and treatment of dermatophytosis in children which is in accordance with evidence - based knowledge on the effectiveness of different therapies .

introductiondermatophytosis is a common skin infection in children . although the epidemiology is relatively unknown it is becoming a major health problem in some countries . we determine the incidence and management of dermatophytosis in dutch general practice in 1987 and 2001.methodswe used data of all children aged 017 years derived from two national surveys performed in dutch general practice in 1987 and 2001 respectively . all diagnoses , prescriptions and referrals were registered over a 12 months period by the participating general practitioners ( gps ) , 161 and 195 respectively . data were stratified for socio - demographic characteristics.resultscompared to 1987 , in 2001 the total reported incidence rate of dermatophytosis in children in general practice increased from 20.8 [ 95%ci 18.922.8 ] to 24.6 [ 95%ci 23.525.7 ] per 1,000 person years . infants ( < 1 year ) , girls , children in rural areas and children of non - western immigrants more often consulted the gp for dermatophytosis in 2001.in both surveys gps treated the majority of children with dermatophytosis with topical drugs , especially with azoles.conclusionsthe reported incidence rate of dermatophytosis in children in general practice increased ; however it is unclear whether this is a consequence of an increasing prevalence in the population or a changing help seeking behaviour . gps generally follow the national guideline for the treatment of dermatophytosis in children .

Introduction Methods Ethical approval Data-analysis Results Study populations in 1987 and 2001 Incidence rate Prescriptions Referrals Discussion Incidence and sociodemographics Prescriptions Referral Strengths and limitations of the study Conclusions

the frequency of tinea capitis is declining in many developed nations ; however in the united kingdom and the united states it is becoming a major public health problem , and afro - caribbean children are particularly affected [ 5 , 913 ] . initial analysis by otters et al showed that the incidence rate of dermatophytosis in children in dutch general practice has increased between 1987 and 2001 . it is unclear whether this increase reflected an increase of the incidence in the population and is related to socio - demographic characteristics . the evaluation of management of dermatophytosis in children in general practice is therefore important . in 1997 , the dutch college of general practitioners issued a clinical guideline for the diagnosis and treatment of dermatophytosis for all ages . in the present study we compared the results of two consecutive surveys in general practice performed in 1987 and 2001 respectively and aimed to answer the following research questions : what was the reported incidence rate of dermatophytosis in children aged 017 in general practice in 1987 and in 2001?were these incidence rates related to sociodemographic characteristics?how did the gp manage dermatophytosis ( prescription , referral ) and did this change between 1987 and 2001 ? what was the reported incidence rate of dermatophytosis in children aged 017 in general practice in 1987 and in 2001 ? we used data from the first and second dutch national surveys of general practice , which were performed by the netherlands institute for health services research ( nivel ) in 1987 and 2001 . for 2001 we calculated the incidence rate of dermatophytosis by dividing the total number of reported new episodes ( numerator ) by the average study population at risk , the mid - time population ( denominator ) . in both surveys this study analyzed data from both surveys for children aged 017 years presenting with dermatophytosis which was coded as s74 ( icpc ) . for 2001 we calculated the incidence rate of dermatophytosis by dividing the total number of reported new episodes ( numerator ) by the average study population at risk , the mid - time population ( denominator ) . in both surveys the study population in 1987 consisted of 86,577 children aged 017 years yielding 21,644 person years . table 1 shows the distribution of reported incidence rates of dermatophytosis in 1987 and 2001 in general practice stratified for several background characteristics . compared to 1987 , in 2001 the total incidence rate of dermatophytosis in general practice table 1incidence rates per 1,000 person years of all new episodes of dermatophytosis in general practice in 1987 and 200119872001p - valueincidence rates95% confidence intervalsincidence rates95% confidence intervalsage categories<1 year12.87.520.535.129.841.1<0.0114 years17.413.721.724.822.627.2<0.0159 years19.716.323.718.917.220.80.691014 years24.820.929.224.022.126.10.731517 years22.718.527.727.624.930.60.07total20.818.922.824.623.525.7<0.01genderboys20.017.422.822.621.224.10.08girls21.718.924.726.024.427.6<0.01urbanization<30,00016.613.919.823.021.524.7<0.0130,00050,00023.320.026.918.216.120.50.01>50,00020.416.425.119.818.121.60.80big cities27.720.836.224.120.128.70.41regionnorthern14.410.020.024.822.027.9<0.01central22.319.924.923.922.525.30.28southern19.816.224.025.723.727.9<0.01seasonwinter19.315.623.722.720.724.90.14spring23.119.627.226.424.228.70.13summer19.916.024.525.423.327.70.02autumn20.316.824.222.220.324.30.36sesnon - manual high21.017.125.621.920.023.90.70non - manual middle20.015.126.024.021.726.50.18non - manual low and farmers23.614.835.825.621.530.30.71manual high / middle21.717.127.023.419.128.40.62manual low26.721.233.325.121.429.30.66ethnic originnatives and western immigrants21.719.424.122.321.023.60.65non - western immigrants29.920.941.433.629.038.70.50 amsterdam , rotterdam , the hague according to father s occupation incidence rates per 1,000 person years of all new episodes of dermatophytosis in general practice in 1987 and 2001 amsterdam , rotterdam , the hague according to father s occupation in 2001 versus 1987 the incidence rate of dermatophytosis in general practice increased in young children ( 04 years ) ; infants ( < 1 year ) showed in 2001 a threefold higher incidence rate ( p < 0.01 ) . in 1987 we found the highest incidence rate in the three big cities and the lowest in the rural areas ( < 30,000 ) whereas in 2001 it was distributed more equally over all urbanization levels . compared to 1987 , in 2001 the incidence rate increased in the rural areas ( < 30,000 ) and decreased in small cities ( 30,00050,000 ) , whereas it remained stable in larger cities ( > 50,000 ) and the three big cities . in 1987 we found the highest incidence rate in the central part of the netherlands compared to the northern part ( p < 0.01 ) whereas in 2001 the incidence rates were not different between regions . in 2001 versus 1987 the incidence rate of dermatophytosis in general practice increased in summer ( p = 0.02 ) . in 2001 , the gp more often diagnosed dermatophytosis in children of non - western immigrants than in children of natives and western immigrants ( p < 0.01 ) . in both surveys about three quarters of the dermatophytosis cases were treated with topical drugs ; gps prescribed in about 50% of the children with dermatophytosis topical antifungals and in about 20% topical antifungals combined with topical steroids . however , compared to 1987 , in 2001 the proportion of oral antifungal prescriptions almost doubled from 3.3% to 5.6% ( p = 0.05 ) . the study population in 1987 consisted of 86,577 children aged 017 years yielding 21,644 person years . table 1 shows the distribution of reported incidence rates of dermatophytosis in 1987 and 2001 in general practice stratified for several background characteristics . compared to 1987 , in 2001 the total incidence rate of dermatophytosis in general practice table 1incidence rates per 1,000 person years of all new episodes of dermatophytosis in general practice in 1987 and 200119872001p - valueincidence rates95% confidence intervalsincidence rates95% confidence intervalsage categories<1 year12.87.520.535.129.841.1<0.0114 years17.413.721.724.822.627.2<0.0159 years19.716.323.718.917.220.80.691014 years24.820.929.224.022.126.10.731517 years22.718.527.727.624.930.60.07total20.818.922.824.623.525.7<0.01genderboys20.017.422.822.621.224.10.08girls21.718.924.726.024.427.6<0.01urbanization<30,00016.613.919.823.021.524.7<0.0130,00050,00023.320.026.918.216.120.50.01>50,00020.416.425.119.818.121.60.80big cities27.720.836.224.120.128.70.41regionnorthern14.410.020.024.822.027.9<0.01central22.319.924.923.922.525.30.28southern19.816.224.025.723.727.9<0.01seasonwinter19.315.623.722.720.724.90.14spring23.119.627.226.424.228.70.13summer19.916.024.525.423.327.70.02autumn20.316.824.222.220.324.30.36sesnon - manual high21.017.125.621.920.023.90.70non - manual middle20.015.126.024.021.726.50.18non - manual low and farmers23.614.835.825.621.530.30.71manual high / middle21.717.127.023.419.128.40.62manual low26.721.233.325.121.429.30.66ethnic originnatives and western immigrants21.719.424.122.321.023.60.65non - western immigrants29.920.941.433.629.038.70.50 amsterdam , rotterdam , the hague according to father s occupation incidence rates per 1,000 person years of all new episodes of dermatophytosis in general practice in 1987 and 2001 amsterdam , rotterdam , the hague according to father s occupation in 2001 versus 1987 the incidence rate of dermatophytosis in general practice increased in young children ( 04 years ) ; infants ( < 1 year ) showed in 2001 a threefold higher incidence rate ( p < 0.01 ) . in 1987 we found the highest incidence rate in the three big cities and the lowest in the rural areas ( < 30,000 ) whereas in 2001 it was distributed more equally over all urbanization levels . compared to 1987 , in 2001 the incidence rate increased in the rural areas ( < 30,000 ) and decreased in small cities ( 30,00050,000 ) , whereas it remained stable in larger cities ( > 50,000 ) and the three big cities . in 1987 we found the highest incidence rate in the central part of the netherlands compared to the northern part ( p < 0.01 ) whereas in 2001 the incidence rates were not different between regions . in 2001 versus 1987 the incidence rate of dermatophytosis in general practice increased in summer ( p = 0.02 ) . in 2001 , the gp more often diagnosed dermatophytosis in children of non - western immigrants than in children of natives and western immigrants ( p < 0.01 ) . in both surveys about three quarters of the dermatophytosis cases were treated with topical drugs ; gps prescribed in about 50% of the children with dermatophytosis topical antifungals and in about 20% topical antifungals combined with topical steroids . however , compared to 1987 , in 2001 the proportion of oral antifungal prescriptions almost doubled from 3.3% to 5.6% ( p = 0.05 ) . table 2prescriptions in the first contact of episodenumber ( % ) 19872001total number of episodes400 ( 100)2,007 ( 100)oral treatmentsantifungals13 ( 3.3)113 ( 5.6)azoles13 ( 3.3)48 ( 2.4)allylamines0 ( 0)60 ( 3.0)others0 ( 0)5 ( 0.2)topical treatmentsantifungals231 ( 57.8)1,098 ( 54.7)azoles209 ( 52.3)1,002 ( 49.9)allylamines0 ( 0)43 ( 2.1)undecenoic acid20 ( 5.0)41 ( 2.0)others2 ( 0.5)12 ( 0.6)combinations ( antifungals and corticosteroids)94 ( 23.5)375 ( 18.7)corticosteroids10 ( 2.5)20 ( 1.0)emollients3 ( 0.8)31 ( 1.5)others37 ( 9.3)78 ( 3.9)no prescription in first contact40 ( 10)451 ( 22.5 ) unweighted number of new episodes number of new episodes in the mid - time population prescriptions in the first contact of episode unweighted number of new episodes number of new episodes in the mid - time population in 1987 twelve ( 2.6% ) and in 2001 thirty - two ( 1.6% ) of the children with dermatophytosis were referred to the dermatologist . the incidence rate of dermatophytosis in general practice increased over the past 14 years which is consistent with a previous study performed in the netherlands . considering the decrease of the overall consultation rate of children in dutch general practice ( second dutch national survey ) probably our finding is a consequence of an increased prevalence in the population as reported by sladden et al . who showed that dermatophytosis is becoming a major health problem in the uk and the usa . a previous study reported that the consultation rate for onychomycosis in dutch general practice increased from 5.9 ( 1999 ) to 8.2 ( 20002001 ) and fell to 4.9 ( 2002 ) following a nationwide information campaign performed by the manufacturer of terbinafine in the netherlands , advising people with onychomycosis to visit their gp . alternatively the increased incidence rate does not reflect an increase in incidence of dermatophytosis in the population but a higher inclination to present this disease to the gp . a previous study performed in children in rural areas in turkey reported that the prevalence of dermatophytosis is higher under poor hygienical conditions . considering the decrease of the overall consultation rate of children in dutch general practice ( second dutch national survey ) it might reflect an increase in incidence rate of dermatophytosis in children in general practice in urban areas . children of non - western immigrants consulted their gp more often with dermatophytosis ; in 2001 this difference became more apparent . this is in accordance with the results of the cochrane reviews [ 15 , 16 ] and the clinical guideline for the treatment of dermatophytosis issued by the dutch college of general practitioners in 1997 . these changes in disease management could be the consequence of the introduction of the clinical guideline for diagnosis and treatment of dermatophytosis issued by the dutch college of general practitioners in 1997 which may have improved the care for patients with dermatophytosis in general practice . as suggested earlier , girls may be more sensitive for the esthetic reasons of the disease and therefore consulted the gp more often than boys in 2001 . to identify a sustained trend of the incidence of dermatophytosis in general practice data of multiple points in time there were small differences in the design of the two national surveys , which might disturb the comparability of data . possibly gps have coded diaper rash as dermatophytosis which may have lead to an overestimation of the incidence rate of dermatophytosis in 2001 . another possibility is that gps have coded dermatophytosis as diaper rash in 1987 , which may have led to an underestimation of the incidence rate of dermatophytosis in 1987 . the incidence rate of dermatophytosis in general practice increased over the past 14 years which is consistent with a previous study performed in the netherlands . considering the decrease of the overall consultation rate of children in dutch general practice ( second dutch national survey ) probably our finding is a consequence of an increased prevalence in the population as reported by sladden et al . who showed that dermatophytosis is becoming a major health problem in the uk and the usa . a previous study reported that the consultation rate for onychomycosis in dutch general practice increased from 5.9 ( 1999 ) to 8.2 ( 20002001 ) and fell to 4.9 ( 2002 ) following a nationwide information campaign performed by the manufacturer of terbinafine in the netherlands , advising people with onychomycosis to visit their gp . alternatively the increased incidence rate does not reflect an increase in incidence of dermatophytosis in the population but a higher inclination to present this disease to the gp . the incidence rate increased in rural areas ( < 30,000 ) and remained stable in urban areas . a previous study performed in children in rural areas in turkey reported that the prevalence of dermatophytosis is higher under poor hygienical conditions . considering the decrease of the overall consultation rate of children in dutch general practice ( second dutch national survey ) it might reflect an increase in incidence rate of dermatophytosis in children in general practice in urban areas . children of non - western immigrants consulted their gp more often with dermatophytosis ; in 2001 this difference became more apparent . this is in accordance with the results of the cochrane reviews [ 15 , 16 ] and the clinical guideline for the treatment of dermatophytosis issued by the dutch college of general practitioners in 1997 . these changes in disease management could be the consequence of the introduction of the clinical guideline for diagnosis and treatment of dermatophytosis issued by the dutch college of general practitioners in 1997 which may have improved the care for patients with dermatophytosis in general practice . as suggested earlier , girls may be more sensitive for the esthetic reasons of the disease and therefore consulted the gp more often than boys in 2001 . to identify a sustained trend of the incidence of dermatophytosis in general practice data of multiple points in time there were small differences in the design of the two national surveys , which might disturb the comparability of data . possibly gps have coded diaper rash as dermatophytosis which may have lead to an overestimation of the incidence rate of dermatophytosis in 2001 . another possibility is that gps have coded dermatophytosis as diaper rash in 1987 , which may have led to an underestimation of the incidence rate of dermatophytosis in 1987 . in general practice the reported incidence rate of dermatophytosis in children increased , especially in girls , children of non - western immigrants , rural areas and in the northern and southern part of the netherlands . this increase in incidence rate could be a consequence of an increasing prevalence in the population . more studies on the population prevalence of dermatophytosis and help seeking behaviour are needed , especially in children of non - western immigrants and rural areas to test this hypothesis . gps generally follow the clinical guideline for diagnosis and treatment of dermatophytosis in children which is in accordance with evidence - based knowledge on the effectiveness of different therapies .

in humans , infection can result from a variety of organisms including bacteria , viruses , parasites and fungi . traditionally , pathogenic infections were defined according to koch 's postulates , i.e. they were organisms that could be found , isolated , cultured and confirmed as a causative agent of disease by inoculation into a new host . apart from the obvious limitations of pathogen isolation and culture techniques , koch 's postulates were criticized and adapted in the face of technological advances , such as the identification of pathogenspecific nucleic acids using molecular biology techniques . with the advent of metagenomic sequencing , it has become clear that the presence of a microbe is not necessarily indicative of pathogenesis and instead may represent a degree of coevolution and symbiosis between the organism and the host.1 accumulating evidence demonstrates that commensal organisms colonizing our mucosal surfaces , particularly the gut microbiome , benefit immune functions at all levels.2 however , opportunistic infections by commensal bacteria and fungi can also occur , usually in the hospital setting of patients who are already ill , resulting in severe nosocomial sepsis or pneumonia . chronic infections are either persistent or latent infections within a host . despite attempts by the immune system to respond to the acute infection , pathogens escape from immune clearance by modulating , or regulating our immune response . for instance , persistence of an unresolved infection can result in immune exhaustion or deviation , for example , by switching of cd4 t cells to interleukin10 ( il10 ) producers in patients with chronic hepatitis c virus ( hcv ) infection,3 or the induction of il10 and interferon coproduction by cd4 t cells following parasitic toxoplasma gondii infection.4 the hijacking of these pathways by pathogens is an effective way for viruses and parasites to avoid immunopathology while establishing a persistent or latent infection.5 , 6 in some instances , this immune regulation may also be coupled to significant benefits for the host , dampening underlying inflammatory disorders and resulting in a survival advantage . below we will consider examples of this mutualistic symbiosis , focusing on examples from chronic viral and parasitic infections . in the case of viruses , metagenomic sequencing of the virome has identified eukaryotic viruses , including acute infections , chronic replicating infections and chronic latent infections . furthermore , in a complex multicellular eukaryotic host , the virome also includes bacteriophages infecting the microbiota , and virusderived genetic elements incorporated into eukaryotic chromosomes , e.g. retrovirusencoded elements , which are thought to make up 8% of the genome.7 it is difficult to know how stably associated the overall virome is with the host , particularly as viruses relying solely on a lytic life cycle , such as influenza virus , respiratory syncytial virus or dengue virus , cause cell death but tend to produce shortlived acute infections . however , noncytopathic viruses , which do not cause such profound cell death , can infect and persist for the lifespan of the host . it is estimated that humans carry between 8 and 12 persistent viral infections , including herpesviruses , retroviruses , allenoviridae and in fewer individuals : papillomaviruses , hepatitis viruses and human immunodeficiency virus ( hiv).8 for most of the lifetime of the host , persistent viral infections cause no overt clinical disease . this pathogeninduced immune regulation is also employed by other chronic infections , including parasites ( discussed in the next section ) , to enhance pathogen survival and often confer a survival advantage to the host . this viral mutualistic symbiosis is well recognized in invertebrates and plants , for example infection with cucumber mosaic virus imparts drought resistance to the nicotiana benthamiana plant.9 some viruses operate through a bacterial gobetween to exert a symbiotic effect , for example aphids are protected from invading wasp larvae by infection with a heritable symbiotic bacteria that harbours a bacteriophage encoding an antiwasp toxin.10 in these examples , the virus alters gene expression in the host and confers an advantage , which in the case of a chronically infected host , results in an increased chance of competitiveness and survival . so can we find examples of mutualistic symbiosis in human ( or vertebrate ) hosts ? the success of the balance between the replication and survival of the invading virus and the immune response and survival of the host can be defined by four key clinical factors : does the presence of viral infection lead to tissue , and hence organ , dysfunction ; does the virus compromise the immunity of the host allowing secondary pathogens to invade ; conversely does the immune system maintain a state of persistent activation leading to immunopathology ; and lastly , is it possible that a persistent viral infection may in some manner benefit the host ? virus relationships.11 although these viruses grow and replicate in the liver , some hepatotropic viruses spread by the oral faecal route ( hepatitis a virus and hepatitis e virus ) . they are adapted to rapid hosttohost horizontal spread by passing from the liver to the gut and the faeces , using the ideal conduit : the biliary tract . however , hepatitis b virus ( hbv ) and hcv are bloodborne infections , whose natural route of infection is perinatal , from the mother to the neonate . for these viruses , the chances of transmission are rarer , and to maximize the likelihood of success , an ideal scenario is persistent infection accompanied by high levels of viraemia . after initial infection with hcv , 80% of people develop chronic infection,12 exhibit detectable viraemia ( ranging from about 10 to 10 copies / ml ) but no evidence of liver disease.13 both hbv and hcv have evolved a series of strategies to avoid or evade the innate and adaptive immune responses , including the modulation of antigen presentation and expansion of antiinflammatory regulatory t cells.14 , 15 the maintenance of the host in this state provides a circulating reservoir of infective virus , increasing the statistical chance of viral transmission to a new host . once chronically infected with hbv or hcv , there is an astonishingly long period in most hosts , usually of decades , where there is no detriment to health . in some individuals attempted immune control of the virus by the host immune system over time drives liver inflammation and complications , such as liver cirrhosis or cancer . in these patients , there is a direct correlation between the degree of chronic inflammation ( in this case the expression of the natural cytotoxicity receptor nkp46 ) and steadily worsening liver pathology.16 during chronic infection , hcv and hbv also employ strategies to escape from ongoing host immunological attack by key adaptive immune cells such as cytotoxic cd8 t cells ; for instance mutation of key viral epitopes , presented to these cd8 t cells by mhc class i molecules.17 other diverse immune evasion strategies include interference with the signalling pathways required for production of proinflammatory cytokines , the upregulation of inhibitory molecules such as pd1 on virusspecific cd8 t cells and the expansion of cd4 regulatory t cells that can inhibit virusspecific cd8 t cells ( reviewed in park and rehermann18 ) ( fig . 1 . ) . these modifications can have a profound impact on the ability of the host to generate an immune response , or on underlying inflammatory conditions . the good ( light ) and bad ( dark ) side of chronic virus infection . infection of tissues by viruses and subsequent chronic inflammation can lead to tissue damage , e.g. hepatitis . in chronic hepatitis c virus ( hcv ) infection , progression to disease is associated with the expression of the natural killer ( nk ) cell receptor nkp46 . many other viruses directly infect antigenpresenting cells ( apcs ) or have a number of molecules that can downregulate mhc class i expression expressed by these cells ; this molecule is important for viral recognition by cd8 t cells but is also important for elimination of tumour cells . the nk cell killing of cells expressing low mhc class i is prevented by the dismantling of nk activating receptors . viral interleukin10 ( vil10 ) can transform b cells and help the virus to establish a chronic infection . chronic virus infection can promote antiinflammatory responses , including the expansion of regulatory t ( treg ) cells and , production of transforming growth factor ( tgf ) and is associated with a switch from interferon ( ifn ) to il10producing cd4 t cells . viral infection is also able to reduce antigen presentation and activation of apcs , reducing cd4 tcell activation and inflammatory responses . as the development of hepatitis virus liverrelated illness usually takes many years and does not impact on reproductive fitness , the question arises : is there a benefit of viral immune manipulation on the human host population ? parasitaemia and exhibit asymptomatic malaria , characterized by decreased inflammatory cytokine production.19 the young children most likely to become infected with hbv are particularly susceptible to malaria infection , illness and death , therefore viral dampening of this antimalaria response would offer a clear selective advantage for the survival and transmission of hbv . eight commonly infect humans : herpes simplex virus types 1 and 2 , varicella zoster virus , cytomegalovirus ( cmv ) , epstein barr virus ( ebv ) , human herpesvirus 6 , human herpesvirus 7 and kaposi 's sarcomaassociated herpesvirus . all herpesviruses can establish latent infection within specific niches ( e.g. nerve cells for herpes simplex virus , haematological cells for ebv , epithelial cells for cmv ) and induce a systemic immune response . immune control of these viruses , and the dynamics of viruses moving between latency and reactivation , results in fluctuating local and systemic inflammatory responses , which may result in injury and damage of infected tissues or a gradual deterioration of the immune system . a case in point is cmv infection of endothelial cells , where cmvinduced effector t cells cause endothelial cell damage and lowgrade longterm vascular injury.20 cmv can have a profound impact on the tcell compartment of the host immune system , driving bursts of functional cmvspecific memory cd8 tcell expansion during chronic infection.21 although the establishment and maintenance of these inflated tcell populations in adults is thought to be critical for the control of persistent viral infection , it is currently unclear how this process impacts on host immune responses to other pathogens and vaccines in otherwise healthy individuals . immunosuppressive medical treatment of immunocompromised patients infected with cmv , can lead to virusrelated clinical disease , such as cmv colitis , hepatitis or organ rejection . underlying cmv infection or the opportunistic outgrowth of viral or bacterial commensal species results in loss of immune responsiveness or containment in these patients.22 the acquisition of cmv during early life has a profound impact on the immune cell subset composition of infants.23 although controversial , some suggest that the natural acquisition of cmv infection by young children may prime and boost the adaptive immune system , moulding it for later life.24 herpesviruses have evolved an extraordinary ability to persist and escape immune detection by dedicating much of their large genomic capacity to subverting a diverse array of innate and adaptive immune responses . a major strategy for viral immune evasion is to target the initiation of the host immune response , by inhibiting host mhc class i antigen presentation of virusspecific epitopes to antiviral cytotoxic cd8 t cells.25 to counteract the possible increased natural killer cell killing of infected cells associated with loss of mhc class i , the machinery that alerts these cells is also dismantled by the virus26 ( fig . , during chronic kaposi 's sarcomaassociated herpesvirus infection , the inhibition of antigen presentation by the virus can lead to the development of the endothelial tumour kaposi 's sarcoma , particularly in immunocompromised patients.27 for the vast majority of us who live harmoniously with these viruses , again the question arises : is there a fitness benefit for the host ? evidence from animal models suggests that the presence of persistent latent ( but not lytic ) herpesvirus infection can confer protection from lethal bacterial infection and lethal lymphoma challenge . protection was associated with increased macrophage activation and the production of the antiviral cytokine interferon 28 or increased natural killer cell activity.29 latent viral infection could also significantly delay type 1 diabetes onset in mice by reducing antigen presentation of autoantigen to cd4 t cells.30 increases in the production of interferon during latent viral infection may underlie protection from other types of inflammatory disease , as supported by epidemiological data demonstrating inverse associations between herpesvirus infection31 , 32 and this skewing of the t helper type 1type 2 balance will be considered in the following section focusing on the possible protective effects of chronic parasitic infection . animal models have also shown that viral cytokine homologues , such as ebv il10 can provide some benefit to the host . ebv il10 can act on multiple cell types and inhibit cytokine synthesis by natural killer and t cells.33 the molecule is thought to play a critical role in the transformation of b cells , enabling time for the virus to establish latency.34 however , it has also been shown to inhibit collageninduced arthritis , autoimmune diabetes and pancreatitis , and to improve survival following sepsis and graft acceptance when expressed or administered in vivo 35 ( fig . 1 . ) . , we are also witnessing emerging new virus infections almost annually , often derived from animal reservoirs . natural elite controllers are found in ~ 3/1000 of untreated hivinfected individuals and are defined by stable cd4 tcell count with very low viral loads ; these patients demonstrate superior antiviral cd4 tcell36 and cytotoxic cd8 tcell responses.37 if effective antiretroviral treatment was not available , it seems inevitable that pathogendriven natural selection would favour these elite controllers , increasing the population frequencies of beneficial genetic polymorphisms such as hlab27 , hlab51 and hlab57.38 polymorphisms such as hlab27 also promote cd8 tcell clearance of hcv but are strongly linked to development of the autoimmune disease ankylosing spondylitis . interestingly , some suggest that plasmodium falciparum infection may have contributed to negative selection of this gene , due to the greater susceptibility of patients expressing this allele to severe forms of malaria.39 genetic selection , the moulding of our immune response and subsequent susceptibility to disease , has occurred as a result of ancient longstanding infections by chronic pathogens , those causing malaria , leprosy , as well as parasitic worms ( discussed in the next section).40 helminths affect over one billion people worldwide , mostly within developing regions , such as subsaharan africa , south america and india . although new epidemiological studies are required , a systematic review in 2011 demonstrated that soiltransmitted helminths are also prevalent in highincome countries.41 helminth infections have coevolved with man and evidence suggests that in a similar manner to chronic viral infections ( discussed in the previous section ) , they have provided selective pressure on the genetic makeup of the host.42 helminth infections are acquired in childhood ; however , in highincome countries zoonotic helminth infections of adults from cats and dogs are most common . infection can cause diarrhoea , abdominal pain , weakness and anaemia and contributes to 14 million disabilityadjusted lifeyears ( or the cumulative number of years lost to illhealth , disability or early death).43 for orally transmitted helminths , mortality is rare and infection is often asymptomatic ; most patients exhibiting low worm burden and limited clinical pathology.44 although gastrointestinal and intravascular helminth infections are reported to have some beneficial effects on inflammationinduced pathology in response to coinfection with other parasites,45 bacterial pathogens46 or viral infections,47 it can also impair protective immunity to concurrent parasite infections,48 , 49 , 50 , 51 promoting anaemia and liver pathology following malaria infection,52 , 53 , 54 exacerbating citrobacter rodentium infection and associated bacterialinduced colitis55 and aggravating virusrelated liver disease.56 this parasite impairment of host immunity has enormous health burden implications , particularly when immune responses to the biggest killers in the developing world , i.e. hiv , tuberculosis and diarrhoeic infections are affected.57 a number of mechanisms of immune suppression of coinfections are at play here , including the impairment of mucosal mastocytosis,58 , 59 the ablation of protective cd8 tcell responses,60 the induction or alteration of regulatory tcell subsets56 , 61 and the alternative activation of colonic macrophages62 ( fig . 2 ) . the good ( light ) and bad ( dark ) side of chronic parasite infection . chronic parasite infection can alter the commensal flora of the gut , resulting in reduced airway inflammation / allergy and inflammatory bowel disease . parasite transforming growth factor homologues ( ptgf ) released into the host can promote regulatory t ( treg ) cell expansion and upregulation of programmed cell death protein 1 ( pd1 ) , which inhibit cd4 tcell expansion and inflammatory cell production , resulting in reduced autoimmunity and allergy . infection also promotes interleukin10 ( il10 ) production in the host , which can also promote treg and reduce inflammation . some helminth infections can promote the expansion of t cells producing il22 ( th22 cells ) , which are able to promote homeostasis of the gut epithelial lining , reducing inflammatory bowel disease . finally , infection is also able to promote the formation of b cells , which can regulate inflammatory disorder such as asthma ( regulatory b cells ) . on the bad side , parasite infection promotes treg cells , which can reduce the efficacy of vaccines and the immune response to other infections . here , il10 produced in the host in response to infection infection with parasites can ablate protective cd8 tcell responses against coinfections such as toxoplasma gondii . the alternative activation of macrophages promoted by parasite infection can also impair host protection against concurrent bacterial infection . apart from an impact on immune responses to primary infection , the presence of chronic parasite infection in endemic areas has important implications for vaccination in the developing world . epidemiological studies have demonstrated that human helminth infections can impair immune responses to tetanus toxoid and cholera toxin b in endemic areas.63 , 64 in murine and large animal models , helminth infection reduces the immune response to malaria and salmonella vaccinations.65 , 66 , 67 as described previously , and in common with virusinduced immune suppression , these effects are related to the production of regulatory cytokines such as il10 or the induction of regulatory t cells during chronic parasite infection . a number of chronic parasites , including schistosoma haematobium , opisthorchis viverrini and clonorchis sinensis , are also classified as group 1 carcinogens , presenting a pertinent health challenge in developing regions . in parasite endemic regions , infection with schistosoma mansoni and schistosoma japonicum resulted in schistosomal colitis and was linked to increased bladder and cervical cancer.68 , 69 , 70 studies in vitro have shown that the production of signature cytokines associated with murine heligmosomoides polygyrus infection or s. mansoni egg administration promoted the replication of a herpesvirus that drives cancer ( kaposi 's sarcoma)71 and recent epidemiological studies associated soiltransmitted helminth infection with an increased prevalence of human papillomavirus ( a major cause of cervical cancer).72 in mouse models , infection with the gastrointestinal helminth h. polygyrus substantially reduced the type 1 inflammatory response associated with helicobacter pylori infection and attenuated gastric atrophy , a premalignant lesion.46 infection with the extraintestinal tapeworm taenia crassiceps reduced the development of colitisassociated tumours in a murine model of colorectal cancer . this effect was associated with increased expression of the type 2 cytokine il4 and with alterations in innate immunity , including macrophage alternative activation , neutrophil attraction and the recruitment of inflammatory monocytes.73 in a similar manner to chronic viral infections , helminths have evolved potent mechanisms to regulate the host immune response , in order to ensure their longterm survival.74 as well as notable effects on specific antiparasite responses , helminth immune regulation is proposed to exert mutualistic symbiosis , similar to the chronic viral infections discussed in the previous section , to further benefit the host by suppressing responses to a number of allergens and autoantigens.75 numerous studies have employed murine models of inflammatory disease to demonstrate that live parasite infection can protect against inflammatory disorders through immunomodulatory mechanisms including the activation of regulatory t cells46 , 49 or the production of il10.50 however , the immuneregulatory pathways that live helminths employ to modulate inflammatory disease are multifaceted , impacting on b cells,76 macrophages,77 innate immunity78 and through alterations to intestinal microbiota and their metabolites.79 , 80 in epidemiological studies , the story is more complex . some studies have shown clear protection against allergen skin test reactivity in individuals infected with intestinal helminth infections such as ascaris lumbricoides , trichuris trichiura , hookworm and schistosomiasis.81 , 82 this protection from pathology is associated with helminthinduced polarization of the inflammatory cytokine response , in a similar manner to chronic viral infections ( as discussed in the previous section ) . a correlation between parasite infection and improvements in multiple sclerosis was associated with increased frequencies of cd4 cd25 foxp3 t cells and increased cellular production of the immuneregulatory cytokines il10 and transforming growth factor.83 resolution of colitis in an individual who infected himself with t. trichiura 84 was associated with an increase in il22producing cd4 cells.85 interestingly , prior parasite infection can mould subsequent immune responses in the longterm ; both increased atopy and eczema were recorded in patients from a parasite endemic region that were treated in a longterm intervention study using the antihelminthic ivermectin.86 use of the antihelminthics albendazole and praziquantal during pregnancy was also associated with an increase rick of eczema in infancy,87 , 88 suggesting that in utero helminth exposure may programme and prime the host immune system of the offspring . these studies clearly demonstrate that helminths can mould the genome of the host , similar to the chronic viral infections discussed in the previous section . perhaps the best example of this for helminth infections is the selection of sickle cells traits , which confer resistance to malaria in endemic regions.89 live helminths are currently being employed in a number of clinical trials in efforts to alleviate allergic and autoimmune disorders.90 successful trials include the treatment of patients with inflammatory bowel disease with eggs from the porcine whipworm trichuris suis.91 , 92 however , not all trials using this strategy have had such a profound outcome , as evidenced by repeated treatment of patients exhibiting grass polleninduced allergic rhinitis with t. suis ova.93 the effectiveness of using parasitic helminth infections to treat inflammatory disorders is called into question in a recent review.94 this publication discusses the limitations of our current understanding of host parasite interactions and cites evidence of causal associations between helminth infections and inflammatory disorders . recent reviews have also revealed the limitations of human studies in poorresource settings,95 and indeed the consensus calls for the identification of helminthderived molecules of therapeutic potential and the use of animal models , alongside appropriately controlled clinical trials , to test these novel treatments of inflammatory disease . some progress has already been made on this front ; individual helminthsecreted products from the human hookworm necator americanus , the filarial nematode acanthocheilonema viteae and the murine gastrointestinal helminth h. polygyrus all suppressed rodent models of inflammatory disease , including arthritis , allergy and asthma.96 , 97 , 98 each of these parasitederived products has a unique capacity to modulate the host immune system at many levels , including suppression of mast cell responses,99 dampening the activation of inflammatory t cells by dendritic cells96 and by promoting the induction of regulatory t cells.100 our increasing understanding of helminth modulation of immune function , has also led to the proposal that helminthtreated populations of cells , such as macrophages , be used in therapeutic applications for inflammatory diseases in humans.101 these results warrant further studies in animal models and possible clinical trials to determine the efficacy of these findings in humans . in conclusion , both chronic viral and parasite infections can have both a beneficial and detrimental impact on the host immune system . a consensus on one conserved mechanism of immune suppression has not been reached , although this is mainly due to the complexity of the immune response , the tissue specificity of infection and the underlying health status of the individual . as for chronic viral infections , intestinal parasites have moulded our genome and have an intimate bidirectional relationship with the host . our increasing understanding of host pathogen interactions and the complexity of our micro and macrobiome will enhance our ability to break the bad consequences of chronic infection and make good through tailored treatment of an individual with an inflammatory disorder , or the development of strategies that increase vaccine efficacy and limit infectionassociated tumour prevalence , in endemic regions .

summaryeukaryotic forms of life have been continually invaded by microbes and larger multicellular parasites , such as helminths . over a billion years ago bacterial endosymbionts permanently colonized eukaryotic cells leading to recognized organelles with a distinct genetic lineage , such as mitochondria and chloroplasts . colonization of our skin and mucosal surfaces with bacterial commensals is now known to be important for host health . however , the contribution of chronic virus and parasitic infections to immune homeostasis is being increasingly questioned . persistent infection does not necessarily equate to exhibiting a chronic illness : healthy hosts ( e.g. humans ) have chronic viral and parasitic infections with no evidence of disease . indeed , there are now examples of complex interactions between these microbes and hosts that seem to confer an advantage to the host at a particular time , suggesting that the relationship has progressed along an axis from parasitic to commensal to one of a mutualistic symbiosis . this concept is explored using examples from viruses and parasites , considering how the relationships may be not only detrimental but also beneficial to the human host .

Introduction Viruses Parasites Disclosures

apart from the obvious limitations of pathogen isolation and culture techniques , koch 's postulates were criticized and adapted in the face of technological advances , such as the identification of pathogenspecific nucleic acids using molecular biology techniques . with the advent of metagenomic sequencing , it has become clear that the presence of a microbe is not necessarily indicative of pathogenesis and instead may represent a degree of coevolution and symbiosis between the organism and the host.1 accumulating evidence demonstrates that commensal organisms colonizing our mucosal surfaces , particularly the gut microbiome , benefit immune functions at all levels.2 however , opportunistic infections by commensal bacteria and fungi can also occur , usually in the hospital setting of patients who are already ill , resulting in severe nosocomial sepsis or pneumonia . despite attempts by the immune system to respond to the acute infection , pathogens escape from immune clearance by modulating , or regulating our immune response . for instance , persistence of an unresolved infection can result in immune exhaustion or deviation , for example , by switching of cd4 t cells to interleukin10 ( il10 ) producers in patients with chronic hepatitis c virus ( hcv ) infection,3 or the induction of il10 and interferon coproduction by cd4 t cells following parasitic toxoplasma gondii infection.4 the hijacking of these pathways by pathogens is an effective way for viruses and parasites to avoid immunopathology while establishing a persistent or latent infection.5 , 6 in some instances , this immune regulation may also be coupled to significant benefits for the host , dampening underlying inflammatory disorders and resulting in a survival advantage . below we will consider examples of this mutualistic symbiosis , focusing on examples from chronic viral and parasitic infections . furthermore , in a complex multicellular eukaryotic host , the virome also includes bacteriophages infecting the microbiota , and virusderived genetic elements incorporated into eukaryotic chromosomes , e.g. retrovirusencoded elements , which are thought to make up 8% of the genome.7 it is difficult to know how stably associated the overall virome is with the host , particularly as viruses relying solely on a lytic life cycle , such as influenza virus , respiratory syncytial virus or dengue virus , cause cell death but tend to produce shortlived acute infections . however , noncytopathic viruses , which do not cause such profound cell death , can infect and persist for the lifespan of the host . it is estimated that humans carry between 8 and 12 persistent viral infections , including herpesviruses , retroviruses , allenoviridae and in fewer individuals : papillomaviruses , hepatitis viruses and human immunodeficiency virus ( hiv).8 for most of the lifetime of the host , persistent viral infections cause no overt clinical disease . this pathogeninduced immune regulation is also employed by other chronic infections , including parasites ( discussed in the next section ) , to enhance pathogen survival and often confer a survival advantage to the host . this viral mutualistic symbiosis is well recognized in invertebrates and plants , for example infection with cucumber mosaic virus imparts drought resistance to the nicotiana benthamiana plant.9 some viruses operate through a bacterial gobetween to exert a symbiotic effect , for example aphids are protected from invading wasp larvae by infection with a heritable symbiotic bacteria that harbours a bacteriophage encoding an antiwasp toxin.10 in these examples , the virus alters gene expression in the host and confers an advantage , which in the case of a chronically infected host , results in an increased chance of competitiveness and survival . so can we find examples of mutualistic symbiosis in human ( or vertebrate ) hosts ? the success of the balance between the replication and survival of the invading virus and the immune response and survival of the host can be defined by four key clinical factors : does the presence of viral infection lead to tissue , and hence organ , dysfunction ; does the virus compromise the immunity of the host allowing secondary pathogens to invade ; conversely does the immune system maintain a state of persistent activation leading to immunopathology ; and lastly , is it possible that a persistent viral infection may in some manner benefit the host ? virus relationships.11 although these viruses grow and replicate in the liver , some hepatotropic viruses spread by the oral faecal route ( hepatitis a virus and hepatitis e virus ) . however , hepatitis b virus ( hbv ) and hcv are bloodborne infections , whose natural route of infection is perinatal , from the mother to the neonate . for these viruses , the chances of transmission are rarer , and to maximize the likelihood of success , an ideal scenario is persistent infection accompanied by high levels of viraemia . after initial infection with hcv , 80% of people develop chronic infection,12 exhibit detectable viraemia ( ranging from about 10 to 10 copies / ml ) but no evidence of liver disease.13 both hbv and hcv have evolved a series of strategies to avoid or evade the innate and adaptive immune responses , including the modulation of antigen presentation and expansion of antiinflammatory regulatory t cells.14 , 15 the maintenance of the host in this state provides a circulating reservoir of infective virus , increasing the statistical chance of viral transmission to a new host . once chronically infected with hbv or hcv , there is an astonishingly long period in most hosts , usually of decades , where there is no detriment to health . in some individuals attempted immune control of the virus by the host immune system over time drives liver inflammation and complications , such as liver cirrhosis or cancer . in these patients , there is a direct correlation between the degree of chronic inflammation ( in this case the expression of the natural cytotoxicity receptor nkp46 ) and steadily worsening liver pathology.16 during chronic infection , hcv and hbv also employ strategies to escape from ongoing host immunological attack by key adaptive immune cells such as cytotoxic cd8 t cells ; for instance mutation of key viral epitopes , presented to these cd8 t cells by mhc class i molecules.17 other diverse immune evasion strategies include interference with the signalling pathways required for production of proinflammatory cytokines , the upregulation of inhibitory molecules such as pd1 on virusspecific cd8 t cells and the expansion of cd4 regulatory t cells that can inhibit virusspecific cd8 t cells ( reviewed in park and rehermann18 ) ( fig . the good ( light ) and bad ( dark ) side of chronic virus infection . infection of tissues by viruses and subsequent chronic inflammation can lead to tissue damage , e.g. viral interleukin10 ( vil10 ) can transform b cells and help the virus to establish a chronic infection . chronic virus infection can promote antiinflammatory responses , including the expansion of regulatory t ( treg ) cells and , production of transforming growth factor ( tgf ) and is associated with a switch from interferon ( ifn ) to il10producing cd4 t cells . as the development of hepatitis virus liverrelated illness usually takes many years and does not impact on reproductive fitness , the question arises : is there a benefit of viral immune manipulation on the human host population ? all herpesviruses can establish latent infection within specific niches ( e.g. a case in point is cmv infection of endothelial cells , where cmvinduced effector t cells cause endothelial cell damage and lowgrade longterm vascular injury.20 cmv can have a profound impact on the tcell compartment of the host immune system , driving bursts of functional cmvspecific memory cd8 tcell expansion during chronic infection.21 although the establishment and maintenance of these inflated tcell populations in adults is thought to be critical for the control of persistent viral infection , it is currently unclear how this process impacts on host immune responses to other pathogens and vaccines in otherwise healthy individuals . immunosuppressive medical treatment of immunocompromised patients infected with cmv , can lead to virusrelated clinical disease , such as cmv colitis , hepatitis or organ rejection . a major strategy for viral immune evasion is to target the initiation of the host immune response , by inhibiting host mhc class i antigen presentation of virusspecific epitopes to antiviral cytotoxic cd8 t cells.25 to counteract the possible increased natural killer cell killing of infected cells associated with loss of mhc class i , the machinery that alerts these cells is also dismantled by the virus26 ( fig . , during chronic kaposi 's sarcomaassociated herpesvirus infection , the inhibition of antigen presentation by the virus can lead to the development of the endothelial tumour kaposi 's sarcoma , particularly in immunocompromised patients.27 for the vast majority of us who live harmoniously with these viruses , again the question arises : is there a fitness benefit for the host ? evidence from animal models suggests that the presence of persistent latent ( but not lytic ) herpesvirus infection can confer protection from lethal bacterial infection and lethal lymphoma challenge . animal models have also shown that viral cytokine homologues , such as ebv il10 can provide some benefit to the host . natural elite controllers are found in ~ 3/1000 of untreated hivinfected individuals and are defined by stable cd4 tcell count with very low viral loads ; these patients demonstrate superior antiviral cd4 tcell36 and cytotoxic cd8 tcell responses.37 if effective antiretroviral treatment was not available , it seems inevitable that pathogendriven natural selection would favour these elite controllers , increasing the population frequencies of beneficial genetic polymorphisms such as hlab27 , hlab51 and hlab57.38 polymorphisms such as hlab27 also promote cd8 tcell clearance of hcv but are strongly linked to development of the autoimmune disease ankylosing spondylitis . interestingly , some suggest that plasmodium falciparum infection may have contributed to negative selection of this gene , due to the greater susceptibility of patients expressing this allele to severe forms of malaria.39 genetic selection , the moulding of our immune response and subsequent susceptibility to disease , has occurred as a result of ancient longstanding infections by chronic pathogens , those causing malaria , leprosy , as well as parasitic worms ( discussed in the next section).40 helminths affect over one billion people worldwide , mostly within developing regions , such as subsaharan africa , south america and india . although new epidemiological studies are required , a systematic review in 2011 demonstrated that soiltransmitted helminths are also prevalent in highincome countries.41 helminth infections have coevolved with man and evidence suggests that in a similar manner to chronic viral infections ( discussed in the previous section ) , they have provided selective pressure on the genetic makeup of the host.42 helminth infections are acquired in childhood ; however , in highincome countries zoonotic helminth infections of adults from cats and dogs are most common . infection can cause diarrhoea , abdominal pain , weakness and anaemia and contributes to 14 million disabilityadjusted lifeyears ( or the cumulative number of years lost to illhealth , disability or early death).43 for orally transmitted helminths , mortality is rare and infection is often asymptomatic ; most patients exhibiting low worm burden and limited clinical pathology.44 although gastrointestinal and intravascular helminth infections are reported to have some beneficial effects on inflammationinduced pathology in response to coinfection with other parasites,45 bacterial pathogens46 or viral infections,47 it can also impair protective immunity to concurrent parasite infections,48 , 49 , 50 , 51 promoting anaemia and liver pathology following malaria infection,52 , 53 , 54 exacerbating citrobacter rodentium infection and associated bacterialinduced colitis55 and aggravating virusrelated liver disease.56 this parasite impairment of host immunity has enormous health burden implications , particularly when immune responses to the biggest killers in the developing world , i.e. the good ( light ) and bad ( dark ) side of chronic parasite infection . parasite transforming growth factor homologues ( ptgf ) released into the host can promote regulatory t ( treg ) cell expansion and upregulation of programmed cell death protein 1 ( pd1 ) , which inhibit cd4 tcell expansion and inflammatory cell production , resulting in reduced autoimmunity and allergy . here , il10 produced in the host in response to infection infection with parasites can ablate protective cd8 tcell responses against coinfections such as toxoplasma gondii . apart from an impact on immune responses to primary infection , the presence of chronic parasite infection in endemic areas has important implications for vaccination in the developing world . epidemiological studies have demonstrated that human helminth infections can impair immune responses to tetanus toxoid and cholera toxin b in endemic areas.63 , 64 in murine and large animal models , helminth infection reduces the immune response to malaria and salmonella vaccinations.65 , 66 , 67 as described previously , and in common with virusinduced immune suppression , these effects are related to the production of regulatory cytokines such as il10 or the induction of regulatory t cells during chronic parasite infection . a number of chronic parasites , including schistosoma haematobium , opisthorchis viverrini and clonorchis sinensis , are also classified as group 1 carcinogens , presenting a pertinent health challenge in developing regions . in parasite endemic regions , infection with schistosoma mansoni and schistosoma japonicum resulted in schistosomal colitis and was linked to increased bladder and cervical cancer.68 , 69 , 70 studies in vitro have shown that the production of signature cytokines associated with murine heligmosomoides polygyrus infection or s. mansoni egg administration promoted the replication of a herpesvirus that drives cancer ( kaposi 's sarcoma)71 and recent epidemiological studies associated soiltransmitted helminth infection with an increased prevalence of human papillomavirus ( a major cause of cervical cancer).72 in mouse models , infection with the gastrointestinal helminth h. polygyrus substantially reduced the type 1 inflammatory response associated with helicobacter pylori infection and attenuated gastric atrophy , a premalignant lesion.46 infection with the extraintestinal tapeworm taenia crassiceps reduced the development of colitisassociated tumours in a murine model of colorectal cancer . this effect was associated with increased expression of the type 2 cytokine il4 and with alterations in innate immunity , including macrophage alternative activation , neutrophil attraction and the recruitment of inflammatory monocytes.73 in a similar manner to chronic viral infections , helminths have evolved potent mechanisms to regulate the host immune response , in order to ensure their longterm survival.74 as well as notable effects on specific antiparasite responses , helminth immune regulation is proposed to exert mutualistic symbiosis , similar to the chronic viral infections discussed in the previous section , to further benefit the host by suppressing responses to a number of allergens and autoantigens.75 numerous studies have employed murine models of inflammatory disease to demonstrate that live parasite infection can protect against inflammatory disorders through immunomodulatory mechanisms including the activation of regulatory t cells46 , 49 or the production of il10.50 however , the immuneregulatory pathways that live helminths employ to modulate inflammatory disease are multifaceted , impacting on b cells,76 macrophages,77 innate immunity78 and through alterations to intestinal microbiota and their metabolites.79 , 80 in epidemiological studies , the story is more complex . some studies have shown clear protection against allergen skin test reactivity in individuals infected with intestinal helminth infections such as ascaris lumbricoides , trichuris trichiura , hookworm and schistosomiasis.81 , 82 this protection from pathology is associated with helminthinduced polarization of the inflammatory cytokine response , in a similar manner to chronic viral infections ( as discussed in the previous section ) . a correlation between parasite infection and improvements in multiple sclerosis was associated with increased frequencies of cd4 cd25 foxp3 t cells and increased cellular production of the immuneregulatory cytokines il10 and transforming growth factor.83 resolution of colitis in an individual who infected himself with t. trichiura 84 was associated with an increase in il22producing cd4 cells.85 interestingly , prior parasite infection can mould subsequent immune responses in the longterm ; both increased atopy and eczema were recorded in patients from a parasite endemic region that were treated in a longterm intervention study using the antihelminthic ivermectin.86 use of the antihelminthics albendazole and praziquantal during pregnancy was also associated with an increase rick of eczema in infancy,87 , 88 suggesting that in utero helminth exposure may programme and prime the host immune system of the offspring . these studies clearly demonstrate that helminths can mould the genome of the host , similar to the chronic viral infections discussed in the previous section . perhaps the best example of this for helminth infections is the selection of sickle cells traits , which confer resistance to malaria in endemic regions.89 live helminths are currently being employed in a number of clinical trials in efforts to alleviate allergic and autoimmune disorders.90 successful trials include the treatment of patients with inflammatory bowel disease with eggs from the porcine whipworm trichuris suis.91 , 92 however , not all trials using this strategy have had such a profound outcome , as evidenced by repeated treatment of patients exhibiting grass polleninduced allergic rhinitis with t. suis ova.93 the effectiveness of using parasitic helminth infections to treat inflammatory disorders is called into question in a recent review.94 this publication discusses the limitations of our current understanding of host parasite interactions and cites evidence of causal associations between helminth infections and inflammatory disorders . some progress has already been made on this front ; individual helminthsecreted products from the human hookworm necator americanus , the filarial nematode acanthocheilonema viteae and the murine gastrointestinal helminth h. polygyrus all suppressed rodent models of inflammatory disease , including arthritis , allergy and asthma.96 , 97 , 98 each of these parasitederived products has a unique capacity to modulate the host immune system at many levels , including suppression of mast cell responses,99 dampening the activation of inflammatory t cells by dendritic cells96 and by promoting the induction of regulatory t cells.100 our increasing understanding of helminth modulation of immune function , has also led to the proposal that helminthtreated populations of cells , such as macrophages , be used in therapeutic applications for inflammatory diseases in humans.101 these results warrant further studies in animal models and possible clinical trials to determine the efficacy of these findings in humans . in conclusion , both chronic viral and parasite infections can have both a beneficial and detrimental impact on the host immune system . a consensus on one conserved mechanism of immune suppression has not been reached , although this is mainly due to the complexity of the immune response , the tissue specificity of infection and the underlying health status of the individual . as for chronic viral infections , intestinal parasites have moulded our genome and have an intimate bidirectional relationship with the host . our increasing understanding of host pathogen interactions and the complexity of our micro and macrobiome will enhance our ability to break the bad consequences of chronic infection and make good through tailored treatment of an individual with an inflammatory disorder , or the development of strategies that increase vaccine efficacy and limit infectionassociated tumour prevalence , in endemic regions .

in the field of neurorehabilitation , kinesthetic illusion1 has been used as a rehabilitation therapy for patients who have suffered a stroke . kinesthetic illusion is used to create the impression of limb movement in the brain , without patients actually having to perform the movement voluntarily1 . kinesthetic illusions , which are induced by sensorimotor information in the brain , involve the excitement of sensorimotor cortical areas and the association of underlying neuronal activity with motion control and skill learning is believed to have beneficial effects in rehabilitation2 . the therapeutic effects of kinesthetic illusion on the motor deficits of patients with stroke have been reported in a number of publications3 , 4 . for example , altschuler et al.7 demonstrated that mirror - induced kinesthetic illusions resulted in recovery of motor function even in patients in the chronic phase of post - stroke recovery ( i.e. , 6 months or longer after the occurrence of stroke ) . in addition , dohle et al.8 examined the effects of a mirror - based treatment on patients suffering severe paralysis after stroke and found improvements in finger motor function after the intervention . when an intervention task entails processing visual information during kinesthetic illusion , various types of brain functions , such as cognitive , visual information processing , and motor functions , related to the unaffected extremity can affect performance of the task . therefore , the stimulus used in the task itself should be as simple and passive as possible . thus , some studies have used vibratory stimulation3 , 9 , 10 as a method for inducing kinesthetic illusion . functional magnetic resonance imaging10 and magnetoencephalography11 have implicated the following regions in kinesthetic illusion - related neural activity : the primary sensorimotor cortex , the sensory association area , the supplementary motor area , and the angular gyrus . during kinesthetic illusion , given that previous sensorimotor experiences need to be simulated in kinesthetic motor imagery12 , it is important that brain function be analyzed based on the degree of kinesthetic illusion achieved . therefore , in order to determine the brain functions occurring during such an illusion , detailed temporal and spatial analyses ( from the beginning of kinesthetic motor image formation ) need to be performed before the induced kinesthetic illusion occurs . the electroencephalographic mu - rhythm13 ( -wave ) is useful as a neurophysiological index for capturing data from such analyses . a -wave is the high - frequency component ranging from 10 to 13 hz of the alpha band14 , which appears in the sensorimotor cortex13 , 15 and the supplementary motor area16 , 17 . since this wave is attenuated not only by actual motion , but also by kinesthetic motor imagery13 , 15 , it reflects neural activity both during kinesthetic illusion and during the generation of a motor image . to date , the association between kinesthetic illusion and -waves has been reported for healthy subjects18 , but not for stroke patients . thus , the purpose of this study was to examine the impact of kinesthetic illusion , produced by vibratory stimulation , on the cerebral neural activity of patients with stroke , through functional neuroimaging of -waves . in accordance with the declaration of helsinki , the scope and contents of this study , as well as the precautions against the disclosure of personal information , were explained to the subjects verbally and in written form . upon their understanding of the explanations , the study was approved by the ethics committee of kurume university school of medicine . this study was conducted on 12 healthy subjects ( six men and six women ; mean age : 60.1 3.2 years ) with no orthopedic or nervous system disease , or motor deficits or sensory disorders ( the healthy group ) , and 13 patients with stroke ( seven men and six women ; mean age : 61.1 3.8 year ; the patient group ) . the dominant hands of the subjects in the healthy group were assessed using the edinburgh inventory19 , and all subjects were confirmed to be right - handed . we confirmed that none of the subjects in the patient group had any cognitive dysfunction or disorder , or any sensory impairment . the attributes of the patients are shown in table 1table 1.attributes of the patient groupcharacteristics of patientsage , yrs ( range)61.1 3.8 ( 5668)male : female ( n)7:6 ( n = 13)diagnosiscerebral infarction lacunarthalamus 2internal capsule2corona radiata4atherothrombosis1cerebral hemorrhage thalamus2putamen2paralysisright13left0dominant handright13left0time from injury , months12.6 7.6ue - fma21.8 5.6mmse26.9 1.8ue - fma : fugl - meyer assesment ( ue ) , mmse : mini - mental state examination . ue - fma : fugl - meyer assesment ( ue ) , mmse : mini - mental state examination the subjects were instructed to relax while reclining on a chair that had a backrest with an adjustable angle , and were asked to position the forearm of their left upper extremity ( that is , the upper extremity on the non - measured side ) so that the elbow touched the chair . it was first explained to subjects that electroencephalographic measurements would be performed under two conditions : a resting state , the control condition ( rest ) , and during the application of a vibratory stimulation , the task condition ( vibratory stimulation ) . the motion targeted in the vibratory stimulation - induced kinesthetic illusion was extension of the right elbow joint . therefore , the bicep tendon of the right upper arm was selected as the stimulation site . because the stimulation was applied to the skin , cutaneous stimulus receptors , in addition to muscle spindles , were excited . the -waves , which change according to sensorimotor information , are also influenced by sensory information received from cutaneous stimuli20 ; therefore , as a control , the subjects were asked to envision that elbow flexion was prevented due to contraction of the bicep muscle during vibratory stimulation20 . in order to ensure that the forearm would not be in contact with the trunk , the measurer fixed a hand massager ( thrive md-01 ; thrive co. , ltd . , osaka , japan ) on the bicep tendon of the subjects right arms . at rest , electroencephalographic measurements were conducted without applying any vibratory stimulation . during the stimulation , in addition , since visual information pertaining to the non - moving upper extremity has been found to reduce kinesthetic illusion , subjects were instructed to close their eyes during the vibratory stimulation9 . data were collected for 3 to 9 days ( mean 4.9 2.0 days ) . vibratory stimulation and rest assessment were both carried out twice a day for 3 consecutive or non - consecutive days . after - effects were taken into consideration by allowing subjects to rest between vibratory stimulation sessions . the average value of the brain wave data of the twelve measurements was calculated , and used as the data for each subject . electroencephalographic measurements were performed during the two conditions , and a neurofax eeg-1200 ( nihon kohden , tokyo , japan ) was used to record the electroencephalograms . measurements were conducted at 18 sites of the international 1020 system ( f3 , f4 , f7 , f8 , fz , c3 , c4 , cz , p3 , p4 , pz , o1 , o2 , oz , t3 , t4 , t5 , and t6 ) , with the earlobes on both sides as the reference electrodes . data were processed with a bandpass filter of 0.5 to 60 hz , and the sampling frequency was 1,000 hz . the duration of the measurement was set to 60 s per session for both the rest and vibratory stimulation conditions10 . for the imaging analysis , 30 epochs were extracted from the data obtained under each condition ( the duration of one epoch was 2 s ) , and a frequency analysis was performed . the data within the -wave frequency range ( 10 to 13 hz ) were calculated and used for intracerebral spatial analyses using the standardized low resolution brain electromagnetic tomography ( sloreta ) analysis22 , a method that allows three - dimensional image display of intracerebral neural activity . the values of each voxel in the brain areas displaying neural activity under each condition were calculated as the neural activity ( current source density [ csd ] ; a / mm*10 ) , and were identified in terms of brodmann s area ( ba ) and montreal neurological institute ( mni ) coordinates23 . by averaging data using the sloreta averager software , the neural activity could be calculated as the global field power ( gfp ) value22 in the two groups under both the rest and vibratory stimulation conditions . in addition , the intracerebral neural activities of the two groups under each condition were compared using the statistics in sloreta snpm24 . in this analysis , the neural areas showing significant differences in activity ( with a statistical significance level of 5% ) were colored , calculated , and drawn . the significance threshold was based on a permutation test with 5000 permutations , using log - transformed loreta values , with subject - wise normalization ( on the assumption that differences in the base - line activity between healthy subjects and patients may exist ) . when -waves manifest in the sensorimotor cortex13 , frontal lobe25 , or occipital lobe26 , the waveform components and the names of the waves can differ ( such as -waves ) depending on their region of occurrence . interestingly , this phenomenon is evident even when these waves have the same frequency range . thus , in this study , the different types of -waves were investigated separately . in accordance with the declaration of helsinki , the scope and contents of this study , as well as the precautions against the disclosure of personal information , were explained to the subjects verbally and in written form . upon their understanding of the explanations , the study was approved by the ethics committee of kurume university school of medicine . this study was conducted on 12 healthy subjects ( six men and six women ; mean age : 60.1 3.2 years ) with no orthopedic or nervous system disease , or motor deficits or sensory disorders ( the healthy group ) , and 13 patients with stroke ( seven men and six women ; mean age : 61.1 3.8 year ; the patient group ) . the dominant hands of the subjects in the healthy group were assessed using the edinburgh inventory19 , and all subjects were confirmed to be right - handed . we confirmed that none of the subjects in the patient group had any cognitive dysfunction or disorder , or any sensory impairment . the attributes of the patients are shown in table 1table 1.attributes of the patient groupcharacteristics of patientsage , yrs ( range)61.1 3.8 ( 5668)male : female ( n)7:6 ( n = 13)diagnosiscerebral infarction lacunarthalamus 2internal capsule2corona radiata4atherothrombosis1cerebral hemorrhage thalamus2putamen2paralysisright13left0dominant handright13left0time from injury , months12.6 7.6ue - fma21.8 5.6mmse26.9 1.8ue - fma : fugl - meyer assesment ( ue ) , mmse : mini - mental state examination . ue - fma : fugl - meyer assesment ( ue ) , mmse : mini - mental state examination the subjects were instructed to relax while reclining on a chair that had a backrest with an adjustable angle , and were asked to position the forearm of their left upper extremity ( that is , the upper extremity on the non - measured side ) so that the elbow touched the chair . it was first explained to subjects that electroencephalographic measurements would be performed under two conditions : a resting state , the control condition ( rest ) , and during the application of a vibratory stimulation , the task condition ( vibratory stimulation ) . the motion targeted in the vibratory stimulation - induced kinesthetic illusion was extension of the right elbow joint . therefore , the bicep tendon of the right upper arm was selected as the stimulation site . because the stimulation was applied to the skin , cutaneous stimulus receptors , in addition to muscle spindles , were excited . the -waves , which change according to sensorimotor information , are also influenced by sensory information received from cutaneous stimuli20 ; therefore , as a control , the subjects were asked to envision that elbow flexion was prevented due to contraction of the bicep muscle during vibratory stimulation20 . in order to ensure that the forearm would not be in contact with the trunk , the measurer fixed a hand massager ( thrive md-01 ; thrive co. , ltd . , osaka , japan ) on the bicep tendon of the subjects right arms . at rest , electroencephalographic measurements were conducted without applying any vibratory stimulation . during the stimulation , in addition , since visual information pertaining to the non - moving upper extremity has been found to reduce kinesthetic illusion , subjects were instructed to close their eyes during the vibratory stimulation9 . data were collected for 3 to 9 days ( mean 4.9 2.0 days ) . vibratory stimulation and rest assessment were both carried out twice a day for 3 consecutive or non - consecutive days . after - effects were taken into consideration by allowing subjects to rest between vibratory stimulation sessions . the average value of the brain wave data of the twelve measurements was calculated , and used as the data for each subject . electroencephalographic measurements were performed during the two conditions , and a neurofax eeg-1200 ( nihon kohden , tokyo , japan ) was used to record the electroencephalograms . measurements were conducted at 18 sites of the international 1020 system ( f3 , f4 , f7 , f8 , fz , c3 , c4 , cz , p3 , p4 , pz , o1 , o2 , oz , t3 , t4 , t5 , and t6 ) , with the earlobes on both sides as the reference electrodes . data were processed with a bandpass filter of 0.5 to 60 hz , and the sampling frequency was 1,000 hz . the duration of the measurement was set to 60 s per session for both the rest and vibratory stimulation conditions10 . for the imaging analysis , 30 epochs were extracted from the data obtained under each condition ( the duration of one epoch was 2 s ) , and a frequency analysis was performed . the data within the -wave frequency range ( 10 to 13 hz ) were calculated and used for intracerebral spatial analyses using the standardized low resolution brain electromagnetic tomography ( sloreta ) analysis22 , a method that allows three - dimensional image display of intracerebral neural activity . the values of each voxel in the brain areas displaying neural activity under each condition were calculated as the neural activity ( current source density [ csd ] ; a / mm*10 ) , and were identified in terms of brodmann s area ( ba ) and montreal neurological institute ( mni ) coordinates23 . by averaging data using the sloreta averager software , the neural activity could be calculated as the global field power ( gfp ) value22 in the two groups under both the rest and vibratory stimulation conditions . in addition , the intracerebral neural activities of the two groups under each condition were compared using the statistics in sloreta snpm24 . in this analysis , the neural areas showing significant differences in activity ( with a statistical significance level of 5% ) were colored , calculated , and drawn . the significance threshold was based on a permutation test with 5000 permutations , using log - transformed loreta values , with subject - wise normalization ( on the assumption that differences in the base - line activity between healthy subjects and patients may exist ) . when -waves manifest in the sensorimotor cortex13 , frontal lobe25 , or occipital lobe26 , the waveform components and the names of the waves can differ ( such as -waves ) depending on their region of occurrence . interestingly , this phenomenon is evident even when these waves have the same frequency range . thus , in this study , the different types of -waves were investigated separately . prior to the analysis , participants were asked what type of feeling they sensed in the elbow joint upon vibratory stimulation , and 23 of the 25 initially recruited subjects answered : i felt as though my elbow kept extending continuously , and i felt as though the elbow continued to move toward the opposite side by itself . however , for two participants in the patient group , a kinesthetic illusion could not be confirmed . therefore , the data of the 12 subjects in the healthy group and the remaining 11 subjects in the patient group were used for the analysis . the mni coordinates and csd values of the areas showing significant differences between the groups under each condition . in the healthy group , intracerebral neural activity in the -wave frequency range was observed at rest in an extended region of motor area ba4 and sensory areas ba3 , 2 , and 1 in the sensorimotor cortex , as well as in supplementary motor area ba6 . on the other hand , -waves were observed in an extended region of the prefrontal cortex and occipital lobe , which showed significantly higher neural activity than other areas of the cerebral cortex ( fig . areas of the brain where the - and -waves significantly increased are indicated in yellow and red . areas of the brain where the - and -waves significantly increased compared to the patient group are indicated in yellow and red . color scale values below the images indicate t - values ( t = 2.288 ) ) . in the patient group , significantly stronger -wave activity was found in the sensorimotor cortical areas , and the supplementary motor area ba6 , while waves were observed in an extended region of the occipital lobe ( fig . in comparison to the patient group , healthy individuals showed a significant increase in neural activity in sensorimotor cortical areas , supplementary motor area ba6 , the occipital lobe , and the prefrontal area ( fig . . areas of the brain where the - and -waves significantly increased are indicated in yellow and red . areas of the brain where the - and -waves significantly increased compared to the patient group are indicated in yellow and red . color scale values below the images indicate t - values ( t = 2.288 ) in both groups , intracerebral neural activity of the -wave frequency band during vibratory stimulation showed significantly higher activity in the occipital lobe than in other areas of the cerebral cortex ; however , sensorimotor cortical areas showed no significant activity in the -wave frequency band during neural activity ( fig . 2-a , bfig . 2.neural activity in the -wave range during vibratory stimulation . a : neural activity in the -wave range of the healthy group . brain areas where the - and -waves significantly increased are indicated in yellow and red .. c : comparison of the neural activities of the healthy and patient groups . areas of brain where the - and -waves significantly increased in the healthy group compared to the patient group are indicated in yellow and red , and areas of the brain where the - and -waves significantly increased in the patient group compared to the healthy group are indicated in light blue and blue . color scale values below the images indicate t - values ( t = 2.264 ) ) . in comparison to the patient group , the healthy group showed a significant increase in activity in the occipital lobe , while the patient group exhibited greater activity in the supplementary motor area ba6 ( fig . brain areas where the - and -waves significantly increased are indicated in yellow and red .. c : comparison of the neural activities of the healthy and patient groups . areas of brain where the - and -waves significantly increased in the healthy group compared to the patient group are indicated in yellow and red , and areas of the brain where the - and -waves significantly increased in the patient group compared to the healthy group are indicated in light blue and blue . color scale values below the images indicate t - values ( t = 2.264 ) this study examined the impact of kinesthetic illusions induced by vibratory stimulation on the intracerebral neural activity in patients with stroke , using functional neuroimaging analysis of electroencephalographic -waves as a neurophysiological index . at rest , the patient group showed - and -waves in the sensorimotor cortical areas , prefrontal areas , and in an extended area over the occipital lobe . these results were consistent with the conventionally known origins of -waves , namely , the sensorimotor cortical areas13 , frontal lobe25 , and occipital lobe26 . the presence of -waves at rest reflected the inhibition of neuronal excitation in sensorimotor areas , and may also reflect the resting state similar to -waves25 . therefore , our results confirm previous reports of brain waves in the frequency range of 10 to 13 hz at rest . our findings also indicate the reliability of the electroencephalographic parameters measured in this study . a comparison of the control and patient groups at rest showed that -waves were significantly decreased in the prefrontal cortex and in the occipital lobe in the patient group compared to the healthy group . recently , dubovik , et al.29 reported that ischemic lesions caused by stroke decrease the synchrony of -waves . moreover , they concluded that this decrease is related to the cognitive and motor deficits observed in stroke patients . in addition , as a characteristic property of electroencephalograms in patients with stroke , the laterality of -waves is not limited to the damaged site , but is often found in the sensorimotor cortical areas and occipital lobe . in the thalamus , -waves are suppressed when the anteromedial region is damaged30 . the lateral edges of lesions in many of our participants were located in the anterior and mediolateral regions of the thalamus . this may have contributed to the significant reductions in - and -waves seen in the patient group compared to the healthy group . furthermore , in the sensorimotor cortex and supplementary motor area ba6 , -waves were significantly decreased in the patient group compared to the healthy group . ba6 conducts oriented processing of information31 , 32 for the sensorimotor cortical areas , suggesting that ba6 may functionally be closely related to the sensorimotor cortex . the neural activity in these areas is known to involve a decrease in -waves during preparation for motor execution , the so - called idling rhythm33 . therefore , the stroke - induced decrease of -waves might include -waves . however , our findings suggest that motor impairment may have affected the process from the stage before the integration of sensorimotor information aimed at executing motion , and that the neural basis of those regions may be functionally different from those of the healthy group . during vibratory stimulation , intracerebral neural activity in the -wave frequency band did not change significantly in the sensorimotor areas of either subject group . this suggests an increase in information from the sensorimotor cortical areas , that plays an important role in the processing of kinesthetic information from the muscle spindles , and in motor perception3 . in this case , -waves may have been attenuated because of the asynchronous nature of neuronal activity on the two sides34 in the sensorimotor cortical areas13 , 15 . neural activity in the motor area ba4 is not only involved in the processing of sensory information from the muscle spindle , but is also related to sensorimotor information per se3 . such information includes the kinesthetic illusion that results from the process ; thus , kinesthetic illusions are greater when the excitation of the motor area is more extensive2 . our results therefore indicate that kinesthetic illusions triggered by vibratory stimuli increased neural activity in the sensorimotor cortical areas . a comparison of the two groups found that activity in ba6 significantly increased during vibratory stimulation in the patient group compared to the healthy group . previous studies have shown that , in patients with stroke , -waves are decreased compared to healthy subjects35 . in addition , our study revealed differences in the neural activity of the supplementary motor area ba6 , but not in the motor area ba4 , which is responsible for perception during kinesthetic illusions . therefore , there may be differences between the healthy subjects and patients in terms of processing of neural information during the generation of kinesthetic illusions . ba6 is involved in spatial cognition of sensorimotor information36 , not only during actual movement , but also during kinesthetic illusion . this area stores simulations of motor imagery based on previous sensorimotor experiences , which are needed for kinesthetic illusion12 , and it is considered to be the area that discriminates sensorimotor information37 , 38 . therefore , it could be hypothesized that patients with stroke functionally differ from healthy individuals in terms of the generation of kinesthetic illusions , and particularly at the simulation stage or in the storage of motor imagery . in this study , electroencephalographic analysis was found to be a useful tool for reflecting38 brain functioning after stroke . we examined the effects of kinesthetic illusion on intracerebral neural activity in patients after stroke using functional neuroimaging analysis based on -waves . our findings suggest that kinesthetic illusions occur in patients with motor deficits due to stroke , and that such patients exhibit elevated neural activity in the sensorimotor areas and in supplementary motor area ba6 . however , this study did not compare or verify neural activity in the sensorimotor , supplementary motor , or related areas . moreover , motor imagery ability or motor function of the subjects was no evaluated . in future , the functional association between sensorimotor areas and related brain regions will require further detailed examination using electroencephalographic analysis . more specifically , these analyses will need to investigate the induction and generation of kinesthetic illusion and neural activity in sensorimotor cortical areas . it will also be important to quantitatively compare healthy subjects and patients with stroke with respect to their abilities to recall motor images , and to assess the association of this process with activity in the supplementary motor area .

[ purpose ] this study evaluated the influence of vibratory stimulation - induced kinesthetic illusion on brain function after stroke . [ subjects ] twelve healthy individuals and 13 stroke patients without motor or sensory loss participated . [ methods ] electroencephalograms were taken at rest and during vibratory stimulation . as a neurophysiological index of brain function , we measured the -rhythm , which is present mainly in the kinesthetic cortex and is attenuated by movement or motor imagery and compared the data using source localization analyses in the standardized low resolution brain electromagnetic tomography ( sloreta ) program . [ results ] at rest , -rhythms appeared in the sensorimotor and supplementary motor cortices in both healthy controls and stroke patients . under vibratory stimulation , no -rhythm appeared in the sensorimotor cortex of either group . moreover , in the supplementary motor area , which stores the motor imagery required for kinesthetic illusions , the -rhythms of patients were significantly stronger than those of the controls , although the -rhythms of both groups were reduced . thus , differences in neural activity in the supplementary motor area were apparent between the subject groups . [ conclusion ] kinesthetic illusions do occur in patients with motor deficits due to stroke . the neural basis of the supplementary motor area in stroke patients may be functionally different from that found in healthy controls .

INTRODUCTION SUBJECTS AND METHODS Subjects Electroencephalography Data analysis RESULTS DISCUSSION

in the field of neurorehabilitation , kinesthetic illusion1 has been used as a rehabilitation therapy for patients who have suffered a stroke . kinesthetic illusions , which are induced by sensorimotor information in the brain , involve the excitement of sensorimotor cortical areas and the association of underlying neuronal activity with motion control and skill learning is believed to have beneficial effects in rehabilitation2 . the therapeutic effects of kinesthetic illusion on the motor deficits of patients with stroke have been reported in a number of publications3 , 4 . for example , altschuler et al.7 demonstrated that mirror - induced kinesthetic illusions resulted in recovery of motor function even in patients in the chronic phase of post - stroke recovery ( i.e. in addition , dohle et al.8 examined the effects of a mirror - based treatment on patients suffering severe paralysis after stroke and found improvements in finger motor function after the intervention . when an intervention task entails processing visual information during kinesthetic illusion , various types of brain functions , such as cognitive , visual information processing , and motor functions , related to the unaffected extremity can affect performance of the task . therefore , the stimulus used in the task itself should be as simple and passive as possible . thus , some studies have used vibratory stimulation3 , 9 , 10 as a method for inducing kinesthetic illusion . functional magnetic resonance imaging10 and magnetoencephalography11 have implicated the following regions in kinesthetic illusion - related neural activity : the primary sensorimotor cortex , the sensory association area , the supplementary motor area , and the angular gyrus . during kinesthetic illusion , given that previous sensorimotor experiences need to be simulated in kinesthetic motor imagery12 , it is important that brain function be analyzed based on the degree of kinesthetic illusion achieved . therefore , in order to determine the brain functions occurring during such an illusion , detailed temporal and spatial analyses ( from the beginning of kinesthetic motor image formation ) need to be performed before the induced kinesthetic illusion occurs . the electroencephalographic mu - rhythm13 ( -wave ) is useful as a neurophysiological index for capturing data from such analyses . a -wave is the high - frequency component ranging from 10 to 13 hz of the alpha band14 , which appears in the sensorimotor cortex13 , 15 and the supplementary motor area16 , 17 . since this wave is attenuated not only by actual motion , but also by kinesthetic motor imagery13 , 15 , it reflects neural activity both during kinesthetic illusion and during the generation of a motor image . to date , the association between kinesthetic illusion and -waves has been reported for healthy subjects18 , but not for stroke patients . thus , the purpose of this study was to examine the impact of kinesthetic illusion , produced by vibratory stimulation , on the cerebral neural activity of patients with stroke , through functional neuroimaging of -waves . in accordance with the declaration of helsinki , the scope and contents of this study , as well as the precautions against the disclosure of personal information , were explained to the subjects verbally and in written form . this study was conducted on 12 healthy subjects ( six men and six women ; mean age : 60.1 3.2 years ) with no orthopedic or nervous system disease , or motor deficits or sensory disorders ( the healthy group ) , and 13 patients with stroke ( seven men and six women ; mean age : 61.1 3.8 year ; the patient group ) . the dominant hands of the subjects in the healthy group were assessed using the edinburgh inventory19 , and all subjects were confirmed to be right - handed . we confirmed that none of the subjects in the patient group had any cognitive dysfunction or disorder , or any sensory impairment . it was first explained to subjects that electroencephalographic measurements would be performed under two conditions : a resting state , the control condition ( rest ) , and during the application of a vibratory stimulation , the task condition ( vibratory stimulation ) . the motion targeted in the vibratory stimulation - induced kinesthetic illusion was extension of the right elbow joint . therefore , the bicep tendon of the right upper arm was selected as the stimulation site . the -waves , which change according to sensorimotor information , are also influenced by sensory information received from cutaneous stimuli20 ; therefore , as a control , the subjects were asked to envision that elbow flexion was prevented due to contraction of the bicep muscle during vibratory stimulation20 . at rest , electroencephalographic measurements were conducted without applying any vibratory stimulation . during the stimulation , in addition , since visual information pertaining to the non - moving upper extremity has been found to reduce kinesthetic illusion , subjects were instructed to close their eyes during the vibratory stimulation9 . the average value of the brain wave data of the twelve measurements was calculated , and used as the data for each subject . the duration of the measurement was set to 60 s per session for both the rest and vibratory stimulation conditions10 . the data within the -wave frequency range ( 10 to 13 hz ) were calculated and used for intracerebral spatial analyses using the standardized low resolution brain electromagnetic tomography ( sloreta ) analysis22 , a method that allows three - dimensional image display of intracerebral neural activity . the values of each voxel in the brain areas displaying neural activity under each condition were calculated as the neural activity ( current source density [ csd ] ; a / mm*10 ) , and were identified in terms of brodmann s area ( ba ) and montreal neurological institute ( mni ) coordinates23 . by averaging data using the sloreta averager software , the neural activity could be calculated as the global field power ( gfp ) value22 in the two groups under both the rest and vibratory stimulation conditions . in addition , the intracerebral neural activities of the two groups under each condition were compared using the statistics in sloreta snpm24 . in this analysis , the neural areas showing significant differences in activity ( with a statistical significance level of 5% ) were colored , calculated , and drawn . the significance threshold was based on a permutation test with 5000 permutations , using log - transformed loreta values , with subject - wise normalization ( on the assumption that differences in the base - line activity between healthy subjects and patients may exist ) . when -waves manifest in the sensorimotor cortex13 , frontal lobe25 , or occipital lobe26 , the waveform components and the names of the waves can differ ( such as -waves ) depending on their region of occurrence . thus , in this study , the different types of -waves were investigated separately . in accordance with the declaration of helsinki , the scope and contents of this study , as well as the precautions against the disclosure of personal information , were explained to the subjects verbally and in written form . upon their understanding of the explanations , the study was approved by the ethics committee of kurume university school of medicine . this study was conducted on 12 healthy subjects ( six men and six women ; mean age : 60.1 3.2 years ) with no orthopedic or nervous system disease , or motor deficits or sensory disorders ( the healthy group ) , and 13 patients with stroke ( seven men and six women ; mean age : 61.1 3.8 year ; the patient group ) . the dominant hands of the subjects in the healthy group were assessed using the edinburgh inventory19 , and all subjects were confirmed to be right - handed . it was first explained to subjects that electroencephalographic measurements would be performed under two conditions : a resting state , the control condition ( rest ) , and during the application of a vibratory stimulation , the task condition ( vibratory stimulation ) . the motion targeted in the vibratory stimulation - induced kinesthetic illusion was extension of the right elbow joint . the -waves , which change according to sensorimotor information , are also influenced by sensory information received from cutaneous stimuli20 ; therefore , as a control , the subjects were asked to envision that elbow flexion was prevented due to contraction of the bicep muscle during vibratory stimulation20 . at rest , electroencephalographic measurements were conducted without applying any vibratory stimulation . during the stimulation , in addition , since visual information pertaining to the non - moving upper extremity has been found to reduce kinesthetic illusion , subjects were instructed to close their eyes during the vibratory stimulation9 . after - effects were taken into consideration by allowing subjects to rest between vibratory stimulation sessions . the average value of the brain wave data of the twelve measurements was calculated , and used as the data for each subject . the duration of the measurement was set to 60 s per session for both the rest and vibratory stimulation conditions10 . the data within the -wave frequency range ( 10 to 13 hz ) were calculated and used for intracerebral spatial analyses using the standardized low resolution brain electromagnetic tomography ( sloreta ) analysis22 , a method that allows three - dimensional image display of intracerebral neural activity . the values of each voxel in the brain areas displaying neural activity under each condition were calculated as the neural activity ( current source density [ csd ] ; a / mm*10 ) , and were identified in terms of brodmann s area ( ba ) and montreal neurological institute ( mni ) coordinates23 . by averaging data using the sloreta averager software , the neural activity could be calculated as the global field power ( gfp ) value22 in the two groups under both the rest and vibratory stimulation conditions . in addition , the intracerebral neural activities of the two groups under each condition were compared using the statistics in sloreta snpm24 . in this analysis , the neural areas showing significant differences in activity ( with a statistical significance level of 5% ) were colored , calculated , and drawn . the significance threshold was based on a permutation test with 5000 permutations , using log - transformed loreta values , with subject - wise normalization ( on the assumption that differences in the base - line activity between healthy subjects and patients may exist ) . when -waves manifest in the sensorimotor cortex13 , frontal lobe25 , or occipital lobe26 , the waveform components and the names of the waves can differ ( such as -waves ) depending on their region of occurrence . thus , in this study , the different types of -waves were investigated separately . prior to the analysis , participants were asked what type of feeling they sensed in the elbow joint upon vibratory stimulation , and 23 of the 25 initially recruited subjects answered : i felt as though my elbow kept extending continuously , and i felt as though the elbow continued to move toward the opposite side by itself . therefore , the data of the 12 subjects in the healthy group and the remaining 11 subjects in the patient group were used for the analysis . the mni coordinates and csd values of the areas showing significant differences between the groups under each condition . in the healthy group , intracerebral neural activity in the -wave frequency range was observed at rest in an extended region of motor area ba4 and sensory areas ba3 , 2 , and 1 in the sensorimotor cortex , as well as in supplementary motor area ba6 . on the other hand , -waves were observed in an extended region of the prefrontal cortex and occipital lobe , which showed significantly higher neural activity than other areas of the cerebral cortex ( fig . in the patient group , significantly stronger -wave activity was found in the sensorimotor cortical areas , and the supplementary motor area ba6 , while waves were observed in an extended region of the occipital lobe ( fig . in comparison to the patient group , healthy individuals showed a significant increase in neural activity in sensorimotor cortical areas , supplementary motor area ba6 , the occipital lobe , and the prefrontal area ( fig . color scale values below the images indicate t - values ( t = 2.288 ) in both groups , intracerebral neural activity of the -wave frequency band during vibratory stimulation showed significantly higher activity in the occipital lobe than in other areas of the cerebral cortex ; however , sensorimotor cortical areas showed no significant activity in the -wave frequency band during neural activity ( fig . 2.neural activity in the -wave range during vibratory stimulation . a : neural activity in the -wave range of the healthy group . areas of brain where the - and -waves significantly increased in the healthy group compared to the patient group are indicated in yellow and red , and areas of the brain where the - and -waves significantly increased in the patient group compared to the healthy group are indicated in light blue and blue . in comparison to the patient group , the healthy group showed a significant increase in activity in the occipital lobe , while the patient group exhibited greater activity in the supplementary motor area ba6 ( fig . brain areas where the - and -waves significantly increased are indicated in yellow and red .. c : comparison of the neural activities of the healthy and patient groups . areas of brain where the - and -waves significantly increased in the healthy group compared to the patient group are indicated in yellow and red , and areas of the brain where the - and -waves significantly increased in the patient group compared to the healthy group are indicated in light blue and blue . color scale values below the images indicate t - values ( t = 2.264 ) this study examined the impact of kinesthetic illusions induced by vibratory stimulation on the intracerebral neural activity in patients with stroke , using functional neuroimaging analysis of electroencephalographic -waves as a neurophysiological index . at rest , the patient group showed - and -waves in the sensorimotor cortical areas , prefrontal areas , and in an extended area over the occipital lobe . therefore , our results confirm previous reports of brain waves in the frequency range of 10 to 13 hz at rest . a comparison of the control and patient groups at rest showed that -waves were significantly decreased in the prefrontal cortex and in the occipital lobe in the patient group compared to the healthy group . moreover , they concluded that this decrease is related to the cognitive and motor deficits observed in stroke patients . in addition , as a characteristic property of electroencephalograms in patients with stroke , the laterality of -waves is not limited to the damaged site , but is often found in the sensorimotor cortical areas and occipital lobe . furthermore , in the sensorimotor cortex and supplementary motor area ba6 , -waves were significantly decreased in the patient group compared to the healthy group . ba6 conducts oriented processing of information31 , 32 for the sensorimotor cortical areas , suggesting that ba6 may functionally be closely related to the sensorimotor cortex . the neural activity in these areas is known to involve a decrease in -waves during preparation for motor execution , the so - called idling rhythm33 . however , our findings suggest that motor impairment may have affected the process from the stage before the integration of sensorimotor information aimed at executing motion , and that the neural basis of those regions may be functionally different from those of the healthy group . during vibratory stimulation , intracerebral neural activity in the -wave frequency band did not change significantly in the sensorimotor areas of either subject group . this suggests an increase in information from the sensorimotor cortical areas , that plays an important role in the processing of kinesthetic information from the muscle spindles , and in motor perception3 . in this case , -waves may have been attenuated because of the asynchronous nature of neuronal activity on the two sides34 in the sensorimotor cortical areas13 , 15 . neural activity in the motor area ba4 is not only involved in the processing of sensory information from the muscle spindle , but is also related to sensorimotor information per se3 . such information includes the kinesthetic illusion that results from the process ; thus , kinesthetic illusions are greater when the excitation of the motor area is more extensive2 . our results therefore indicate that kinesthetic illusions triggered by vibratory stimuli increased neural activity in the sensorimotor cortical areas . a comparison of the two groups found that activity in ba6 significantly increased during vibratory stimulation in the patient group compared to the healthy group . previous studies have shown that , in patients with stroke , -waves are decreased compared to healthy subjects35 . in addition , our study revealed differences in the neural activity of the supplementary motor area ba6 , but not in the motor area ba4 , which is responsible for perception during kinesthetic illusions . therefore , there may be differences between the healthy subjects and patients in terms of processing of neural information during the generation of kinesthetic illusions . this area stores simulations of motor imagery based on previous sensorimotor experiences , which are needed for kinesthetic illusion12 , and it is considered to be the area that discriminates sensorimotor information37 , 38 . therefore , it could be hypothesized that patients with stroke functionally differ from healthy individuals in terms of the generation of kinesthetic illusions , and particularly at the simulation stage or in the storage of motor imagery . in this study , electroencephalographic analysis was found to be a useful tool for reflecting38 brain functioning after stroke . we examined the effects of kinesthetic illusion on intracerebral neural activity in patients after stroke using functional neuroimaging analysis based on -waves . our findings suggest that kinesthetic illusions occur in patients with motor deficits due to stroke , and that such patients exhibit elevated neural activity in the sensorimotor areas and in supplementary motor area ba6 . however , this study did not compare or verify neural activity in the sensorimotor , supplementary motor , or related areas . moreover , motor imagery ability or motor function of the subjects was no evaluated . more specifically , these analyses will need to investigate the induction and generation of kinesthetic illusion and neural activity in sensorimotor cortical areas . it will also be important to quantitatively compare healthy subjects and patients with stroke with respect to their abilities to recall motor images , and to assess the association of this process with activity in the supplementary motor area .

in recent years , the number of people ascending to high altitudes worldwide has clearly increased . however , acute mountain sickness ( ams ) is a major obstacle faced by many people after arrival at high altitudes . ams is a common syndrome encountered particularly by lowlanders after rapidly ascending to altitudes above 2,500 m.1 this syndrome is characterized by headache , dizziness or lightheadedness , gastrointestinal symptoms ( anorexia , nausea , or vomiting ) , weakness or fatigue , and insomnia.2 among these symptoms , headache is the most frequent and essential for final ams diagnosis . although self - limiting , ams may develop into fatal high altitude pulmonary or cerebral edema in a small number of people.3 therefore , prevention of ams is particularly important . research on risk factors for ams ( eg , female sex and younger age)4 is beneficial for the prevention of ams , although further investigations are needed to establish these risk factors.5 with the development of transportation , rapid ascension to high altitudes is becoming common and easy . however , as yet , there has been no large - scale epidemiological investigation into the relationship between age and ams upon rapid ascent to high altitudes . thus , the relationship between age and ams upon acute exposure to high altitudes remains unclear . given that past literature68 indicated a tendency for older adults to be at greater risk of ams than younger adults , we hypothesized that older age would increase the prevalence of ams upon rapid ascent to high altitude . therefore , this study uses a cross - sectional design aiming to explore the relationship between age and ams in subjects suddenly exposed to high altitude . in the present study , 987 adult chinese men aged 18 years were recruited in june 2012 . all subjects were lowlanders , without preceding altitude exposure during the previous half year , and none suffered from cardiopulmonary disease or was currently taking any medicines ( eg , vitamins , cold cure , etc ) . prior to the investigation , all subjects provided written informed consent approved by the ethics review board of xinqiao hospital , second affiliated hospital of third military medical university ( decision number : 2012015 ) . the study protocol was registered in the chinese clinical trial registry ( http://www.chictr.org ; registration no . one week before ascending to 3,700 m , all subjects were interviewed and underwent a routine physical examination ( echocardiogram , electrocardiogram , transcranial doppler , chest x - ray , heart rate [ hr ] , blood pressure [ bp ] , and arterial oxygen saturation [ sao2 ] ) . height and weight were measured with standard methods , and body mass index ( bmi ) was obtained by the formula ( weight [ kg ] divided by height squared [ m ] ) . in addition , sleep status of the subjects was investigated using the athens insomnia scale score ( aiss).9 subjects demographic characteristics ( age , race [ chinese han , miao , man , tibetan , etc ] , education , drinking10 and smoking11 habits , etc ) were recorded in the questionnaire . in the morning , all subjects ascended by plane from 500 m to 3,700 m within 2 hours , in batches ( with a 1-day interval between batches ) . after arrival at 3,700 m , all subjects undertook the same daily regimen and refrained from vigorous exercise . on the morning of the second day at 3,700 m , all subjects completed the athens insomnia scale ( ais)9 and lake louise score ( lls)2 questionnaires . all identifying characteristics ( name , age , etc ) were concealed before data analysis . in the present study , the lls questionnaire comprised five items : headache , dizziness or lightheadedness , gastrointestinal symptoms ( anorexia , nausea , vomiting ) , fatigue and/or weakness , and insomnia . each item was rated on a four - point likert scale ( 0= not at all , 1= mild , 2= moderate , and 3= severe ) , with total scores varying from 0 to 15 . ams was determined when a subject reported a headache and one or more of the other symptoms as well as an lls of 3 . the ams was then classified as mild ( lls 3 or 4 ) or serious ( lls 5 ) . after completion of the lls questionnaire , the hr , sao2 , and bp values of the subjects were measured after a 15-minute rest in a comfortable chair . hr and sao2 values were obtained with finger - pulse oximetry ( onyx 9500 ; nonin medical , inc . , bp was measured with a sphygmomanometer ( omron hem-6200 ; omron health care , inc . , three consecutive measurements for all parameters were obtained , and the mean value of the three measurements was used in the analysis . sleep status of the subjects in the past month was assessed using the ais.9 the ais includes eight items : the first five items assess sleep procedure ( sleep induction , night awakening , awakening in the early morning , total sleep duration , and total quality of sleep ) , and the last three items estimate decreased sense and functioning , and sleepiness in the next day . each item is rated from 0 to 3 using a four - point likert scale ( 0= no event , 1= mild , 2= moderate , and 3= severe ) . subjects with an ais score 6 were considered to be suffering from insomnia.12 the ais is considered an excellent instrument for assessing subjective sleep quality in both clinical and study settings as it has good reliability and validity9 and is easy to complete.13 data are shown as mean standard deviation , median ( interquartile range ) , or n ( % ) , and were analyzed using spss version 13.0 ( ibm corporation , armonk , ny , usa ) . all tests were two - tailed , and a p - value < 0.05 was considered significant . whitney u - test was used for the comparison of non - normally distributed data . relationship between lls and age , bmi , and other variables , as well as between age and bmi , bp , and other variables , was determined using the spearman correlation test . before the multivariate analysis , an odds ratio ( or ) with 95% confidence intervals ( cis ) for ams associated with risk factors on univariate analysis , factors with a p - value 0.10 were entered into multivariate logistic regression analysis . the backward stepwise likelihood ratio test was used to determine the independent factors associated with ams . in the present study , the dependent variable was ams and recorded as 0= no and 1= yes . the independent variables were age , bmi , race , education , smoking status , drinking status , physiological parameters , and aiss . we recorded the age value as 0 ( 20 years ) , 1 ( 25 years ) , 2 ( 30 years ) , and 3 ( 35 years ) . in the present study , 987 adult chinese men aged 18 years were recruited in june 2012 . all subjects were lowlanders , without preceding altitude exposure during the previous half year , and none suffered from cardiopulmonary disease or was currently taking any medicines ( eg , vitamins , cold cure , etc ) . prior to the investigation , all subjects provided written informed consent approved by the ethics review board of xinqiao hospital , second affiliated hospital of third military medical university ( decision number : 2012015 ) . the study protocol was registered in the chinese clinical trial registry ( http://www.chictr.org ; registration no . one week before ascending to 3,700 m , all subjects were interviewed and underwent a routine physical examination ( echocardiogram , electrocardiogram , transcranial doppler , chest x - ray , heart rate [ hr ] , blood pressure [ bp ] , and arterial oxygen saturation [ sao2 ] ) . height and weight were measured with standard methods , and body mass index ( bmi ) was obtained by the formula ( weight [ kg ] divided by height squared [ m ] ) . in addition , sleep status of the subjects was investigated using the athens insomnia scale score ( aiss).9 subjects demographic characteristics ( age , race [ chinese han , miao , man , tibetan , etc ] , education , drinking10 and smoking11 habits , etc ) were recorded in the questionnaire . in the morning , all subjects ascended by plane from 500 m to 3,700 m within 2 hours , in batches ( with a 1-day interval between batches ) . after arrival at 3,700 m , all subjects undertook the same daily regimen and refrained from vigorous exercise . on the morning of the second day at 3,700 m , all subjects completed the athens insomnia scale ( ais)9 and lake louise score ( lls)2 questionnaires . all identifying characteristics ( name , age , etc ) were concealed before data analysis . in the present study , the lls system was used for the diagnosis of ams in subjects . the lls questionnaire comprised five items : headache , dizziness or lightheadedness , gastrointestinal symptoms ( anorexia , nausea , vomiting ) , fatigue and/or weakness , and insomnia . each item was rated on a four - point likert scale ( 0= not at all , 1= mild , 2= moderate , and 3= severe ) , with total scores varying from 0 to 15 . ams was determined when a subject reported a headache and one or more of the other symptoms as well as an lls of 3 . the ams was then classified as mild ( lls 3 or 4 ) or serious ( lls 5 ) . after completion of the lls questionnaire , the hr , sao2 , and bp values of the subjects were measured after a 15-minute rest in a comfortable chair . hr and sao2 values were obtained with finger - pulse oximetry ( onyx 9500 ; nonin medical , inc . , bp was measured with a sphygmomanometer ( omron hem-6200 ; omron health care , inc . , three consecutive measurements for all parameters were obtained , and the mean value of the three measurements was used in the analysis . sleep status of the subjects in the past month was assessed using the ais.9 the ais includes eight items : the first five items assess sleep procedure ( sleep induction , night awakening , awakening in the early morning , total sleep duration , and total quality of sleep ) , and the last three items estimate decreased sense and functioning , and sleepiness in the next day . each item is rated from 0 to 3 using a four - point likert scale ( 0= no event , 1= mild , 2= moderate , and 3= severe ) . subjects with an ais score 6 were considered to be suffering from insomnia.12 the ais is considered an excellent instrument for assessing subjective sleep quality in both clinical and study settings as it has good reliability and validity9 and is easy to complete.13 data are shown as mean standard deviation , median ( interquartile range ) , or n ( % ) , and were analyzed using spss version 13.0 ( ibm corporation , armonk , ny , usa ) . all tests were two - tailed , and a p - value < 0.05 was considered significant . whitney u - test was used for the comparison of non - normally distributed data . relationship between lls and age , bmi , and other variables , as well as between age and bmi , bp , and other variables , was determined using the spearman correlation test . before the multivariate analysis , an odds ratio ( or ) with 95% confidence intervals ( cis ) for ams associated with risk factors was determined using logistic regression . on univariate analysis , factors with a p - value 0.10 were entered into multivariate logistic regression analysis . the backward stepwise likelihood ratio test was used to determine the independent factors associated with ams . in the present study , the independent variables were age , bmi , race , education , smoking status , drinking status , physiological parameters , and aiss . we recorded the age value as 0 ( 20 years ) , 1 ( 25 years ) , 2 ( 30 years ) , and 3 ( 35 years ) . we enrolled 987 participants at 500 m , 79 of whom were excluded for incomplete data or preceding exposure to high altitude . in addition , 22 tibetan subjects were removed from the study because of the small sample size . after arrival at 3,700 m , one subject withdrew from the study because of high altitude pulmonary edema , and a further 29 subjects withdrew due to severe headache , diarrhea , or other unknown reasons . of these , 195 subjects were aged 1820 years , 314 were aged 2125 years , 238 were aged 2630 years , and 109 were aged 3135 years . the mean age in the groups aged 20 , 2125 , 2630 , and 3135 years was 19 , 22 , 27 , and 33 years , respectively . increase in bmi was consistent with increase in age ( 21.12.4 , 22.02.3 , 22.32.5 , and 23.62.8 kg / m in the four age groups , respectively ) . except for age ( p<0.001 ) and bmi ( p<0.001 ) , no statistically significant differences in smoking and drinking status , race , and education were observed between the four age groups . at an altitude of 3,700 m , the lls in the age groups 20 , 2125 , 2630 , and 3135 years was 3.42.1 , 3.32.1 , 3.92.3 , and 4.62.1 , respectively . differences in lls between the four groups ( p<0.05 or p<0.01 ) were significant , except for between the age groups 20 years and 2125 years ( p=0.68 ) . accordingly , the increased prevalence of ams was consistent with the increase in lls ( 54.36% , 59.55% , 68.07% , and 73.39% in the four groups , respectively ) . the prevalence of ams in the 20 and 2125 years age groups was significantly lower than in the 2630 and 3135 years age groups ( p<0.05 or p<0.01 ) . notably , the prevalence of severe ams increased with increasing age ( table 1 ) . in addition , the overall prevalence of ams at the 3,700 m altitude was 62.38% . in terms of ams symptoms , the most common were headache ( 74.88% ) , followed by weakness or fatigue ( 73.36% ) , dizziness or lightheadedness ( 73.23% ) , difficulty sleeping ( 66.59% ) , and the prevalence of gastrointestinal symptoms is relatively lower than the prevalence of other ams symptoms ( 22.43% ) . moreover , the prevalence of ams symptoms clearly increased with age ( figure 1 ) , and the difference in the prevalence of headache , insomnia , and weakness or fatigue reached statistical significance ( p<0.001 , p<0.001 , and p<0.01 , respectively ) . at 500 m , the hr and sao2 for the whole group were 689 beats / minute and 98.1%0.9% , respectively . no significant differences between the four age groups were observed in both hr and sao2 ( both p>0.05 ) ( figure 2 ) . after arrival at 3,700 m , the mean hr for the whole group increased to 8412 beats / minute . conversely , the mean sao2 decreased to 88.8%2.9% . this tendency was observed in all four age groups . furthermore , significant differences between these groups were observed in hr ( figure 3 ) , and there was a tendency for sao2 to decrease with increasing age . aiss increased with increasing age at both 500 m and 3,700 m , and the aiss obtained at 3,700 m was significantly higher than that obtained at 500 m ( each p<0.001 ) . the same tendency was observed in insomnia prevalence . upon rapid ascent from 500 m to 3,700 m , the increase in insomnia prevalence in the group aged 3135 years ( 2.79 times ) was a little more than in the other three groups ( 2.74 , 2.58 , and 2.69 times in groups aged 20 , 2125 , and 2630 years , respectively ) . consequently , the prevalence of insomnia was significantly higher in the group aged 3135 years than in the other three groups ( each p<0.001 , table 2 ) . insomnia prevalence in the whole group at 500 m and 3,700 m was 18.57% and 49.88% , respectively . on simple univariate analysis , age , hr , and aiss were positively correlated with lls , whereas sao2 was negatively correlated with lls ( table 3 ) . no significant correlations were found between lls and other variables ( bmi , race , etc ) . additionally , age was significantly positively correlated with bmi , hr , and aiss , but significantly negatively correlated with sao2 ( data not shown ) . on univariate logistic regression analysis , ams was associated with age ( or 1.035 , 95% ci 1.0031.068 , p<0.05 ) , smoking ( or 0.633 , 95% ci 0.4620.868 , p<0.01 ) , sao2 ( or 0.933 , 95% ci 0.8900.978 , p<0.01 ) , and aiss ( or 1.411 , 95% ci 1.3331.495 , p<0.001 ) , but not with bmi and other variables ( race , education , drinking , etc ) . a multivariate analysis that considered previous age , smoking , sao2 , and aiss showed that all variables except smoking and sao2 were significantly associated with ams ( table 4 ) . we enrolled 987 participants at 500 m , 79 of whom were excluded for incomplete data or preceding exposure to high altitude . in addition , 22 tibetan subjects were removed from the study because of the small sample size . after arrival at 3,700 m , one subject withdrew from the study because of high altitude pulmonary edema , and a further 29 subjects withdrew due to severe headache , diarrhea , or other unknown reasons . of these , 195 subjects were aged 1820 years , 314 were aged 2125 years , 238 were aged 2630 years , and 109 were aged 3135 years . the mean age in the groups aged 20 , 2125 , 2630 , and 3135 years was 19 , 22 , 27 , and 33 years , respectively . increase in bmi was consistent with increase in age ( 21.12.4 , 22.02.3 , 22.32.5 , and 23.62.8 kg / m in the four age groups , respectively ) . except for age ( p<0.001 ) and bmi ( p<0.001 ) , no statistically significant differences in smoking and drinking status , race , and education were observed between the four age groups . at an altitude of 3,700 m , the lls in the age groups 20 , 2125 , 2630 , and 3135 years was 3.42.1 , 3.32.1 , 3.92.3 , and 4.62.1 , respectively . differences in lls between the four groups ( p<0.05 or p<0.01 ) were significant , except for between the age groups 20 years and 2125 years ( p=0.68 ) . accordingly , the increased prevalence of ams was consistent with the increase in lls ( 54.36% , 59.55% , 68.07% , and 73.39% in the four groups , respectively ) . the prevalence of ams in the 20 and 2125 years age groups was significantly lower than in the 2630 and 3135 years age groups ( p<0.05 or p<0.01 ) . notably , the prevalence of severe ams increased with increasing age ( table 1 ) . in addition , the overall prevalence of ams at the 3,700 m altitude was 62.38% . in terms of ams symptoms , the most common were headache ( 74.88% ) , followed by weakness or fatigue ( 73.36% ) , dizziness or lightheadedness ( 73.23% ) , difficulty sleeping ( 66.59% ) , and the prevalence of gastrointestinal symptoms is relatively lower than the prevalence of other ams symptoms ( 22.43% ) . moreover , the prevalence of ams symptoms clearly increased with age ( figure 1 ) , and the difference in the prevalence of headache , insomnia , and weakness or fatigue reached statistical significance ( p<0.001 , p<0.001 , and p<0.01 , respectively ) . at 500 m , the hr and sao2 for the whole group were 689 beats / minute and 98.1%0.9% , respectively . no significant differences between the four age groups were observed in both hr and sao2 ( both p>0.05 ) ( figure 2 ) . after arrival at 3,700 m , the mean hr for the whole group increased to 8412 beats / minute . furthermore , significant differences between these groups were observed in hr ( figure 3 ) , and there was a tendency for sao2 to decrease with increasing age . aiss increased with increasing age at both 500 m and 3,700 m , and the aiss obtained at 3,700 m was significantly higher than that obtained at 500 m ( each p<0.001 ) . the same tendency was observed in insomnia prevalence . upon rapid ascent from 500 m to 3,700 m , the increase in insomnia prevalence in the group aged 3135 years ( 2.79 times ) was a little more than in the other three groups ( 2.74 , 2.58 , and 2.69 times in groups aged 20 , 2125 , and 2630 years , respectively ) . consequently , the prevalence of insomnia was significantly higher in the group aged 3135 years than in the other three groups ( each p<0.001 , table 2 ) . insomnia prevalence in the whole group at 500 m and 3,700 m was 18.57% and 49.88% , respectively . on simple univariate analysis , age , hr , and aiss were positively correlated with lls , whereas sao2 was negatively correlated with lls ( table 3 ) . no significant correlations were found between lls and other variables ( bmi , race , etc ) . additionally , age was significantly positively correlated with bmi , hr , and aiss , but significantly negatively correlated with sao2 ( data not shown ) . on univariate logistic regression analysis , ams was associated with age ( or 1.035 , 95% ci 1.0031.068 , p<0.05 ) , smoking ( or 0.633 , 95% ci 0.4620.868 , p<0.01 ) , sao2 ( or 0.933 , 95% ci 0.8900.978 , p<0.01 ) , and aiss ( or 1.411 , 95% ci 1.3331.495 , p<0.001 ) , but not with bmi and other variables ( race , education , drinking , etc ) . a multivariate analysis that considered previous age , smoking , sao2 , and aiss showed that all variables except smoking and sao2 were significantly associated with ams ( table 4 ) . to our knowledge , this is the first large - scale epidemiological investigation of the relationship between age and ams upon rapid ascent to high altitude . the main finding of the present study is the significantly higher prevalence of ams in the older , young adults than in the younger , young adults after rapid ascent from 500 m to 3,700 m. furthermore , older age , one of the risk factors for ams , was identified by univariate and multivariable analyses in the present study . additionally , pre - existing subjective poor sleep quality was an important contributor to the increase of the prevalence of ams in the older , young adults . prevalence of ams was higher in the present study than in two other similar altitude studies,7,8 but lower than in the studies by ren et al14 and murdoch.6 differences between our study and others in terms of duration of hypoxic exposure , ascent rate , and age of subjects may account for this result . the prevalence of ams in the study by li et al8 was 40% ; perceptibly lower than our result ( 54.36% in the group aged 20 years ) . the first clear difference between our study and that of li et al8 is the duration of hypoxic exposure , which was 24 hours in our study and 48 hours in that of li et al.8 it is known that ams is present within 612 hours after exposure to altitudes above 2,500 m , and that ams symptoms gradually resolve within 48 hours if 1 day of rest or other interventions are undertaken.4 thus , the prevalence of ams would be substantially affected by the duration of hypoxic exposure . in our study , the mean age in the groups aged 20 , 2125 , 2630 , and 3135 years was 19 , 22 , 27 , and 33 years , respectively ; whereas that of li et al8 was 18 years . notably , with the same ascent rate and altitude , as well as a big sample size , there is good comparability and strength between our study and that of li et al.8 in the study by moraga et al7 the prevalence of ams was 56% , which was a little lower than in the group aged 2125 years ( 59.55% ) in our study . the discrepancy may be partially because of the slower ascent rate ( approximately 435 m / hour ) in moraga et al.7 moreover , in other research with a large sample size , murdoch6 reported an ams prevalence of 84% at 3,860 m , wherein subjects flew directly to 3,740 m and slept at 3,860 m. age of the subjects in murdoch s study6 was clearly higher ( mean age 26 versus 45 years ) than in our study , which may at least partly explain the difference in prevalence of ams between the two studies . at the same time , we also noted one other similar altitude and ascent rate study ( ren et al).14 with an average subject age of 18 years , the ams prevalence in the study by ren et al14 was 57.2% , which was higher than that for the group aged 20 years ( 54.36% ) in the present study . the discrepancy may be mainly due to the different scoring system for the assessment of ams used in our study and that of ren et al.14 through comparison of the studies by li et al8 and ren et al14 we can clearly see that the prevalence of ams diagnosed by the chinese criteria is higher than the lake louise criteria . in the present study , we found that higher age was a risk factor for ams ( adjusted or 1.07 , 95% ci 1.011.13 , p=0.03 ) , adjusted for smoking , sao2 , and aiss . however , this is in contrast to a previous study.5 after careful assessment , we think the difference in ascent profile between our study and that of wu et al5 may mainly account for the result . on the other hand , after arrival at high altitude , the subjects in the present study mainly rested in their rooms . the subjects in the study by wu et al5 did not rest , and the authors thought younger people were at higher risk of ams than older people because of vigorous physical activity after arrival at high altitude . notably , another two recent studies15,16 have suggested that older subjects may experience higher ams prevalence upon rapid ascent to high altitudes . a significant increase in hr was observed in the whole group after arrival at altitude , and , furthermore , a more marked increase was observed in older subjects . this is supported by the literature.17,18 conversely , sao2 decreased in each group , and a slightly larger decrease was observed in older subjects . at the same time , correlational analysis showed that there was a weak correlation between age and hr ( r 0.064 , p=0.048 ) , as well as sao2 ( r 0.072 , p=0.029 ) . therefore , the hr increased more markedly in the older subjects in response to the more severe hypoxia . poor subjective sleep in the general population is very common in modern societies , and can be caused by diversiform factors ( eg , aging , physical and mental illness , etc).19 in terms of different definitions , the prevalence of insomnia ranges from 5% to 50%,20 and the symptoms of insomnia increase with age in men.21 however , whether the sleep status of the subjects prior to the ascent would affect the prevalence of ams is unclear , and furthermore , the sleep status of the subjects can not be completely presented by the lake louise diagnostic criteria for ams . therefore , the ais was used in our study to assess the sleep status of the subjects at both 500 m and 3,700 m. consistent with previous studies,2123 we found that aiss and prevalence of insomnia increased with age among young adult chinese men at 500 m ( table 3 ) , and the prevalence of insomnia was within the reported range ( 5%50% ) . after high altitude exposure , the prevalence of insomnia markedly increased in each age group , which is consistent with a previous study conducted with the ais.24 furthermore , increase in insomnia in the older age group was more marked ( 2.79 versus 2.74 , 2.58 , and 2.69 times ) ( table 3 ) . high altitude hypoxia is one important reason for sleep impairment;25,26 however , existing poor subjective sleep status before the rapid ascent may be another important contributor to the increase in insomnia in the present study . moreover , the aiss was positively correlated with age and lls , and was a risk factor for ams ( adjusted or 1.39 , 95% ci 1.281.51 , p<0.001 ) . in combination , our data showed that poor subjective sleep quality was one important reason why the older , young adult chinese men were at high risk of ams upon rapid ascent to high altitude . in the present study , we also observed that smoking and high sao2 were significant protective factors against ams by univariate analysis ; however , the significance disappeared when adjusted for other significant predictors in the multivariate analysis . these results may reveal that there remain correlations between ams and smoking status and sao2 ; however , further work is needed to explore whether smoking status and sao2 level are sensitive predictors for ams . the strengths of the present study are the large sample sizes , the identical ascent profile , and controlled physical activity . first , the participants were all male , which could probably introduce a sex bias . second , no participants were older than 35 years , which , therefore , makes it hard to extend our results to other age groups . therefore , further research involving females and older subjects should be conducted to confirm these findings . the present study suggests that older age is an independent risk factor for ams upon acute exposure to high altitude among young adult chinese men , and sustained poor subjective sleep prior to ascent may be an important contributor to an increased prevalence of ams in the older subjects . with the development of transportation in our modern society , it is realistic to expect that more and more people could rapidly ascend to high altitudes . a better understanding of the relationship between age and ams could lead to highly effective prevention of ams . thus , the present study may have implications for the prevention of ams among older populations upon rapid ascent to high altitudes . further research involving females and subjects older than 36 years should be conducted to confirm the present results .

backgroundthe aim of this study was to explore the relationship between age and acute mountain sickness ( ams ) when subjects are exposed suddenly to high altitude.methodsa total of 856 young adult men were recruited . before and after acute altitude exposure , the athens insomnia scale score ( aiss ) was used to evaluate the subjective sleep quality of subjects . ams was assessed using the lake louise scoring system . heart rate ( hr ) and arterial oxygen saturation ( sao2 ) were measured.resultsresults showed that , at 500 m , aiss and insomnia prevalence were higher in older individuals . after acute exposure to altitude , the hr , aiss , and insomnia prevalence increased sharply , and the increase in older individuals was more marked . the opposite trend was observed for sao2 . at 3,700 m , the prevalence of ams increased with age , as did severe ams , and ams symptoms ( except gastrointestinal symptoms ) . multivariate logistic regression analysis showed that age was a risk factor for ams ( adjusted odds ratio [ or ] 1.07 , 95% confidence interval [ ci ] 1.011.13 , p<0.05 ) , as well as aiss ( adjusted or 1.39 , 95% ci 1.281.51 , p<0.001).conclusionthe present study is the first to demonstrate that older age is an independent risk factor for ams upon rapid ascent to high altitude among young adult chinese men , and pre - existing poor subjective sleep quality may be a contributor to increased ams prevalence in older subjects .

Introduction Methods Subjects and design Acute mountain sickness assessment Physiological parameter measurements Sleep assessment Statistical analyses Results Subjects Acute mountain sickness Physiological parameters Athens Insomnia Scale score Correlational analysis Logistic regression analysis Discussion Conclusion

however , acute mountain sickness ( ams ) is a major obstacle faced by many people after arrival at high altitudes . ams is a common syndrome encountered particularly by lowlanders after rapidly ascending to altitudes above 2,500 m.1 this syndrome is characterized by headache , dizziness or lightheadedness , gastrointestinal symptoms ( anorexia , nausea , or vomiting ) , weakness or fatigue , and insomnia.2 among these symptoms , headache is the most frequent and essential for final ams diagnosis . research on risk factors for ams ( eg , female sex and younger age)4 is beneficial for the prevention of ams , although further investigations are needed to establish these risk factors.5 with the development of transportation , rapid ascension to high altitudes is becoming common and easy . however , as yet , there has been no large - scale epidemiological investigation into the relationship between age and ams upon rapid ascent to high altitudes . thus , the relationship between age and ams upon acute exposure to high altitudes remains unclear . given that past literature68 indicated a tendency for older adults to be at greater risk of ams than younger adults , we hypothesized that older age would increase the prevalence of ams upon rapid ascent to high altitude . therefore , this study uses a cross - sectional design aiming to explore the relationship between age and ams in subjects suddenly exposed to high altitude . one week before ascending to 3,700 m , all subjects were interviewed and underwent a routine physical examination ( echocardiogram , electrocardiogram , transcranial doppler , chest x - ray , heart rate [ hr ] , blood pressure [ bp ] , and arterial oxygen saturation [ sao2 ] ) . in addition , sleep status of the subjects was investigated using the athens insomnia scale score ( aiss).9 subjects demographic characteristics ( age , race [ chinese han , miao , man , tibetan , etc ] , education , drinking10 and smoking11 habits , etc ) were recorded in the questionnaire . on the morning of the second day at 3,700 m , all subjects completed the athens insomnia scale ( ais)9 and lake louise score ( lls)2 questionnaires . in the present study , the lls questionnaire comprised five items : headache , dizziness or lightheadedness , gastrointestinal symptoms ( anorexia , nausea , vomiting ) , fatigue and/or weakness , and insomnia . after completion of the lls questionnaire , the hr , sao2 , and bp values of the subjects were measured after a 15-minute rest in a comfortable chair . sleep status of the subjects in the past month was assessed using the ais.9 the ais includes eight items : the first five items assess sleep procedure ( sleep induction , night awakening , awakening in the early morning , total sleep duration , and total quality of sleep ) , and the last three items estimate decreased sense and functioning , and sleepiness in the next day . relationship between lls and age , bmi , and other variables , as well as between age and bmi , bp , and other variables , was determined using the spearman correlation test . before the multivariate analysis , an odds ratio ( or ) with 95% confidence intervals ( cis ) for ams associated with risk factors on univariate analysis , factors with a p - value 0.10 were entered into multivariate logistic regression analysis . one week before ascending to 3,700 m , all subjects were interviewed and underwent a routine physical examination ( echocardiogram , electrocardiogram , transcranial doppler , chest x - ray , heart rate [ hr ] , blood pressure [ bp ] , and arterial oxygen saturation [ sao2 ] ) . in addition , sleep status of the subjects was investigated using the athens insomnia scale score ( aiss).9 subjects demographic characteristics ( age , race [ chinese han , miao , man , tibetan , etc ] , education , drinking10 and smoking11 habits , etc ) were recorded in the questionnaire . on the morning of the second day at 3,700 m , all subjects completed the athens insomnia scale ( ais)9 and lake louise score ( lls)2 questionnaires . in the present study , the lls system was used for the diagnosis of ams in subjects . the lls questionnaire comprised five items : headache , dizziness or lightheadedness , gastrointestinal symptoms ( anorexia , nausea , vomiting ) , fatigue and/or weakness , and insomnia . after completion of the lls questionnaire , the hr , sao2 , and bp values of the subjects were measured after a 15-minute rest in a comfortable chair . sleep status of the subjects in the past month was assessed using the ais.9 the ais includes eight items : the first five items assess sleep procedure ( sleep induction , night awakening , awakening in the early morning , total sleep duration , and total quality of sleep ) , and the last three items estimate decreased sense and functioning , and sleepiness in the next day . subjects with an ais score 6 were considered to be suffering from insomnia.12 the ais is considered an excellent instrument for assessing subjective sleep quality in both clinical and study settings as it has good reliability and validity9 and is easy to complete.13 data are shown as mean standard deviation , median ( interquartile range ) , or n ( % ) , and were analyzed using spss version 13.0 ( ibm corporation , armonk , ny , usa ) . relationship between lls and age , bmi , and other variables , as well as between age and bmi , bp , and other variables , was determined using the spearman correlation test . before the multivariate analysis , an odds ratio ( or ) with 95% confidence intervals ( cis ) for ams associated with risk factors was determined using logistic regression . we enrolled 987 participants at 500 m , 79 of whom were excluded for incomplete data or preceding exposure to high altitude . after arrival at 3,700 m , one subject withdrew from the study because of high altitude pulmonary edema , and a further 29 subjects withdrew due to severe headache , diarrhea , or other unknown reasons . at an altitude of 3,700 m , the lls in the age groups 20 , 2125 , 2630 , and 3135 years was 3.42.1 , 3.32.1 , 3.92.3 , and 4.62.1 , respectively . accordingly , the increased prevalence of ams was consistent with the increase in lls ( 54.36% , 59.55% , 68.07% , and 73.39% in the four groups , respectively ) . the prevalence of ams in the 20 and 2125 years age groups was significantly lower than in the 2630 and 3135 years age groups ( p<0.05 or p<0.01 ) . notably , the prevalence of severe ams increased with increasing age ( table 1 ) . in terms of ams symptoms , the most common were headache ( 74.88% ) , followed by weakness or fatigue ( 73.36% ) , dizziness or lightheadedness ( 73.23% ) , difficulty sleeping ( 66.59% ) , and the prevalence of gastrointestinal symptoms is relatively lower than the prevalence of other ams symptoms ( 22.43% ) . moreover , the prevalence of ams symptoms clearly increased with age ( figure 1 ) , and the difference in the prevalence of headache , insomnia , and weakness or fatigue reached statistical significance ( p<0.001 , p<0.001 , and p<0.01 , respectively ) . at 500 m , the hr and sao2 for the whole group were 689 beats / minute and 98.1%0.9% , respectively . after arrival at 3,700 m , the mean hr for the whole group increased to 8412 beats / minute . furthermore , significant differences between these groups were observed in hr ( figure 3 ) , and there was a tendency for sao2 to decrease with increasing age . aiss increased with increasing age at both 500 m and 3,700 m , and the aiss obtained at 3,700 m was significantly higher than that obtained at 500 m ( each p<0.001 ) . upon rapid ascent from 500 m to 3,700 m , the increase in insomnia prevalence in the group aged 3135 years ( 2.79 times ) was a little more than in the other three groups ( 2.74 , 2.58 , and 2.69 times in groups aged 20 , 2125 , and 2630 years , respectively ) . insomnia prevalence in the whole group at 500 m and 3,700 m was 18.57% and 49.88% , respectively . on univariate logistic regression analysis , ams was associated with age ( or 1.035 , 95% ci 1.0031.068 , p<0.05 ) , smoking ( or 0.633 , 95% ci 0.4620.868 , p<0.01 ) , sao2 ( or 0.933 , 95% ci 0.8900.978 , p<0.01 ) , and aiss ( or 1.411 , 95% ci 1.3331.495 , p<0.001 ) , but not with bmi and other variables ( race , education , drinking , etc ) . we enrolled 987 participants at 500 m , 79 of whom were excluded for incomplete data or preceding exposure to high altitude . after arrival at 3,700 m , one subject withdrew from the study because of high altitude pulmonary edema , and a further 29 subjects withdrew due to severe headache , diarrhea , or other unknown reasons . accordingly , the increased prevalence of ams was consistent with the increase in lls ( 54.36% , 59.55% , 68.07% , and 73.39% in the four groups , respectively ) . the prevalence of ams in the 20 and 2125 years age groups was significantly lower than in the 2630 and 3135 years age groups ( p<0.05 or p<0.01 ) . notably , the prevalence of severe ams increased with increasing age ( table 1 ) . in addition , the overall prevalence of ams at the 3,700 m altitude was 62.38% . in terms of ams symptoms , the most common were headache ( 74.88% ) , followed by weakness or fatigue ( 73.36% ) , dizziness or lightheadedness ( 73.23% ) , difficulty sleeping ( 66.59% ) , and the prevalence of gastrointestinal symptoms is relatively lower than the prevalence of other ams symptoms ( 22.43% ) . moreover , the prevalence of ams symptoms clearly increased with age ( figure 1 ) , and the difference in the prevalence of headache , insomnia , and weakness or fatigue reached statistical significance ( p<0.001 , p<0.001 , and p<0.01 , respectively ) . at 500 m , the hr and sao2 for the whole group were 689 beats / minute and 98.1%0.9% , respectively . after arrival at 3,700 m , the mean hr for the whole group increased to 8412 beats / minute . furthermore , significant differences between these groups were observed in hr ( figure 3 ) , and there was a tendency for sao2 to decrease with increasing age . aiss increased with increasing age at both 500 m and 3,700 m , and the aiss obtained at 3,700 m was significantly higher than that obtained at 500 m ( each p<0.001 ) . upon rapid ascent from 500 m to 3,700 m , the increase in insomnia prevalence in the group aged 3135 years ( 2.79 times ) was a little more than in the other three groups ( 2.74 , 2.58 , and 2.69 times in groups aged 20 , 2125 , and 2630 years , respectively ) . consequently , the prevalence of insomnia was significantly higher in the group aged 3135 years than in the other three groups ( each p<0.001 , table 2 ) . insomnia prevalence in the whole group at 500 m and 3,700 m was 18.57% and 49.88% , respectively . additionally , age was significantly positively correlated with bmi , hr , and aiss , but significantly negatively correlated with sao2 ( data not shown ) . on univariate logistic regression analysis , ams was associated with age ( or 1.035 , 95% ci 1.0031.068 , p<0.05 ) , smoking ( or 0.633 , 95% ci 0.4620.868 , p<0.01 ) , sao2 ( or 0.933 , 95% ci 0.8900.978 , p<0.01 ) , and aiss ( or 1.411 , 95% ci 1.3331.495 , p<0.001 ) , but not with bmi and other variables ( race , education , drinking , etc ) . a multivariate analysis that considered previous age , smoking , sao2 , and aiss showed that all variables except smoking and sao2 were significantly associated with ams ( table 4 ) . to our knowledge , this is the first large - scale epidemiological investigation of the relationship between age and ams upon rapid ascent to high altitude . the main finding of the present study is the significantly higher prevalence of ams in the older , young adults than in the younger , young adults after rapid ascent from 500 m to 3,700 m. furthermore , older age , one of the risk factors for ams , was identified by univariate and multivariable analyses in the present study . additionally , pre - existing subjective poor sleep quality was an important contributor to the increase of the prevalence of ams in the older , young adults . prevalence of ams was higher in the present study than in two other similar altitude studies,7,8 but lower than in the studies by ren et al14 and murdoch.6 differences between our study and others in terms of duration of hypoxic exposure , ascent rate , and age of subjects may account for this result . the first clear difference between our study and that of li et al8 is the duration of hypoxic exposure , which was 24 hours in our study and 48 hours in that of li et al.8 it is known that ams is present within 612 hours after exposure to altitudes above 2,500 m , and that ams symptoms gradually resolve within 48 hours if 1 day of rest or other interventions are undertaken.4 thus , the prevalence of ams would be substantially affected by the duration of hypoxic exposure . notably , with the same ascent rate and altitude , as well as a big sample size , there is good comparability and strength between our study and that of li et al.8 in the study by moraga et al7 the prevalence of ams was 56% , which was a little lower than in the group aged 2125 years ( 59.55% ) in our study . the discrepancy may be partially because of the slower ascent rate ( approximately 435 m / hour ) in moraga et al.7 moreover , in other research with a large sample size , murdoch6 reported an ams prevalence of 84% at 3,860 m , wherein subjects flew directly to 3,740 m and slept at 3,860 m. age of the subjects in murdoch s study6 was clearly higher ( mean age 26 versus 45 years ) than in our study , which may at least partly explain the difference in prevalence of ams between the two studies . at the same time , we also noted one other similar altitude and ascent rate study ( ren et al).14 with an average subject age of 18 years , the ams prevalence in the study by ren et al14 was 57.2% , which was higher than that for the group aged 20 years ( 54.36% ) in the present study . the discrepancy may be mainly due to the different scoring system for the assessment of ams used in our study and that of ren et al.14 through comparison of the studies by li et al8 and ren et al14 we can clearly see that the prevalence of ams diagnosed by the chinese criteria is higher than the lake louise criteria . in the present study , we found that higher age was a risk factor for ams ( adjusted or 1.07 , 95% ci 1.011.13 , p=0.03 ) , adjusted for smoking , sao2 , and aiss . on the other hand , after arrival at high altitude , the subjects in the present study mainly rested in their rooms . the subjects in the study by wu et al5 did not rest , and the authors thought younger people were at higher risk of ams than older people because of vigorous physical activity after arrival at high altitude . notably , another two recent studies15,16 have suggested that older subjects may experience higher ams prevalence upon rapid ascent to high altitudes . a significant increase in hr was observed in the whole group after arrival at altitude , and , furthermore , a more marked increase was observed in older subjects . at the same time , correlational analysis showed that there was a weak correlation between age and hr ( r 0.064 , p=0.048 ) , as well as sao2 ( r 0.072 , p=0.029 ) . poor subjective sleep in the general population is very common in modern societies , and can be caused by diversiform factors ( eg , aging , physical and mental illness , etc).19 in terms of different definitions , the prevalence of insomnia ranges from 5% to 50%,20 and the symptoms of insomnia increase with age in men.21 however , whether the sleep status of the subjects prior to the ascent would affect the prevalence of ams is unclear , and furthermore , the sleep status of the subjects can not be completely presented by the lake louise diagnostic criteria for ams . therefore , the ais was used in our study to assess the sleep status of the subjects at both 500 m and 3,700 m. consistent with previous studies,2123 we found that aiss and prevalence of insomnia increased with age among young adult chinese men at 500 m ( table 3 ) , and the prevalence of insomnia was within the reported range ( 5%50% ) . after high altitude exposure , the prevalence of insomnia markedly increased in each age group , which is consistent with a previous study conducted with the ais.24 furthermore , increase in insomnia in the older age group was more marked ( 2.79 versus 2.74 , 2.58 , and 2.69 times ) ( table 3 ) . high altitude hypoxia is one important reason for sleep impairment;25,26 however , existing poor subjective sleep status before the rapid ascent may be another important contributor to the increase in insomnia in the present study . moreover , the aiss was positively correlated with age and lls , and was a risk factor for ams ( adjusted or 1.39 , 95% ci 1.281.51 , p<0.001 ) . in combination , our data showed that poor subjective sleep quality was one important reason why the older , young adult chinese men were at high risk of ams upon rapid ascent to high altitude . the present study suggests that older age is an independent risk factor for ams upon acute exposure to high altitude among young adult chinese men , and sustained poor subjective sleep prior to ascent may be an important contributor to an increased prevalence of ams in the older subjects . a better understanding of the relationship between age and ams could lead to highly effective prevention of ams . thus , the present study may have implications for the prevention of ams among older populations upon rapid ascent to high altitudes .

methyl ethyl ketone ( mek ) , methyl isobuthyl ketone ( mibk ) , cyclohexanone ( chone ) , 2,5 hexanedione ( 2,5 hd ) , and acetone ( ace ) are ketones monitored in urine to obtain an indication of occupational exposure or environmental contamination to mek , mibk , cyclohexane , hexane , methyl butyl ketone , and ace . these solvents are widely used in mixtures rather than as an unmixed single solvent , and various authors have indicated that metabolic interactions exist in acute or chronic coexposure . all these urinary biological exposure indices ( beis ) are widely considered by the american conference of governmental industrial hygienists ( acgih ) that indicated for them health - based values . they have been used as indicators of human exposure to these solvents even though it is not clear if these substances are the most suitable for this purpose , because of a large variability in the threshold concentrations of these five ketones excreted by nonoccupationally exposed individuals . so the specificity of unmetabolized ace as a marker for workplace ace exposure may be limited since this molecule may derive from ketone bodies that include also 3-hydroxybutyrate ( bohb ) and acetoacetate ( acac ) , present in the urine of all humans , but in elevated concentrations when associated with diabetic- , alcoholic- , and starvation - ketoacidoses . also the validity of the use of total 2,5 hd ( obtained from the acid hydrolysis of urine at ph 0.1 , which corresponds to the free 2,5 hd plus 4,5-dihydroxy-2-hexanedione and 5-hydroxy-2-hexanone converted into 2,5 hd during hydrolysis ) as opposed to free 2,5 hd , for biological monitoring , has been the basis of a considerable controversy . for many years , acgih recommended the determination of total 2,5 hd , with bei of 5.0 mg / l . in 2003 acgih began to provide additional information on the status of this chemical substance and bei of 0.4 mg / simultaneous and more sensitive assay methods than those available were needed and the necessity for standardization of analytical factors affecting these ketones determination has been indicated in a number of researches [ 57 ] . apart from biomonitoring applications , these ketones show different concentrations in urine and thus , either gas chromatography ( gc ) , gc - mass spectrometry ( ms ) , liquid chromatography ( lc)-tandem ms , selected ion flow tube - ms , or dipstick methods were developed for measuring single or multiple compounds for similar urinary concentration levels [ 814 ] . however , no single automated procedure is currently capable of quantization the following urinary ketones : mek , mibk , chone , free 2,5 hd , ace , bohb , and acac . furthermore , in these previous works , the use of solvents and/or clean - up steps were often necessary to extract and eliminate interfering compounds from the urine . this implies an increase of the required manual operations , bigger costs , uncertainty in the analysis determination , and the possible loss of the analyte . accordingly , we sought to simplify the situation by developing new ketones preparation methods where the ability of direct immersion ( di ) solid phase microextraction ( spme ) techniques by on - sample and on - fiber derivatization has been demonstrated also theoretically , together with the quantitative determination by fast gc / negative chemical ionization ( nci)-electron ionization ( ei)/ms in order to propose a new robotic friendly use ms apparatus able to analyze great quantities of samples . the fast gc techniques are based on the use of short capillary column , called narrow bore , that allows the achievement of high - speed separations while maintaining excellent resolution . in the last decade this technique has found routine application by the achievement of adequate specific conditions , as the increased inlet pressures , rapid oven heating rates , and fast electronics for detection and data collection . however , high split ratio is necessary when liquids are injected , because of low sample narrow - bore capillary column capacities that may have an affect on the efficiency . this problem can be easily overcome by the use of spme , a solvent - free technique that incorporates sampling , isolation , and enrichment into one step . within analytical chemistry , the spme analysis of biological samples is an important technology , it is considered one of major ideas that shaped 20th - century analytical chemistry , capable of producing powerful diagnostic and identifying important toxicology and disease biomarkers . as interest in this area grows , and the number of such applications continues to increase , the need for efficient , comprehensive , reproducible methods has developed within both experts and nonexperts . in this work we design an automated multiple assay which meets this need through the development of a new robotic system called multifiber exchange ( mfx ) installed on a xyz gc autosampler . as , this increased versatility of mfx allows on - sample and on - fiber spme derivatization approaches in fully automated mode , the authors are interested in both o-(2,3,4,5,6 , pentafluorobenzyl)-hydroxylamine ( pfbha)-oxime derivatives of carbonyl group , where selectivity and sensitivity of the analytical method may be greatly improved by use of nci , and towards methyl ester and tert - butyldimethylsilyl ( tbdms ) derivatives of carboxylic functional groups , where the reactions occur under mild conditions . the object of this work is obtained assays in automated very short time windows , with consequently more sensitivity power and high discrimination , by structurally informative ms fragmentation pattern , than other techniques for routine employed in toxicological and clinical chemistry laboratories . o-(2,3,4,5,6 , pentafluorobenzyl)-hydroxylamine ( 98% ) , 2,4-dinitrophenylhydrazine ( 98% ) , pentafluorophenylhydrazine ( 98% ) , trimethyloxonium tetrafluoroborate , n - tert .- butyldimethylsilyl - n - methyltrifluoroacetamide ( bstfa , > 98% ) , methyl ethyl ketone ( > 99% ) , methyl isobuthyl ketone , cyclohexanone ( > 99% ) , acetone ( > 99.9% ) , 2,5 hexanedione ( 98% ) , sodium dl-3-hydroxybutyrate , lithium acetoacetate , methanol ( > 99% ) , and internal standard , 2,4 pentanedione ( 99% ) were purchased from aldrich ( sigma - aldrich , milan , italy ) . ketone - pfbha - oximes ( > 99% ) , 3-hydroxybutyrate - methylester ( > 99% ) , acetoacetate - pfbha - oxime - methylester ( > 99% ) , and internal standard acetoacetate d5 ( > 99% ) were synthesized from protera ( sesto fiorentino , italy ) . internal standard sodium dl-3-hydroxybutyrate-2,2-d2 was obtained from c / d / n isotopes ( chemical research 2000 , rome , italy ) . urine control for ace and acac ( liquichek urinalysis control ) was purchased from bio - rad laboratories ( milan , italy ) . a multicompound stock solution was prepared in methanol , and by appropriate dilution working standard solutions were obtained . c5-c28 n - alkanes ( supelco , sigma - aldrich ) were used for evaluation of the linear temperature - programmed retention index ( ltpri ) . two spme sampling techniques , on - sample derivatization by pfbha for mek , mibk , chone , free 2,5 hd , ace , plus trimethyloxonium tetrafluoroborate ( tmo ) for acac and bohb , and on - fiber derivatization by n - tert .- butyldimethylsilyl - n - methyltrifluoroacetamide ( mtbstfa ) for bohb and acac were investigated . two ml of urine were transferred into a 10 ml autosampler vial with a magnetic stirring bar and mixed with 10 l 2,4 pentanedione solution 240 g / ml plus 100 l of 40 mg / ml pfbha aqueous solution . the condition used for full reaction was 20 hours at room temperature . to convert the acac - oxime and bohb into their methyl esters , derivatization with tmo was performed at room temperature in two steps . while stirring about 20 mg of na2co3 were added and within 4 minutes approximately 30 mg of solid tmo were added in two aliquots . finally , for head space ( hs)-spme sodium chloride ( 0.5 g ) was added while for di - spme the vial was filled by 8 ml of water ketones - free and incubated for 2 minutes at 100c . the appropriate spme fiber ( see results and discussion ) was directly immersed for 5 minutes at 50c , previous 30 minutes incubation time for hs - spme , under continuous agitation at 500 rpm and desorbing into gc injector port for 1 minute . spme extraction was performed by immersing the 85 m polyacrilate ( pa ) spme fiber into 2 ml autosampler vial filled with diluted urine ( 1 : 5 v / v ) in temperature - controlled agitation ( 50c and 500 rpm ) for 15 minutes , after addition of 10 l internal standards solution ( bohb d2 and acac d5 25 mg / ml methanol ) . on - fiber silylation of bohb and acac were performed by exposing the fiber to the hs in a 2 ml autosampler vial containing 50 l of mtbstfa for 15 minutes at 50c . since the determination of the seven ketones requires different fibers , the object of this paper is focused on the automation of the spme extraction procedure and on the performance of the fast gc / ms apparatus . the extraction efficiency for 85 m pa , and 7 , 30 , and 100 m polydimethylsiloxane ( pdms ) fibers was investigated for temperature , agitation , and mass loaded at equilibrium , by spme mfx . new fully automation of the procedure was achieved using a gerstel mps2 autosampler equipped with 3-position trays mfx ( sra instruments , milan , italy ) and fast fit assemblies ( ffas)-spme fibers ( supelco , sigma - aldrich , milan , italy ) patented by chromline ( prato , italy ) . it is formed by adaptors that make the fiber much more robust and identifiable by its barcode . thanks to ffa it is possible to change spme fibers in automatic mode by mfx ( figure 1 ) . in this option the fibers are transported between the 3-position tray , the vials , containing urine or derivatization agents , and the injector by new spme holder equipped with a plunger / magnetic system . at the end of the analysis desorbed fiber is moved back to the tray and the cycle is repeated . moreover , the autosampler parameters allowed to complete the analysis cycle by conditioning and sealing in the teflon septum inserted into the tray . the automatically change of spme fibers and processing using xyz autosampler is indicated as flowchart in figure 2 . the shimadzu gc 2010 ( shimadzu italia , milan , italy ) was equipped with slb5-ms costumizer column ( 5 m 0.10 mm internal diameter 0.4 m film thickness , supelco , sigma - aldrich , milan , italy ) ; oven setting was 65c hold for 0.3 minutes , with a ramp of 80c / minute up to 100c and 50c / minute up to 275c ( total program time 5.24 minutes ) . inlet pressure , column flow , and average linear velocity were , respectively , 313.7 kpa , 1.00 ml / minute , and 89.6 cm / s . the ms detector was a shimadzu qp 2010 series with the system acquisition gc solution software 2.5 su3 version operating in ei for tbdms - boho / acac derivatives , methane nci for pfbha - oxime-/methylester - pfbha - oximes , and methane positive chemical ionization ( pci ) mode for molecular weights identification . spectra were collected at 0.03 sec event time and detector voltage was set to 8001300 v. the ms detector was autotimed weekly . ms analysis was operated by the new fast automated scan / selective ion monitoring ( sim ) technique ( fasst ) acquisition mode with scan a range of m / z 40550 . for ltpri the pdms column was used with the same condition and indicated for fast gc / ei mode . effective use of the theory minimizes the number of experiments that need to be performed and to simplify mathematical relationship the theory assumes idealized condition . many of these data are now readily available on line and in this way it is possible to know in advance whether or not the di - spme method offers some advantages . national library of medicine , chemldplus , http://toxnet.nlm.nih.gov/ ( accessed november 18 , 2009 ) ; u.s . environmental protection agency , epa online tools for site assessment calculation , http://www.epa.gov/ ( accessed november 18 , 2009 ) ; dortmund data bank ( ddb ) , http://www.ddbst.de/new/default.htm ( accessed november 18 , 2009 ) ; universal functional activity coefficient ( unifac ) , http://www.unifac.org/ ( accessed november 18 , 2009 ) , physical properties database ( physprop ) by syracuse research corporation , http://www.srcinc.com/ ( accessed november 18 , 2009 ) , as well as publications and texts were looked up in [ 1722 ] . calibration samples , prepared and analyzed as described above in section 2 , to obtain a calibration curve . a multi - compound stock solution was prepared in methanol , and by appropriate dilution working standard solution was obtained . six calibrations sample and blank , were obtained ; five analysis for each concentration were performed . least squares linear digression analysis was used to estimate slopes ( m ) and intercepts ( b ) of calibration curves y = mx + b , where y is the ratio between the chromatographic area of the analyte and the relative internal standard , and x the urinary concentration of the analyte ( mg the limit of detection ( lod ) of the assay was calculated according to the expression ( 1)lod=3seb+bm , where seb is the internal standard error of the intercept . the precision of the assay ( as a coefficient of variation , c.v.% ) was estimated both a within session and as intersession repeatability by analysis of urine spiked with ketones . accuracy was evaluated by recoveries ( calculated from the ratio percent between the measured and the nominal concentrations in spiked blank urines ) at all concentrations used for the calibration plot . values of the accuracy were compared with the requirement of us food and drug administration for the bioanalytical method validation . sampling of urine by di - spme sampling and following fast gc / ms analysis has aroused interest in the authors of this paper and has been investigated as a possible alternative to conventional methods . the aim of this paper is to provide a simple , fast , sensitive , and organic - solvent free innovative procedure for analysis of ketones in urine . so , to achieve successful method , three fundamental requisites were satisfied by the authors . the first objective was to develop the derivatization conditions onto di - spme technologies to obtain compounds which are stable under a variety of conditions and easily amenable to sampling , to gc separation , and to ms identification . several papers indicate the on - fiber hs spme to determinate carbonyl functional groups . by this paper , the authors point out the reasons of the rejection of this derivatization technique for the ketones : ( i ) long periods of equilibrium time for hs ; ( ii ) the range of kh of the involved ketones is too wide , ace and 2,5 hd 3.88 10 and 4.43 10 atm m / mol at 25c , respectively ; and ( iii ) the excess of pfbha is an interference on chromatographic separation system . in - sample derivatization of ketone to hydrazones and oximes is frequently used ; the procedure involves derivatization of the analyte with 2,4-dinitrophenylhydrazine , 2,4,6-trichlorophenylhydrazine , pentafluorophenylhydrazine , benzyloxime , or pentafluoro benzyloxime . for example , the carbonyl of the ketones is hindered , and , thus , the compounds react very slowly with 2,4-dinitrophenylhydrazine . the method potentially allows specific quantitation of ketones by lc but not through gc , since for many hydrazones the maximum product yield was attained at ph 1.01.5 . pfbha reacts in weakly acidic media ( ph 46 ) with carbonyl functional groups to produce the corresponding oxime . in fact , the reactivity of chloroketone with pfbha is triggered by the chloro - substitution in the principal product . herein to verify the drawbacks with indicated above methods and in light of previous works [ 29 , 30 ] , the authors investigated the derivatization with pfbha . the reaction was evaluated concerning the effect of the reaction time and product yield attained by the determination of the amount of ketones converting to corresponding oximes . briefly , 5 ml of water containing 2 g of mek , mibk , chone , 2,5 hd , and ace were doped with 1 ml of pfbha solution ( 40 mg / ml ) . the samples were left to three experimental derivatization reaction settings ( 20 hours at 25c , 1 hour at 25c , and 30 minutes to 65c ) for establishing the conditions that minimize the time needed for preparation . after incubations , the oximes were extracted twice with 1 ml of hexane and 0.3 l was analyzed . the gc area counts were compared with the amount of ace- , mek- , mibk- , chone- , and 2,5 hd - pfbha - oxime standards previously injected by fast gc / nci / ms . for 20 hours at 25c incubation ketones were converted in their corresponding derivatives with values of 99% and no decomposition of pfbha - oximes was observed ( figure 3 ) . however , the reaction yield after 30 minutes to 65c is enough for the formation of products also ensuring fastness in the application of the method . if higher sensitivity is aimed , the reaction time and exposure fiber can be prolonged based on the profile of the reaction and absorption progress ( figure 4 ) . no decomposition of the excess of derivatization agent by adding 5 l of 9 m sulfuric acid was necessary if overnight incubation was employed . eventually , the selective reduction of this bond with pyridine : borane reagent leads to single gc peak for each compound . for following sampling by di , the effect of agitation and the absorption - time profiles on the spme extraction efficiency of the derivatives of these carbonyl compounds were examined . the agitation of the solution can strongly improve the spme extraction process and the equilibrium times increased with increasing molecular mass of the analytes ( table 1 ) . since the extraction with spme is based on an equilibrium between the analyte concentrations in the liquid and fiber coating , it is not necessary to reach an absorption equilibrium for quantitative analysis if the absorption time and mixing conditions are held constant throughout the experiment . thus , a 5-min extraction time was employed because this yielded sufficient extraction and excellent precision data ( table 2 ) . except for ace - pfbha - oxime , with a single chromatographic peak , for the other ketone - pfbha - oximes the formation of a double peak , corresponding to syn- and anti - isomer , was observed ( figure 5 ) and the total area of the two peaks was used for their quantitative determination . the pfbha by on - sample derivatization was so chosen for this work , because it is commercially avaiable and , under these conditions , the method allows for the selective detection of ketones at a very low levels without interferences . several reagents can be found in the literature for the derivatisation of carboxylic compounds for allowing different derivatisation approaches : 1-pyrenyldiazomethane , 1-(pentafluorphenyl ) diazomethane , diazomethane , 2,3,4,5,6,-pentafluorobenzyl bromide , bstfa , or isobutyl chloroformate [ 32 , 33 ] . the addition of modifiers , such as ion - pairing agents which activate the analytes during derivatization in the injection port , increases esterification yields . for carboxylic functional groups of acac and boho the authors confirm the same critical factors more than those observed by the employed of the derivatizations above indicated , ( i ) very expensive reagents ; ( ii ) presence of moisture led to production of artefacts ; ( iii ) effect of the injector temperature on the efficiency of derivatization ; ( iv ) capability to derivatize only the aliphatic molecule with low molecular weight ; and ( v ) damaging of the coating of the fiber after only few derivatizations . in this work , a new and different approach to this problems is implemented by the use of mtbstfa and of tmo , as a silylating and methylating agents for boho and acac urinary determination . mtbstfa as a tbdms derivatizing agents was used in gc analysis of amino acids and in gc - ms analysis of fatty acids , ketone bodies , and hydroxylated fluorenes , and it was shown that tbdms derivatives were thermally stable and had favourable fragmentations upon ei ionization [ 35 , 36 ] . these spectra were simple and gave intense fragment ion peaks ( boho- , m / z 275 and acac- tbdms , and m / z 273 ) , corresponding to the [ m-57 ] ion due to the loss of a tert - butyl fragment from the molecular ion . it was found that the intensity due the base peaks of boho-/acac - tbdms was about five times higher than that of boho-/acac - trimethylsilyl ( tms ) even if the tbdms derivatives had later eluting times than the corresponding tms derivatives . for spme on - fiber derivatization , the pa fiber was selected as the most appropriate since the coating of pdms fiber was stripped off after only 35 derivatizations by the mtbstfa . so , the effects of time , temperature and volume of urine , and derivatizating reagent for automated and high - throughput quantitative analysis were evaluated . an excellent spme extraction sensitivity for the urinary boho and acac was generally achieved by immerging the fiber in the diluted urine ( 1 : 5 v / v ) with temperature - controlled agitation ( 50c and 500 rpm ) . dilution of the urine with distilled water reduces the sensitivity of the method but increases the precision and the fiber lifetime . the better results were obtained with di times up to 2 hours . moreover , since the reduction in vial diameter by a factor of 3 resulted in an order of magnitude decrease in extraction time , where t , the average time of the diffusion through the aqueous layer is expected to be proportional to the square of the migration distance , x , and inversely proportional to the diffusion coefficient in dwater , ( 2)t = x22d for high - throughput analysis 2 ml vial was used instead of 10 ml vial . for on - fiber derivatization low and high values for three variables ( 20100 l mtbstfa , 2550c , and 1060 minutes ) the volume of mtbstfa and the derivatization time did not show significant effects and so they were fixed to 20 l and 15 minutes , respectively . in order to avoid contamination problems between consecutive samples , on - fiber derivatization was performed in different 2 ml vials and the fibers were additionally desorbed after the analysis during 1 minute at 300c . in spite of these slow kinetics rates , the extraction and derivatization time were limited to 15 minutes to speed up sample preparation ( figure 6 ) . trialkyloxonium ions ( meerwein salts ) , r3o , with various counterions such as sbf6 , bf4 , sbcl6 , and pf6 are excellent alkylating agents for nucleophiles containing heteroatoms such as n , o , or s . with tmo a methyl group of the oxonium ion reacts with the anion of the carboxylic functional groups to form the methyl ester : the fast gc / ms was used for verifing conversion of the boho and acac into the corresponding methylester and methylester - pfbha - oxime derivative . briefly , the urine was treated in the same manner as indicated in experimental data and extracted with 3 ml of hexane . after the extraction , the solvent was evaporated to dryness and the residue was dissolved in 0.2 ml of hexane for gc analysis . conversion of acac and boho was carried out using calibration graph from known amounts of boho - methylester and acac - methylester - pfbha - oxime and data gave an indication of the yield of the derivatisation reaction more than 95% . the second objective to the verify the ability of di - spme technique to determine the seven urinary ketones . the absortive liquid coatings pdms and pa were choice for sampling of a very complex matrix as to urine , because there is no competition between analytes and so no labeled compounds were used as internal standards . for pdms , where 100 , 30 , and 7 m thickness fiber coatings were commercially available , better result was obtained with 30 m pdms fiber , accorded with what is indicated from the theory where thin thickness has more efficient extraction for high molecular weight in fast equilibrium time . since the polar coating pa has better affinity toward methylester than nonpolar pdms - coated , pa fiber was employed for boho determination ( figure 4 ) . a decrease in the coating thickness by a factor 3 from 100 m to about 30 m results in a ca . 3-fold decrease in extraction time , as well as the extraction time profiles are dramatically affected by the stirring rate ( ca . the extraction time in the unagitated case is limited by the transport of analyte in the aqueous phase . a decrease in the diffusion coefficient of the analyte in water by an order of magnitude produced about an order of magnitude increase in equilibration time as discussed by louch et al . . moreover , the equilibrium and kinetics of the seven ketones versus spme fiber with liquid coating were investigated theoretically . table 1 illustrated the physicochemical constants of the ketones - oxime-/methyl ester - ketone - oxime - derivatives and pdms obtained by literature [ 1722 ] to anticipate trends in sampling extraction . for bohb and acac the derivatisations were also performed on - fiber after the preconcentration step by di - spme with pa liquid phase , and so kinetics of the underivatised bohb and acac versus spme coating were evaluated . because of the properties liquid coating , which is applied in di - spme analysis , the extraction obeys the rules of liquid - liquid equilibrium ( 3)n = c0v1v2kkv1+v2 where k is the partition constant spme fiber liquid polymeric coating / sample , c0 is the initial concentration of the analyte in the aqueous solution , v1 and v2 are the volumes of the coating and the aqueous solution , in the equilibrium concentration of the analyte in the aqueous matrix . thus , it is also evident that di - spme will mainly have a low or very low recovery with small k1 ( < 1.08510 ) , high henry 's constant ( > 34 atm cm / mol ) , and high hydrophobicity ( octanol - water partitioning , kow up to 81.2 ) values . as indicated in table 1 kow is a good estimated of k , however , the correlation has to be confirmed for the group of substances from a number of investigators . k values of the analytes are often very close to the gas phase partition coefficient / aqueous matrix partition coefficient ( k2 = kh / rt ) and to the spme coating / gas phase partition coefficient ( k1 ) ; k = k2 k1 , where it is more practical to say that both k1 and k2 values allowed to know in advance whether or not the spme method offers the advantages . the linear relationship with solute activity coefficient and ltpri , as in ( 4 ) and ( 5 ) , respectively , can be used further to estimate the k1 values ( 4)log k1=at+b , where a = h/2.303r and b = [ log ( rt/ pvap ) h/2.303r * ] ; h ( j / moli ) is the analyte heat of vaporization , r ( 8.314 j / moli k ) is the gas constant , t ( k ) is the sampling temperature , solute activity coefficient , pvap ( pa ) is the vapour pressure , and t * is the known temperature of coefficient , ( 5)log k1=0.0042ltpri0.188 . the ltpri = 100 ( tr(a ) tr(n)/tr(n+1 ) tr(n ) ) + 100 n ) was experimentally calculated by pdms capillary column where , tr(a ) is the analyte retention time , tr(n ) is the retention time of n - alkane eluting directly before tr(a ) , tr(n+1 ) is the retention time of the n - alkane eluting directly after tr(a ) , and n is the number of carbon atoms for tr(n ) . however , this is virtually impossible , at least for ( 5 ) , as no chromatographic columns with pa phases are commercially available to date . the significant correlation found in ( table 1 ) seems to indicate the evaluated k1 values of seven substances were essentially identical with experimental procedures and this alternative theoretical approach could allow to simplify the use of spme for qualification of compounds without the need for fiber calibration . accordingly , we developed a methodology where the properties of spme technique after on - sample and on - fiber derivatisation were coupled to the quantitative determination by fast gc / nci - ei / ms . so , the third objective was the effective chromatographic separation of the seven substances in the minimum time and the investigation of the structurally informative ms fragmentation pattern . in this investigation , the gc analysis of ketones - oxime-/methyl ester - ketone - oxime - derivatives is carried out in less than 3.50 minutes and 2 minutes between two different chromatographic runs , with a speed gain of almost 8 times in comparison with traditional gc procedure to maintain sufficient resolving power for separation of the compounds of interest . regards to pfbha - oxime - derivates the mass spectrum ( table 3 ) shows the advantages in using nci rather than the usual ei , that is , more detailed information about the molecular structure and not only a conspicuous increase in sensibility as due to a sensible lowering of the chemical background ( chemical noise ) . as a matter of fact , the ei rarely allows to detect the molecular peak of the pfbha - oxime - ketones , the only peak detected often being that of m / z 181 , related to the pfb portion . with ms detector which registers selected negative ions generated in the ion source by chemical ionization , it is also possible to detect not only the signals related to pbf ( m / z 197 , 196 , 181 , 178 ) , but also the [ m - hf ] signal . moreover , this analysis allows to distinguish a methyl - ketone from another kind of ketones , thanks to the presence of anion : this technology allows to analyze 96 samples in 24 hours , for a total 627 determinations / day . since boho and acac the on - fiber derivatization method was better for less analysis time than hs - spme of boho - methylester and di - spme for acac - methylester - pfbha - oxime , for fully automated analysis , priority samples when high ace concentrations can be added to the system at any point of the analysis sequence of boho / acac - tbdms . the quality of the fast gc / ms approach allowed for excellent resolution , even with very short analysis time , to resolve the analytes of interest from similar compounds that would interfere with the assay . in light of what indicated above the authors the resulting calibration curves were linear , in the investigated range for all the considerated ketones , with correlation coefficients > 0.998 . the precision of the assay ( reported as a coefficient of variation , c.v.% ) , estimated both as withinsession and as intersession repeatability resulted in the range 2.536.98 and 1.145.32% , respectively . accuracy was within 15% of the theoretical concentration , in line with the requirement of us food and drug administration for the bioanalytical methods validation . to demonstrate the applicability of the method to urinary samples , the content of these compounds in human urines ( n = 20 ) of no - exposed the urinary excretion of mek , mibk , chone and free 2,5 hd in control subjects was in the range 1.25.0 g / l , while ace , bohb , and acac were excreted at a high concentration ( 0.110.38 mg mg / l ) , acac ( 50872 mg / l ) , and ac ( 10431 mg / l ) in urine test stick positive ( score + ++++ ) were obtained . this paper reports the first contribution to the automated and high - throughput analysis of seven ketones in urine by spme / ms system . our data suggest that automated spme extraction coupled with fast gc / nci - ei / ms may be a viable alternative for ketones analyses in human urine . a noninvasive method has been developed for occupational health and diagnosis of ketoacidoses where it is critically important to rapidly differentiate between relative benign and life - threatening conditions ( i.e. , point of care testing ) . the accurate determination of the ketone body ratio is important in the diagnosis of diabetes mellitus and of order metabolic disorders . clinicians are encouraged to consider and utilize this noninvasive assay to guide early clinical diagnosis and management of metabolic acidosis and works are in progress along this line . so , the introduction of dedicated , automated , and robotic systems allowed a friendly use of ms apparatus for high - throughput screening so as to reduce the costs of the monitoring campaigns . the specific ms instrumentation suitable for this aim , such as the changeover from nci to ei modes and the automation of the preparation procedure with mfx , that allowed the change of the fiber by robotic device , allowed a friendly use of ms apparatus with a number of advantages including reduced analyst time both for routine analysis and method development , and greater reproducibility .

the present research is focused on automation , miniaturization , and system interaction with high throughput for multiple and specific direct immersion - solid phase microextraction / fast gas chromatography analysis of the urinary ketones . the specific mass spectrometry instrumentation , capable of supporting such the automated changeover from negative chemical to electron ionization mode , as well as the automation of the preparation procedure by new device called multifiber exchange , through change of the fibers , allowed a friendly use of mass spectrometry apparatus with a number of advantages including reduced analyst time and greater reproducibility ( 2.015.32% ) . the detection limits for the seven ketones were less than 0.004 mg / l . for an innovative powerful meaning in high - throughput routine , the generality of the structurally informative mass spectrometry fragmentation patterns together with the chromatographic separation and software automation are also investigated .

1. Introduction 2. Experimental 3. Results and Discussion 4. Conclusion

also the validity of the use of total 2,5 hd ( obtained from the acid hydrolysis of urine at ph 0.1 , which corresponds to the free 2,5 hd plus 4,5-dihydroxy-2-hexanedione and 5-hydroxy-2-hexanone converted into 2,5 hd during hydrolysis ) as opposed to free 2,5 hd , for biological monitoring , has been the basis of a considerable controversy . for many years , acgih recommended the determination of total 2,5 hd , with bei of 5.0 mg / l . in 2003 acgih began to provide additional information on the status of this chemical substance and bei of 0.4 mg / simultaneous and more sensitive assay methods than those available were needed and the necessity for standardization of analytical factors affecting these ketones determination has been indicated in a number of researches [ 57 ] . apart from biomonitoring applications , these ketones show different concentrations in urine and thus , either gas chromatography ( gc ) , gc - mass spectrometry ( ms ) , liquid chromatography ( lc)-tandem ms , selected ion flow tube - ms , or dipstick methods were developed for measuring single or multiple compounds for similar urinary concentration levels [ 814 ] . however , no single automated procedure is currently capable of quantization the following urinary ketones : mek , mibk , chone , free 2,5 hd , ace , bohb , and acac . furthermore , in these previous works , the use of solvents and/or clean - up steps were often necessary to extract and eliminate interfering compounds from the urine . this implies an increase of the required manual operations , bigger costs , uncertainty in the analysis determination , and the possible loss of the analyte . accordingly , we sought to simplify the situation by developing new ketones preparation methods where the ability of direct immersion ( di ) solid phase microextraction ( spme ) techniques by on - sample and on - fiber derivatization has been demonstrated also theoretically , together with the quantitative determination by fast gc / negative chemical ionization ( nci)-electron ionization ( ei)/ms in order to propose a new robotic friendly use ms apparatus able to analyze great quantities of samples . the fast gc techniques are based on the use of short capillary column , called narrow bore , that allows the achievement of high - speed separations while maintaining excellent resolution . in the last decade this technique has found routine application by the achievement of adequate specific conditions , as the increased inlet pressures , rapid oven heating rates , and fast electronics for detection and data collection . this problem can be easily overcome by the use of spme , a solvent - free technique that incorporates sampling , isolation , and enrichment into one step . within analytical chemistry , the spme analysis of biological samples is an important technology , it is considered one of major ideas that shaped 20th - century analytical chemistry , capable of producing powerful diagnostic and identifying important toxicology and disease biomarkers . as interest in this area grows , and the number of such applications continues to increase , the need for efficient , comprehensive , reproducible methods has developed within both experts and nonexperts . in this work we design an automated multiple assay which meets this need through the development of a new robotic system called multifiber exchange ( mfx ) installed on a xyz gc autosampler . as , this increased versatility of mfx allows on - sample and on - fiber spme derivatization approaches in fully automated mode , the authors are interested in both o-(2,3,4,5,6 , pentafluorobenzyl)-hydroxylamine ( pfbha)-oxime derivatives of carbonyl group , where selectivity and sensitivity of the analytical method may be greatly improved by use of nci , and towards methyl ester and tert - butyldimethylsilyl ( tbdms ) derivatives of carboxylic functional groups , where the reactions occur under mild conditions . two ml of urine were transferred into a 10 ml autosampler vial with a magnetic stirring bar and mixed with 10 l 2,4 pentanedione solution 240 g / ml plus 100 l of 40 mg / ml pfbha aqueous solution . since the determination of the seven ketones requires different fibers , the object of this paper is focused on the automation of the spme extraction procedure and on the performance of the fast gc / ms apparatus . new fully automation of the procedure was achieved using a gerstel mps2 autosampler equipped with 3-position trays mfx ( sra instruments , milan , italy ) and fast fit assemblies ( ffas)-spme fibers ( supelco , sigma - aldrich , milan , italy ) patented by chromline ( prato , italy ) . in this option the fibers are transported between the 3-position tray , the vials , containing urine or derivatization agents , and the injector by new spme holder equipped with a plunger / magnetic system . the shimadzu gc 2010 ( shimadzu italia , milan , italy ) was equipped with slb5-ms costumizer column ( 5 m 0.10 mm internal diameter 0.4 m film thickness , supelco , sigma - aldrich , milan , italy ) ; oven setting was 65c hold for 0.3 minutes , with a ramp of 80c / minute up to 100c and 50c / minute up to 275c ( total program time 5.24 minutes ) . the ms detector was a shimadzu qp 2010 series with the system acquisition gc solution software 2.5 su3 version operating in ei for tbdms - boho / acac derivatives , methane nci for pfbha - oxime-/methylester - pfbha - oximes , and methane positive chemical ionization ( pci ) mode for molecular weights identification . spectra were collected at 0.03 sec event time and detector voltage was set to 8001300 v. the ms detector was autotimed weekly . for ltpri the pdms column was used with the same condition and indicated for fast gc / ei mode . effective use of the theory minimizes the number of experiments that need to be performed and to simplify mathematical relationship the theory assumes idealized condition . environmental protection agency , epa online tools for site assessment calculation , http://www.epa.gov/ ( accessed november 18 , 2009 ) ; dortmund data bank ( ddb ) , http://www.ddbst.de/new/default.htm ( accessed november 18 , 2009 ) ; universal functional activity coefficient ( unifac ) , http://www.unifac.org/ ( accessed november 18 , 2009 ) , physical properties database ( physprop ) by syracuse research corporation , http://www.srcinc.com/ ( accessed november 18 , 2009 ) , as well as publications and texts were looked up in [ 1722 ] . a multi - compound stock solution was prepared in methanol , and by appropriate dilution working standard solution was obtained . least squares linear digression analysis was used to estimate slopes ( m ) and intercepts ( b ) of calibration curves y = mx + b , where y is the ratio between the chromatographic area of the analyte and the relative internal standard , and x the urinary concentration of the analyte ( mg the limit of detection ( lod ) of the assay was calculated according to the expression ( 1)lod=3seb+bm , where seb is the internal standard error of the intercept . the precision of the assay ( as a coefficient of variation , c.v.% ) was estimated both a within session and as intersession repeatability by analysis of urine spiked with ketones . values of the accuracy were compared with the requirement of us food and drug administration for the bioanalytical method validation . the aim of this paper is to provide a simple , fast , sensitive , and organic - solvent free innovative procedure for analysis of ketones in urine . by this paper , the authors point out the reasons of the rejection of this derivatization technique for the ketones : ( i ) long periods of equilibrium time for hs ; ( ii ) the range of kh of the involved ketones is too wide , ace and 2,5 hd 3.88 10 and 4.43 10 atm m / mol at 25c , respectively ; and ( iii ) the excess of pfbha is an interference on chromatographic separation system . for example , the carbonyl of the ketones is hindered , and , thus , the compounds react very slowly with 2,4-dinitrophenylhydrazine . the reaction was evaluated concerning the effect of the reaction time and product yield attained by the determination of the amount of ketones converting to corresponding oximes . briefly , 5 ml of water containing 2 g of mek , mibk , chone , 2,5 hd , and ace were doped with 1 ml of pfbha solution ( 40 mg / ml ) . the gc area counts were compared with the amount of ace- , mek- , mibk- , chone- , and 2,5 hd - pfbha - oxime standards previously injected by fast gc / nci / ms . however , the reaction yield after 30 minutes to 65c is enough for the formation of products also ensuring fastness in the application of the method . if higher sensitivity is aimed , the reaction time and exposure fiber can be prolonged based on the profile of the reaction and absorption progress ( figure 4 ) . for following sampling by di , the effect of agitation and the absorption - time profiles on the spme extraction efficiency of the derivatives of these carbonyl compounds were examined . the agitation of the solution can strongly improve the spme extraction process and the equilibrium times increased with increasing molecular mass of the analytes ( table 1 ) . except for ace - pfbha - oxime , with a single chromatographic peak , for the other ketone - pfbha - oximes the formation of a double peak , corresponding to syn- and anti - isomer , was observed ( figure 5 ) and the total area of the two peaks was used for their quantitative determination . the pfbha by on - sample derivatization was so chosen for this work , because it is commercially avaiable and , under these conditions , the method allows for the selective detection of ketones at a very low levels without interferences . in this work , a new and different approach to this problems is implemented by the use of mtbstfa and of tmo , as a silylating and methylating agents for boho and acac urinary determination . mtbstfa as a tbdms derivatizing agents was used in gc analysis of amino acids and in gc - ms analysis of fatty acids , ketone bodies , and hydroxylated fluorenes , and it was shown that tbdms derivatives were thermally stable and had favourable fragmentations upon ei ionization [ 35 , 36 ] . for spme on - fiber derivatization , the pa fiber was selected as the most appropriate since the coating of pdms fiber was stripped off after only 35 derivatizations by the mtbstfa . so , the effects of time , temperature and volume of urine , and derivatizating reagent for automated and high - throughput quantitative analysis were evaluated . an excellent spme extraction sensitivity for the urinary boho and acac was generally achieved by immerging the fiber in the diluted urine ( 1 : 5 v / v ) with temperature - controlled agitation ( 50c and 500 rpm ) . moreover , since the reduction in vial diameter by a factor of 3 resulted in an order of magnitude decrease in extraction time , where t , the average time of the diffusion through the aqueous layer is expected to be proportional to the square of the migration distance , x , and inversely proportional to the diffusion coefficient in dwater , ( 2)t = x22d for high - throughput analysis 2 ml vial was used instead of 10 ml vial . for on - fiber derivatization low and high values for three variables ( 20100 l mtbstfa , 2550c , and 1060 minutes ) the volume of mtbstfa and the derivatization time did not show significant effects and so they were fixed to 20 l and 15 minutes , respectively . with tmo a methyl group of the oxonium ion reacts with the anion of the carboxylic functional groups to form the methyl ester : the fast gc / ms was used for verifing conversion of the boho and acac into the corresponding methylester and methylester - pfbha - oxime derivative . briefly , the urine was treated in the same manner as indicated in experimental data and extracted with 3 ml of hexane . conversion of acac and boho was carried out using calibration graph from known amounts of boho - methylester and acac - methylester - pfbha - oxime and data gave an indication of the yield of the derivatisation reaction more than 95% . the second objective to the verify the ability of di - spme technique to determine the seven urinary ketones . for pdms , where 100 , 30 , and 7 m thickness fiber coatings were commercially available , better result was obtained with 30 m pdms fiber , accorded with what is indicated from the theory where thin thickness has more efficient extraction for high molecular weight in fast equilibrium time . 3-fold decrease in extraction time , as well as the extraction time profiles are dramatically affected by the stirring rate ( ca . moreover , the equilibrium and kinetics of the seven ketones versus spme fiber with liquid coating were investigated theoretically . for bohb and acac the derivatisations were also performed on - fiber after the preconcentration step by di - spme with pa liquid phase , and so kinetics of the underivatised bohb and acac versus spme coating were evaluated . because of the properties liquid coating , which is applied in di - spme analysis , the extraction obeys the rules of liquid - liquid equilibrium ( 3)n = c0v1v2kkv1+v2 where k is the partition constant spme fiber liquid polymeric coating / sample , c0 is the initial concentration of the analyte in the aqueous solution , v1 and v2 are the volumes of the coating and the aqueous solution , in the equilibrium concentration of the analyte in the aqueous matrix . as indicated in table 1 kow is a good estimated of k , however , the correlation has to be confirmed for the group of substances from a number of investigators . the linear relationship with solute activity coefficient and ltpri , as in ( 4 ) and ( 5 ) , respectively , can be used further to estimate the k1 values ( 4)log k1=at+b , where a = h/2.303r and b = [ log ( rt/ pvap ) h/2.303r * ] ; h ( j / moli ) is the analyte heat of vaporization , r ( 8.314 j / moli k ) is the gas constant , t ( k ) is the sampling temperature , solute activity coefficient , pvap ( pa ) is the vapour pressure , and t * is the known temperature of coefficient , ( 5)log k1=0.0042ltpri0.188 . the ltpri = 100 ( tr(a ) tr(n)/tr(n+1 ) tr(n ) ) + 100 n ) was experimentally calculated by pdms capillary column where , tr(a ) is the analyte retention time , tr(n ) is the retention time of n - alkane eluting directly before tr(a ) , tr(n+1 ) is the retention time of the n - alkane eluting directly after tr(a ) , and n is the number of carbon atoms for tr(n ) . so , the third objective was the effective chromatographic separation of the seven substances in the minimum time and the investigation of the structurally informative ms fragmentation pattern . in this investigation , the gc analysis of ketones - oxime-/methyl ester - ketone - oxime - derivatives is carried out in less than 3.50 minutes and 2 minutes between two different chromatographic runs , with a speed gain of almost 8 times in comparison with traditional gc procedure to maintain sufficient resolving power for separation of the compounds of interest . regards to pfbha - oxime - derivates the mass spectrum ( table 3 ) shows the advantages in using nci rather than the usual ei , that is , more detailed information about the molecular structure and not only a conspicuous increase in sensibility as due to a sensible lowering of the chemical background ( chemical noise ) . as a matter of fact , the ei rarely allows to detect the molecular peak of the pfbha - oxime - ketones , the only peak detected often being that of m / z 181 , related to the pfb portion . the quality of the fast gc / ms approach allowed for excellent resolution , even with very short analysis time , to resolve the analytes of interest from similar compounds that would interfere with the assay . the precision of the assay ( reported as a coefficient of variation , c.v.% ) , estimated both as withinsession and as intersession repeatability resulted in the range 2.536.98 and 1.145.32% , respectively . accuracy was within 15% of the theoretical concentration , in line with the requirement of us food and drug administration for the bioanalytical methods validation . to demonstrate the applicability of the method to urinary samples , the content of these compounds in human urines ( n = 20 ) of no - exposed the urinary excretion of mek , mibk , chone and free 2,5 hd in control subjects was in the range 1.25.0 g / l , while ace , bohb , and acac were excreted at a high concentration ( 0.110.38 mg mg / l ) , acac ( 50872 mg / l ) , and ac ( 10431 mg / l ) in urine test stick positive ( score + ++++ ) were obtained . this paper reports the first contribution to the automated and high - throughput analysis of seven ketones in urine by spme / ms system . so , the introduction of dedicated , automated , and robotic systems allowed a friendly use of ms apparatus for high - throughput screening so as to reduce the costs of the monitoring campaigns . the specific ms instrumentation suitable for this aim , such as the changeover from nci to ei modes and the automation of the preparation procedure with mfx , that allowed the change of the fiber by robotic device , allowed a friendly use of ms apparatus with a number of advantages including reduced analyst time both for routine analysis and method development , and greater reproducibility .

juvenile hormone ( jh ) is a sesquiterpenoid hormone that cooperates with 20-hydroxyecdysone ( 20e ) to regulate many aspects of insect physiology , including growth , development , and reproduction [ 1 , 2 ] . in insects , jh is generally synthesized by the corpora allata and contributes to maintain larval growth [ 2 , 3 ] . 20e is transformed from ecdysone produced in the prothoracic glands and triggers larval molting and metamorphosis with the larval - pupal transition . higher levels of jh during larval stages prevent metamorphosis , whereas lower levels or an absence of jh at the end of the larval or pupal stages allows 20e to promote metamorphosis [ 46 ] . recently , some studies have reported that jh can induce the transcription of a large number of genes in vivo or in vitro [ 714 ] , and specific jh - response elements ( jhres ) in some jh - regulated genes have been identified . however , little is known about the genomewide response to jh and the crosstalk between jh and 20e in insect larvae . importantly , a transcription factor krppel homolog 1 ( kr - h1 ) is transcriptionally regulated by jh and is involved in transducing the jh signal as a repressor of insect metamorphosis [ 1519 ] . the binding motif of kr - h1 in the promoter of kr - h1 target has been identified . therefore , kr - h1 is regarded as an excellent indicator of jh sensitivity in insects . jh is involved in maintaining metabolic homeostasis in insects , which is consistent with its function of keeping insects in the larval growth phase . for example , early evidence from the silkworm ( bombyx mori ) showed that jh analogue ( jha ) application impairs atp production . recent reports found that jh regulates lipolysis and trehalose homeostasis in insects [ 13 , 23 , 24 ] . interestingly , an evolutionarily conserved insulin / insulin - like growth factor signaling ( iis ) pathway is also involved in mediating nutrition signaling during insect growth . the iis pathway participates in the nutritional regulation of jh biosynthesis in the yellow fever mosquito ( aedes aegypti ) [ 2628 ] . in addition , jh controls trehalose homeostasis to modulate starvation resistance by regulating the synthesis of insulin - like peptide 2 ( ilp2 ) in the red flour beetle ( tribolium castaneum ) . however , our understanding of the metabolic pathways regulated by jh and their interconnections with the iis pathway is limited . the jh titer is high in the early stage of each larval instar whereas the 20e titer is elevated at the later stage . this fluctuation suggests an essential interplay between jh and 20e . to our current knowledge , some molecules , including met , ecr , broad complex , and kr - h1 , have been confirmed to orchestrate the crosstalk between jh and 20e during insect growth and development [ 9 , 16 , 2931 ] . these findings raise the possibility that jh may affect the 20e levels by modulating the activity of the nutrition - dependent iis pathway . the silkworm is an excellent model for studying crosstalk between jh and 20e because of the obvious boundary between feeding and molting during larval stages . for example , jh has been demonstrated to be a potent stimulator of the secretion of ptth , a peptide that promotes ecdysteroidogenesis . the overexpression of the juvenile hormone esterase gene that is involved in jh metabolism , the mutation of the cyp15c1 gene that is involved in jh biosynthesis , or the direct downregulation of jh biosynthesis in the silkworm result in precocious metamorphosis and a reduction in larval molting times [ 3638 ] . the application of the jha methoprene at the beginning of the fourth larval instar induces perfectly the emergence of superlarvae with an additional larval instar . jh is also involved in glycolysis and the immune response [ 11 , 13 , 40 ] . however , the genomewide response to jh and the genetic basis for the crosstalk between jh and other signaling pathways , such as the 20e signaling and iis pathways , are still unclear in the silkworm . to uncover the genomewide response to jh in vivo and to find new clues to the effect of jh on the signaling pathways related to larval growth and development , we investigated the genomewide changes of gene expression following jha treatment . we measured gene expression in the integument ( including muscle ) of the silkworm using a silkworm microarray and a silkworm superlarvae model induced by jha application . our results show that jha application increases the expression levels of genes involved in various metabolic processes and the iis pathway in the silkworm . the silkworm strain dazao ( p50 ) with a characteristic of tetramolter ( four molting times during larval stages ) was used in our experiments . the larvae were reared on fresh mulberry leaves at 25c under a 12-hour light/12-hour dark photoperiod . we collected individual larvae one hour after the completion of the third larval molt and reared together to use for hormone treatment experiments . the jha methoprene ( sigma , usa ) was dissolved in acetone . according to previous report [ 35 , 38 , 39 ] , doses of 1030 g per larva were applied to newly molted fourth instar larvae along the dorsal midline of the larval thorax . silkworm larvae treated with the same dose of pure acetone were used as a control . larval integument ( including muscle ) is considered a peripheral tissue targeted by jh in insects . to analyze the genomewide responses to jha application in the silkworm integument , we separately isolated the integument from 20 larval individuals at different time points after jha treatment , including 12 hours after treatment ( hat ) with jha or acetone ( control ) , 24 hat , 36 hat , and 48 hat . integument samples at each time point were immediately snap - frozen in liquid nitrogen and stored at 80c . we further surveyed the changes of gene expression after jha application using silkworm genomewide microarray , which has been widely used to profile gene expression in many studies [ 41 , 43 ] . total rna extraction , microarray hybridization , raw data normalization , and microarray data analysis were performed as described in previous reports [ 40 , 41 ] . in brief , total rna was extracted from the prepared sample using trizol reagent ( invitrogen , usa ) . because of the high cost and the high reproducibility of microarray hybridization , we mixed equal amounts of total rna from three biological replicates at each time point to create one sample for the hybridization experiment . cdna for the microarray hybridization was prepared from each mixed rna template , labeled with fluorescent dyes ( cy5 for the test samples and cy3 for the control samples ) , and finally hybridized to the silkworm genome oligonucleotide microarray . the raw signal intensity data for gene expression was extracted from the image obtained via scanning each hybridized array and normalized to the confirmed housekeeping genes . for the microarray data analysis , we concluded that a gene was expressed in any sample at any time point if its expression signal intensity was greater than 400 units . the ratio of expression change for a gene in each time point after jha application was evaluated by comparing with that in the control . this ration of expression change for a gene at each time point was further used to profile its time course expression change . finally , a gene was considered as up- or downregulated after jha application if it displayed at least 2.0-fold change in expression level compared with the control . hierarchical clustering of gene expression was performed using the cluster 3.0 program . to analyze parallel gene expression changes of the jha - modulated genes across four selected time points all of the microarray data presented in this study have been deposited in the geo database under accession number gse53374 . using the online wego program , we functionally predicted the gene ontology ( go ) terms of the jha - modulated genes . in addition , we used the probe sequences of all up- and downregulated genes to blast search against the silkworm gene collection to obtain the probe - matched genes . the online kegg pathway database ( http://www.genome.jp/kegg/ ) and sequence similarities enabled these probe - matched genes to be mapped to different kegg pathways using the kaas ( kegg automatic annotation server ) tool . using quantitative real - time rt - pcr experiments , we examined the changes in the mrna expression of selected genes in the integument at 12 , 24 , 36 , and 48 hours after jha application . for an additional comparison , we evaluated the expression of the same gene collection in another jh - targeted tissue , the fat body , after jha application . because dissecting the fat body at the early stage of the fourth larval instar is difficult , we only dissected the fat body at 36 and 48 hours after jha application . sample collection , rna extraction , and cdna synthesis were performed according to previously described procedures . real - time rt - pcr was performed with the sybr premix ex taq system ( takara biotech , japan ) . each pcr reaction was conducted in a final volume of 20 l containing 70 ng of cdna ( 2 l ) , sybr premix ex taq ( 10 l ) , and 0.4 m primers . the pcr amplification conditions were set to the following : 95c for 30 s , followed by 40 cycles of 95c for 30 s and 60c for 30 s. the mrna expression levels of the selected genes were normalized to the control of translation initiation factor 4a ( eif-4a ) gene . the pearson correlation coefficient between microarray data and real time rt - pcr results was further calculated . we fetched the sequences from the approximately 3 kb upstream untranslated region ( utr ) of translation initiation site of genes that their mrna expressions were upregulated with at least 1.5-fold at 12 hours after jha application . these sequences were used to search the conserved recognition and binding motif of transcription factor kr - h1 , a key transducer of jh signal , by the online matinspector program ( http://www.genomatix.de/ ) [ 20 , 48 ] . to determine the gene expression changes in silkworm in response to jh , we used the jha methoprene to induce silkworm superlarvae according to previous reports [ 38 , 39 ] . after several doses of jha were applied topically to newly molted fourth instar larvae , we found that 10 or 20 g of jha per larva induced silkworm superlarvae with a high probability of 93.3% . however , a dose of 30 g per larva resulted in approximately 50% larval death before the fourth molting ( figure 1(a ) ) . therefore , we applied 20 g of jha per larva to hundreds of silkworm larvae for further analysis . compared with pure acetone treatment as control , the jha application initiated premature fourth molting and led to an additional fifth molting , with the consequence of becoming superlarvae ( figures 1(b ) and 1(c ) ) . although the developmental duration of the fourth and fifth larval instars of silkworm superlarvae was shortened owing to an additional sixth larval instar , the entire larval stage was extended by approximately six days , and the average weight per individual was elevated before wandering ( figure 1(d ) ) . furthermore , quantitative rt - pcr examination showed that in silkworm superlarvae , the jh - regulated marker gene kr - h1 displayed a significantly upregulated expression in the integument at 12 , 24 , 36 , and 48 hat ( figure 1(e ) ) and in the fat body at 36 and 48 hat . based on the model of jha - induced silkworm superlarvae , we used a genomewide silkworm microarray to analyze the global gene expression pattern in the integument following jha application . as shown in table s2 and figure s1 , a total of 8,670 genes were expressed in the integuments with both treatments from jha and pure acetone . scatter plot analysis demonstrated that the expression levels of many genes were altered by jha application ( figure 2(a ) ) . intriguingly , 2,143 genes exhibited an expression change of at least 2.0-fold after jha application ( table s3 ) . for convenience in the following description , we defined these genes as jha - modulated genes . in detail , at 12 hat , 49 and 87 genes were up- and downregulated , respectively . at 24 hat , 69 and 74 genes were up- and downregulated , respectively . at 36 and 48 hat , respectively , 450 and 857 genes were upregulated , while 165 and 956 genes were downregulated . further real - time rt - pcr experiments confirmed that most of the examined genes , whose expressions were deregulated after jha application , showed good correlations ( pearson correlation coefficient , r > 0.7 ) between microarray data and rt - pcr results ( table s4 ) . go annotation showed that jha - modulated genes could be functionally classified into 32 categories ( figure 2(b ) ) . the number of jh - modulated genes belonging to the categories of catalytic activity and metabolic process was the largest . in addition , kegg analysis revealed that jha - modulated genes were mainly related to metabolism , genetic information processing , environmental information processing , cellular processes , and organismal systems ( figure 2(c ) ; table s5 ) . remarkably , after a redundancy subtraction for probes , we noted that jha - modulated genes involved in basic metabolic processes have the largest number ( i.e. , 302 ) . genecluster - based coexpression analysis revealed that the expression changes of most jha - modulated genes in the integument exhibited a similar time course dependence . as shown in figure 2(d ) and table s6 , the dynamic expression of jha - modulated genes could be grouped into eight clusters . jha - modulated genes from the c0 , c1 , c2 , and c6 clusters were upregulated at 48 hat . the c3 and c4 clusters showed an upregulation at 12 and 36 hat as well as a downregulation at 24 and 48 hat . we used matinspector program to search the binding motifs of transcription factors in the upstream utr regions of the translation start sites of 157 genes whose expression showed upregulation with at least 1.5-fold at 12 hat after jha application . our results found that the conserved binding motif of kr - h1 , which is a key transcription factor for mediating jh signal , was present within 3 kb upstream utr regions of 95 of the 157 jha - induced genes at 12 hat ( table s7 ) . importantly , as shown in figure 2(e ) , the kr - h1 binding motifs of 67 jha - induced genes revealed the highest core similarity ( core sim ) of 1 with the binding motif of kr - h1 , which all were comprised of a consensus sequence gggt . this indicates that these jha - induced genes may be the direct targets of silkworm kr - h1 . because the number of jha - modulated genes involved in metabolic pathways was the highest and the main function of jh is to maintain insect larval growth , we further investigated jha - modulated genes related to metabolic pathways . our results from the kegg analysis showed that 99 jha - modulated genes were involved in carbohydrate metabolism ( figure 3 ; table s8 ) , with 74 genes that were upregulated in the integument after jha application and that could be classified as members of the c0 , c1 , c2 , c5 , or c6 clusters ( table s6 ) . interestingly , we observed that jha - modulated genes involved in three pathways of carbohydrate metabolism were all upregulated , including ten genes for the pentose phosphate pathway ( e.g. , ribose - phosphate pyrophosphokinase , 6-phosphogluconate dehydrogenase , 6-phosphogluconolactonase , phosphoglucomutase , transketolase , fructose-1,6-bisphosphatase i , fructose - bisphosphate aldolase , and glucose-6-phosphate isomerase ) , five genes involved in propanoate metabolism ( i.e. , 4-aminobutyrate aminotransferase , aldehyde dehydrogenase , aldehyde dehydrogenase family 7 member a1 , acetyl - coa c - acetyltransferase , and enoyl - coa hydratase ) , and five genes involved in butanoate metabolism ( i.e. , 3-oxoacid coa - transferase , 4-aminobutyrate aminotransferase , enoyl - coa hydratase , hydroxymethylglutaryl - coa lyase , and acetyl - coa c - acetyltransferase ) . similarly , most of the genes implicated in other several carbohydrate metabolism pathways ( such as glycolysis , pentose and glucuronate interconversions , galactose metabolism , starch and sucrose metabolism , amino sugar and nucleotide sugar metabolism , pyruvate metabolism , and glyoxylate and dicarboxylate metabolism ) were also upregulated by jh application . the kegg analysis showed that 47 jha - modulated genes are involved in energy metabolism ( figure 4 ; table s8 ) . in addition , 25 jha - modulated genes were upregulated , with most ( 18 ) of them belonging to cluster c6 , which is upregulated at 48 hat . in detail , more than half of the genes involved in each of the four pathways related to energy metabolism , including methane metabolism , nitrogen metabolism , carbon fixation in photosynthetic organisms , and carbon fixation pathways in prokaryotes , were upregulated . intriguingly , we found that all 15 jh - modulated genes involved in the program of oxidative phosphorylation ( oxphos ) were downregulated , including 13 types of v - type h - transporting atpase subunits and two succinate dehydrogenase ( ubiquinone ) subunits . there are 58 enzyme - encoding genes involved in 14 pathways related to lipid metabolism that were modulated by jha ( figure 4 ; table s8 ) . of these genes , all 11 genes catalyzing the biosynthesis of fatty acids and unsaturated fatty acids were upregulated after jha induction , such as fatty acid synthase , 3-oxoacyl-[acyl - carrier protein ] reductase , very - long - chain 3-oxoacyl - coa reductase , stearoyl - coa desaturase ( delta-9 desaturase ) , and acyl - coa oxidase . similarly , among 18 genes involved in the elongation and metabolism of fatty acids , 14 genes were upregulated after jha application , including acetyl - coa acyltransferase , palmitoyl - protein thioesterase , aldehyde dehydrogenase , long - chain acyl - coa synthetase , very - long - chain acyl - coa dehydrogenase , and enoyl - coa hydratase . furthermore , four genes participating in the synthesis and degradation of ketone bodies were also induced by jha . among the 37 jha - modulated genes involved in glycogen biosynthesis and metabolism , notably , all jha - modulated genes involved in the biosynthesis of several types of glycans ( e.g. , n - glycan , mucin - type o - glycan , glycosaminoglycan , and lipopolysaccharide ) were upregulated . in addition , jh - modulated genes for glycosaminoglycan degradation all increased their expression after jha application , including alpha-1,2-mannosyltransferase , arylsulfatase b , hexosaminidase , xylosylprotein 4-beta - galactosyltransferase , mannosyl - oligosaccharide alpha-1,2-mannosidase , beta - galactosidase , and oligosaccharyltransferase complex subunit beta . our kegg analysis indicated that 117 jha - modulated genes were involved in 20 pathways related to amino acid metabolism , with 96 being upregulated after jha application ( figure 5 ; table s8 ) . these pathways involved a large number of jha - modulated enzymes mainly included the metabolism of glycine , serine , and threonine , the degradation of valine , leucine , and isoleucine , the metabolism of arginine and proline , and the metabolism of tryptophan . the number of jha - modulated genes for these four pathways was 23 , 23 , 19 , and 17 , respectively . in addition , 12 enzyme - coding genes involved in beta - alanine metabolism were all upregulated after jha application , including dihydropyrimidine dehydrogenase , 4-aminobutyrate aminotransferase , aldehyde dehydrogenase , spermine synthase , beta - ureidopropionase , enoyl - coa hydratase , and aldehyde dehydrogenase family 7 member a1 . we also noted that 26 jha - modulated genes were responsible for catalyzing the metabolism of two types of nucleotides : purine and pyrimidine ( figure 5 ; table s8 ) . importantly , most ( 24 ) of these genes were upregulated after jha application , including 5-nucleotidase , adenylate kinase , polyribonucleotide nucleotidyltransferase , urate oxidase , pyruvate kinase , ribose - phosphate pyrophosphokinase , phosphoglucomutase , dihydropyrimidine dehydrogenase , adenylosuccinate lyase , phosphoribosylformylglycinamidine synthase , phosphoribosylaminoimidazole carboxylase , beta - ureidopropionase , purine - nucleoside phosphorylase , and ribonucleoside - diphosphate reductase subunit . given that that jh is a key regulator for controlling insect growth and that jha application enhanced a variety of basic metabolic processes , we evaluated the jha - induced response of factors involved in protein processing pathways including synthesis and degradation . the kegg analysis revealed that 97 jha - modulated genes were involved in protein synthesis and degradation , with 79 being upregulated after jha application ( figure 6 ; table s8 ) . among the 41 jh - modulated genes involved in protein processing in the endoplasmic reticulum , 34 were upregulated following jha application , including transitional endoplasmic reticulum atpase , protein disulfide - isomerase , mannosyl - oligosaccharide alpha-1,2-mannosidase , protein transport protein sec6 , and translocation and translocation - associated proteins . the expression of eight jha - modulated genes related to protein export were increased after jha application ( figure 6 ; table s8 ) , including protein transport protein sec61 subunit alpha , protein transport protein sec61 subunit gamma , translocation protein sec63 , signal peptidase complex subunit 2 , signal recognition particle subunit srp54 , and signal recognition particle receptor subunit alpha . we noted that the expression levels of most jha - modulated genes associated with protein degradation were also upregulated after jha application ( figure 6 ; table s8 ) , including five genes related to ubiquitin - mediated proteolysis ( i.e. , e3 ubiquitin - protein ligase synoviolin , ww domain - containing e3 ubiquitin protein ligase 1 , e3 ubiquitin - protein ligase herc2 , dna damage - binding protein 1 , and f - box and wd-40 domain protein ) , two genes involved in proteasome formation ( i.e. , 26s proteasome regulatory subunits n1 and n2 ) , 22 genes associated with the lysosome ( including four cathepsin family members , hexosaminidase , arylsulfatase b , glucosylceramidase , and beta - galactosidase ) , and 13 genes associated with the peroxisome ( including catalase , phytanoyl - coa hydroxylase , hydroxymethylglutaryl - coa lyase , ( s)-2-hydroxy - acid oxidase , fatty acyl - coa reductase , isocitrate dehydrogenase , and superoxide dismutase ) . intriguingly , our microarray data showed that the expression level of the insulin receptor gene inr ( probe i d : sw20525 ) was increased in the integument following jha application ( figure 7(a ) ; table s8 ) , which was also confirmed in the integument and fat body using quantitative real - time rt - pcr ( figure 7(b ) ) . we further examined the expression of other jha - modulated genes related to the iis pathway in our microarray data . the result showed that 21 of 29 jh - modulated genes involved in the iis pathway were upregulated after jha application , including two genes for insulin secretion ( i.e. , solute carrier family 2 and sodium / potassium - transporting atpase subunit beta ) , five genes for insulin signaling ( receptor - type tyrosine - protein phosphatase f , starch phosphorylase , fatty acid synthase , fructose-1,6-bisphosphatase i , and protein phosphatase 1-regulatory ( inhibitor ) subunit 3 ) , and six genes for pi3k - akt signaling ( heat shock protein 90 kda beta , laminin alpha , laminin beta , laminin gamma , protein phosphatase 2 regulatory subunit a , and thrombospondin ) . importantly , real - time rt - pcr examination revealed that three key genes involved in the iis pathway , pi3k , akt , and 4e - bp , were also mediated by jha application ( figure 7(b ) ) . after jha application , pi3k and akt were upregulated , whereas 4e - bp was downregulated in both the integument and the fat body . given that insulin signaling pathways are implicated in ecdysteroidogenesis in insects [ 32 , 33 ] and larval molting is controlled by a 20e pulse , it was necessary to check whether genes involved in 20e - related pathways were also upregulated in jha - induced silkworm superlarvae . as expected , the cytochrome p450 enzyme gene cyp314a1 ( shade ) , which is responsible for converting ecdysone to active 20e , was upregulated by jha from 24 to 48 hat ( figure 7(a ) ; table s8 ) . the rt - pcr examination showed that the 20e receptor gene ecr was also upregulated after jha induction ( figure 7(b ) ) . the larval stage in most holometabolic insects is characterized by nutrient - dependent growth and several times of larval molting . insect larval growth and molting are mainly coordinated by two endocrine hormones , jh and 20e . jh maintains larval growth whereas nutrient - sensitive 20e signal triggers the initiation of larval molting . therefore , larval growth and molting are excellent biological events for studying the crosstalk between jh and 20e in insects . however , which metabolic processes are regulated by jh during larval growth and how jh contributes to 20e signaling have not been comprehensively examined . in this study , we used the model of jha - induced silkworm superlarvae and microarray - based gene expression profiling to investigate the functions of jh in both the maintenance of metabolic homeostasis and the 20e - based regulation of larval molting during silkworm larval stages . previous studies reported that the application of the jha methoprene to silkworm larvae at the beginning of the fourth larval instar will induce superlarvae with a supernumerary fifth molting . to understand the mechanisms underlying the crosstalk between jh and 20e during the larval stage , we induced silkworm superlarvae by jha application and observed that the onset of the fourth molt in superlarvae was approximately one day earlier than that in the control larvae treated with pure acetone ( figure 1 ) . the jh - responsive gene kr - h1 also showed increased expression in two jh - targeted tissues of superlarvae , the integument and fat body , further confirming that the induction of silkworm superlarvae could be the consequence of jha application . to determine how silkworm larvae respond to jh during larval growth and molting , we performed a microarray - based analysis of gene expression changes after jha application in the integument . a genomewide silkworm microarray has been previously used to profile gene expression in the silkworm [ 11 , 40 , 41 ] . in addition , the identification of jh - responsive genes has been performed in some specific cell lines or tissues as well as at different developmental stages in some insects , including the fruit fly ( drosophila melanogaster ) [ 8 , 9 , 12 ] , yellow fever mosquito , red flour beetle , pine engraver beetle ( ips pini ) , and silkworm [ 11 , 13 ] . comparatively , some new and interesting findings were identified by our microarray analysis of the jh response in the silkworm integument . our studies first provide systematic insights into jh modulation on the programs controlling basic metabolism and protein processing during silkworm larval growth and molting . comparative analysis showed that the expression levels of thousands of genes were significantly changed after jha application ( table s3 ) . interestingly , most of these jha - modulated genes are involved in basic metabolic pathways ( e.g. , glycolysis , other carbohydrate metabolic programs , and lipid metabolism ) and protein processing pathways . furthermore , most jha - modulated genes were upregulated after jha application ( table s8 ) , indicating that jha application could promote most metabolic processes and protein processing programs . previous reports have confirmed that a reduction in jh biosynthesis could decrease the levels of carbohydrate and lipid metabolism in the red flour beetle and that jh application could upregulate both the mrna levels and enzymatic activity of proteins related to glycolysis in the silkworm fat body . taken together , we propose that a jh - induced increase in basic metabolic activities may produce more nutritional supplies and further facilitate the growth of silkworm larvae . an interesting observation of our study is that the mrna level of trehalose-6-phosphate synthase ( tps ) , an enzyme controlling starch and sucrose metabolism , showed an obvious increase in the silkworm integument after jha application , which is different from a previous finding showing no significant changes in tps expression in the silkworm fat body after jha application . this finding raises the prospect that the jh - induced regulation of tps transcription might be tissue specific in the silkworm . intriguingly , our data also revealed that most of jh - modulated genes that were involved in oxphos exhibited significantly reduced expression in the integument ( figure 4 ; table s8 ) . generally , the oxphos pathway mediates energy metabolism and produces atp via the oxidation of nutrients in the mitochondria of animal muscles [ 50 , 51 ] . thus , the reduction of expression level of genes involved in the oxphos pathway in the silkworm integument before larval molting indicates that oxphos - mediated atp production or requirements might be decreased after jha application . the iis pathway acts as a nutrient sensor in the regulation of metabolic homeostasis and growth . previous evidences showed that the iis pathway is stimulated by high glucose and regulates multiple metabolic pathways related to normal growth through various ways , for example , attenuating sugar levels , promoting glycolysis , and synthesizing fatty acids and proteins . surprisingly , our microarray data revealed that jha application increased the expression of the insulin receptor gene inr in the integument and upregulated other genes involved in iis - related pathways , including insulin secretion , insulin signaling , and the pi3k - akt signaling pathway ( figure 7(a ) ) . real - time rt - pcr examination further confirmed that among several key regulators involved in the iis pathway , inr , pi3k , and akt showed an expected upregulation after jha application , whereas 4e - bp expression was decreased ( figure 7(b ) ) , indicating that jha application can increase the activity of the iis pathway . moreover , the fact that jha application impaired the oxphos pathway also indicated an increased activity of the iis pathway after jha application because oxphos processes can be attenuated by abnormal nutritional signals ( such as high insulin levels or a high - fat diet ) in human skeletal muscle [ 5457 ] . in combination with our observation of the jha - induced modulation of carbohydrate metabolism , we speculate that jh might regulate carbohydrate metabolism to increase sugar levels in hemocytes , which could further activate the iis pathway that is required for regulating glycolysis , lipid metabolism , and protein processing . insect larval molting is triggered by a pulse of 20e when the larvae grow to an appropriate body size under normal nutrient conditions [ 58 , 59 ] . thus , jha - induced premature larval molting indicates that jha application might accelerate the emergence of 20e pulse in the fourth larval instar . in fact , 20e biosynthesis was previously reported to be catalyzed by several cytochrome p450 enzymes . our data showed that the expression of two 20e - related genes , cyp314a1 ( shade ) , which encodes a p450 enzyme for transforming inactive ecdysone into active 20e , and ecr , which encodes ecdysone receptor that is involved in 20e signaling , was significantly elevated following jha application . this further confirmed that premature larval molting was likely resulted from the early emergence of 20e pulse after jha application in the end of the fourth larval instar , which consequently contributed to shortening the developmental duration of the fourth larval instar and to subsequently reducing the body weight . in addition , we noted that compared to the early stages ( 12 and 24 hat ) after jha application , a large number of genes showed more dramatic changes at the late stage of 48 hat in expression levels , indicating that these jha - modulated genes with dramatic expression changes may be just the indirect targets of jh , but the targets of 20e to a great extent . furthermore , it has been known that insect metamorphosis with larval - pupal transition is also determined by a critical weight that is related to body size . given that the body weight of the jha - induced superlarvae in the fifth larval instar was also reduced , we suggested that in the fifth larval instar , jha - induced silkworm superlarvae did not reach to critical weight as same with that in the control , so it should experience an additional larval molting , which may be the same as the fourth instar of the control . moreover , previous reports have demonstrated that 20e biosynthesis is also positively mediated by insulin signaling [ 32 , 34 , 62 ] . together with our observation that genes involved in the iis pathway were significantly upregulated after jha application , we therefore suggest that jh may regulate 20e signaling by mediating basic metabolic activities and the nutrient - dependent iis pathway . in this study , we profiled the genomewide alteration of gene expression in the integument of silkworm larvae after jha application and found the cues about jh regulation on metabolic homeostasis , iis pathway , and 20e signaling . further studies should be conducted and may be helpful for deciphering the mechanism underlying the interaction between jh and 20e during silkworm larval growth .

juvenile hormone ( jh ) coordinates with 20-hydroxyecdysone ( 20e ) to regulate larval growth and molting in insects . however , little is known about how this cooperative control is achieved during larval stages . here , we induced silkworm superlarvae by applying the jh analogue ( jha ) methoprene and used a microarray approach to survey the mrna expression changes in response to jha in the silkworm integument . we found that jha application significantly increased the expression levels of most genes involved in basic metabolic processes and protein processing and decreased the expression of genes associated with oxidative phosphorylation in the integument . several key genes involved in the pathways of insulin / insulin - like growth factor signaling ( iis ) and 20e signaling were also upregulated after jha application . taken together , we suggest that jh may mediate the nutrient - dependent iis pathway by regulating various metabolic pathways and further modulate 20e signaling .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

juvenile hormone ( jh ) is a sesquiterpenoid hormone that cooperates with 20-hydroxyecdysone ( 20e ) to regulate many aspects of insect physiology , including growth , development , and reproduction [ 1 , 2 ] . higher levels of jh during larval stages prevent metamorphosis , whereas lower levels or an absence of jh at the end of the larval or pupal stages allows 20e to promote metamorphosis [ 46 ] . however , little is known about the genomewide response to jh and the crosstalk between jh and 20e in insect larvae . jh is involved in maintaining metabolic homeostasis in insects , which is consistent with its function of keeping insects in the larval growth phase . for example , early evidence from the silkworm ( bombyx mori ) showed that jh analogue ( jha ) application impairs atp production . recent reports found that jh regulates lipolysis and trehalose homeostasis in insects [ 13 , 23 , 24 ] . interestingly , an evolutionarily conserved insulin / insulin - like growth factor signaling ( iis ) pathway is also involved in mediating nutrition signaling during insect growth . in addition , jh controls trehalose homeostasis to modulate starvation resistance by regulating the synthesis of insulin - like peptide 2 ( ilp2 ) in the red flour beetle ( tribolium castaneum ) . however , our understanding of the metabolic pathways regulated by jh and their interconnections with the iis pathway is limited . these findings raise the possibility that jh may affect the 20e levels by modulating the activity of the nutrition - dependent iis pathway . the silkworm is an excellent model for studying crosstalk between jh and 20e because of the obvious boundary between feeding and molting during larval stages . the overexpression of the juvenile hormone esterase gene that is involved in jh metabolism , the mutation of the cyp15c1 gene that is involved in jh biosynthesis , or the direct downregulation of jh biosynthesis in the silkworm result in precocious metamorphosis and a reduction in larval molting times [ 3638 ] . however , the genomewide response to jh and the genetic basis for the crosstalk between jh and other signaling pathways , such as the 20e signaling and iis pathways , are still unclear in the silkworm . to uncover the genomewide response to jh in vivo and to find new clues to the effect of jh on the signaling pathways related to larval growth and development , we investigated the genomewide changes of gene expression following jha treatment . we measured gene expression in the integument ( including muscle ) of the silkworm using a silkworm microarray and a silkworm superlarvae model induced by jha application . our results show that jha application increases the expression levels of genes involved in various metabolic processes and the iis pathway in the silkworm . the silkworm strain dazao ( p50 ) with a characteristic of tetramolter ( four molting times during larval stages ) was used in our experiments . to analyze the genomewide responses to jha application in the silkworm integument , we separately isolated the integument from 20 larval individuals at different time points after jha treatment , including 12 hours after treatment ( hat ) with jha or acetone ( control ) , 24 hat , 36 hat , and 48 hat . the ratio of expression change for a gene in each time point after jha application was evaluated by comparing with that in the control . using quantitative real - time rt - pcr experiments , we examined the changes in the mrna expression of selected genes in the integument at 12 , 24 , 36 , and 48 hours after jha application . for an additional comparison , we evaluated the expression of the same gene collection in another jh - targeted tissue , the fat body , after jha application . because dissecting the fat body at the early stage of the fourth larval instar is difficult , we only dissected the fat body at 36 and 48 hours after jha application . the pcr amplification conditions were set to the following : 95c for 30 s , followed by 40 cycles of 95c for 30 s and 60c for 30 s. the mrna expression levels of the selected genes were normalized to the control of translation initiation factor 4a ( eif-4a ) gene . we fetched the sequences from the approximately 3 kb upstream untranslated region ( utr ) of translation initiation site of genes that their mrna expressions were upregulated with at least 1.5-fold at 12 hours after jha application . to determine the gene expression changes in silkworm in response to jh , we used the jha methoprene to induce silkworm superlarvae according to previous reports [ 38 , 39 ] . after several doses of jha were applied topically to newly molted fourth instar larvae , we found that 10 or 20 g of jha per larva induced silkworm superlarvae with a high probability of 93.3% . furthermore , quantitative rt - pcr examination showed that in silkworm superlarvae , the jh - regulated marker gene kr - h1 displayed a significantly upregulated expression in the integument at 12 , 24 , 36 , and 48 hat ( figure 1(e ) ) and in the fat body at 36 and 48 hat . based on the model of jha - induced silkworm superlarvae , we used a genomewide silkworm microarray to analyze the global gene expression pattern in the integument following jha application . scatter plot analysis demonstrated that the expression levels of many genes were altered by jha application ( figure 2(a ) ) . remarkably , after a redundancy subtraction for probes , we noted that jha - modulated genes involved in basic metabolic processes have the largest number ( i.e. genecluster - based coexpression analysis revealed that the expression changes of most jha - modulated genes in the integument exhibited a similar time course dependence . we used matinspector program to search the binding motifs of transcription factors in the upstream utr regions of the translation start sites of 157 genes whose expression showed upregulation with at least 1.5-fold at 12 hat after jha application . because the number of jha - modulated genes involved in metabolic pathways was the highest and the main function of jh is to maintain insect larval growth , we further investigated jha - modulated genes related to metabolic pathways . our results from the kegg analysis showed that 99 jha - modulated genes were involved in carbohydrate metabolism ( figure 3 ; table s8 ) , with 74 genes that were upregulated in the integument after jha application and that could be classified as members of the c0 , c1 , c2 , c5 , or c6 clusters ( table s6 ) . interestingly , we observed that jha - modulated genes involved in three pathways of carbohydrate metabolism were all upregulated , including ten genes for the pentose phosphate pathway ( e.g. intriguingly , we found that all 15 jh - modulated genes involved in the program of oxidative phosphorylation ( oxphos ) were downregulated , including 13 types of v - type h - transporting atpase subunits and two succinate dehydrogenase ( ubiquinone ) subunits . similarly , among 18 genes involved in the elongation and metabolism of fatty acids , 14 genes were upregulated after jha application , including acetyl - coa acyltransferase , palmitoyl - protein thioesterase , aldehyde dehydrogenase , long - chain acyl - coa synthetase , very - long - chain acyl - coa dehydrogenase , and enoyl - coa hydratase . among the 37 jha - modulated genes involved in glycogen biosynthesis and metabolism , notably , all jha - modulated genes involved in the biosynthesis of several types of glycans ( e.g. in addition , jh - modulated genes for glycosaminoglycan degradation all increased their expression after jha application , including alpha-1,2-mannosyltransferase , arylsulfatase b , hexosaminidase , xylosylprotein 4-beta - galactosyltransferase , mannosyl - oligosaccharide alpha-1,2-mannosidase , beta - galactosidase , and oligosaccharyltransferase complex subunit beta . our kegg analysis indicated that 117 jha - modulated genes were involved in 20 pathways related to amino acid metabolism , with 96 being upregulated after jha application ( figure 5 ; table s8 ) . in addition , 12 enzyme - coding genes involved in beta - alanine metabolism were all upregulated after jha application , including dihydropyrimidine dehydrogenase , 4-aminobutyrate aminotransferase , aldehyde dehydrogenase , spermine synthase , beta - ureidopropionase , enoyl - coa hydratase , and aldehyde dehydrogenase family 7 member a1 . importantly , most ( 24 ) of these genes were upregulated after jha application , including 5-nucleotidase , adenylate kinase , polyribonucleotide nucleotidyltransferase , urate oxidase , pyruvate kinase , ribose - phosphate pyrophosphokinase , phosphoglucomutase , dihydropyrimidine dehydrogenase , adenylosuccinate lyase , phosphoribosylformylglycinamidine synthase , phosphoribosylaminoimidazole carboxylase , beta - ureidopropionase , purine - nucleoside phosphorylase , and ribonucleoside - diphosphate reductase subunit . given that that jh is a key regulator for controlling insect growth and that jha application enhanced a variety of basic metabolic processes , we evaluated the jha - induced response of factors involved in protein processing pathways including synthesis and degradation . the kegg analysis revealed that 97 jha - modulated genes were involved in protein synthesis and degradation , with 79 being upregulated after jha application ( figure 6 ; table s8 ) . among the 41 jh - modulated genes involved in protein processing in the endoplasmic reticulum , 34 were upregulated following jha application , including transitional endoplasmic reticulum atpase , protein disulfide - isomerase , mannosyl - oligosaccharide alpha-1,2-mannosidase , protein transport protein sec6 , and translocation and translocation - associated proteins . the expression of eight jha - modulated genes related to protein export were increased after jha application ( figure 6 ; table s8 ) , including protein transport protein sec61 subunit alpha , protein transport protein sec61 subunit gamma , translocation protein sec63 , signal peptidase complex subunit 2 , signal recognition particle subunit srp54 , and signal recognition particle receptor subunit alpha . we noted that the expression levels of most jha - modulated genes associated with protein degradation were also upregulated after jha application ( figure 6 ; table s8 ) , including five genes related to ubiquitin - mediated proteolysis ( i.e. intriguingly , our microarray data showed that the expression level of the insulin receptor gene inr ( probe i d : sw20525 ) was increased in the integument following jha application ( figure 7(a ) ; table s8 ) , which was also confirmed in the integument and fat body using quantitative real - time rt - pcr ( figure 7(b ) ) . we further examined the expression of other jha - modulated genes related to the iis pathway in our microarray data . the result showed that 21 of 29 jh - modulated genes involved in the iis pathway were upregulated after jha application , including two genes for insulin secretion ( i.e. importantly , real - time rt - pcr examination revealed that three key genes involved in the iis pathway , pi3k , akt , and 4e - bp , were also mediated by jha application ( figure 7(b ) ) . after jha application , pi3k and akt were upregulated , whereas 4e - bp was downregulated in both the integument and the fat body . given that insulin signaling pathways are implicated in ecdysteroidogenesis in insects [ 32 , 33 ] and larval molting is controlled by a 20e pulse , it was necessary to check whether genes involved in 20e - related pathways were also upregulated in jha - induced silkworm superlarvae . the rt - pcr examination showed that the 20e receptor gene ecr was also upregulated after jha induction ( figure 7(b ) ) . the larval stage in most holometabolic insects is characterized by nutrient - dependent growth and several times of larval molting . insect larval growth and molting are mainly coordinated by two endocrine hormones , jh and 20e . therefore , larval growth and molting are excellent biological events for studying the crosstalk between jh and 20e in insects . however , which metabolic processes are regulated by jh during larval growth and how jh contributes to 20e signaling have not been comprehensively examined . in this study , we used the model of jha - induced silkworm superlarvae and microarray - based gene expression profiling to investigate the functions of jh in both the maintenance of metabolic homeostasis and the 20e - based regulation of larval molting during silkworm larval stages . to understand the mechanisms underlying the crosstalk between jh and 20e during the larval stage , we induced silkworm superlarvae by jha application and observed that the onset of the fourth molt in superlarvae was approximately one day earlier than that in the control larvae treated with pure acetone ( figure 1 ) . the jh - responsive gene kr - h1 also showed increased expression in two jh - targeted tissues of superlarvae , the integument and fat body , further confirming that the induction of silkworm superlarvae could be the consequence of jha application . to determine how silkworm larvae respond to jh during larval growth and molting , we performed a microarray - based analysis of gene expression changes after jha application in the integument . comparatively , some new and interesting findings were identified by our microarray analysis of the jh response in the silkworm integument . our studies first provide systematic insights into jh modulation on the programs controlling basic metabolism and protein processing during silkworm larval growth and molting . comparative analysis showed that the expression levels of thousands of genes were significantly changed after jha application ( table s3 ) . interestingly , most of these jha - modulated genes are involved in basic metabolic pathways ( e.g. , glycolysis , other carbohydrate metabolic programs , and lipid metabolism ) and protein processing pathways . furthermore , most jha - modulated genes were upregulated after jha application ( table s8 ) , indicating that jha application could promote most metabolic processes and protein processing programs . previous reports have confirmed that a reduction in jh biosynthesis could decrease the levels of carbohydrate and lipid metabolism in the red flour beetle and that jh application could upregulate both the mrna levels and enzymatic activity of proteins related to glycolysis in the silkworm fat body . taken together , we propose that a jh - induced increase in basic metabolic activities may produce more nutritional supplies and further facilitate the growth of silkworm larvae . an interesting observation of our study is that the mrna level of trehalose-6-phosphate synthase ( tps ) , an enzyme controlling starch and sucrose metabolism , showed an obvious increase in the silkworm integument after jha application , which is different from a previous finding showing no significant changes in tps expression in the silkworm fat body after jha application . this finding raises the prospect that the jh - induced regulation of tps transcription might be tissue specific in the silkworm . intriguingly , our data also revealed that most of jh - modulated genes that were involved in oxphos exhibited significantly reduced expression in the integument ( figure 4 ; table s8 ) . thus , the reduction of expression level of genes involved in the oxphos pathway in the silkworm integument before larval molting indicates that oxphos - mediated atp production or requirements might be decreased after jha application . surprisingly , our microarray data revealed that jha application increased the expression of the insulin receptor gene inr in the integument and upregulated other genes involved in iis - related pathways , including insulin secretion , insulin signaling , and the pi3k - akt signaling pathway ( figure 7(a ) ) . real - time rt - pcr examination further confirmed that among several key regulators involved in the iis pathway , inr , pi3k , and akt showed an expected upregulation after jha application , whereas 4e - bp expression was decreased ( figure 7(b ) ) , indicating that jha application can increase the activity of the iis pathway . moreover , the fact that jha application impaired the oxphos pathway also indicated an increased activity of the iis pathway after jha application because oxphos processes can be attenuated by abnormal nutritional signals ( such as high insulin levels or a high - fat diet ) in human skeletal muscle [ 5457 ] . in combination with our observation of the jha - induced modulation of carbohydrate metabolism , we speculate that jh might regulate carbohydrate metabolism to increase sugar levels in hemocytes , which could further activate the iis pathway that is required for regulating glycolysis , lipid metabolism , and protein processing . thus , jha - induced premature larval molting indicates that jha application might accelerate the emergence of 20e pulse in the fourth larval instar . our data showed that the expression of two 20e - related genes , cyp314a1 ( shade ) , which encodes a p450 enzyme for transforming inactive ecdysone into active 20e , and ecr , which encodes ecdysone receptor that is involved in 20e signaling , was significantly elevated following jha application . this further confirmed that premature larval molting was likely resulted from the early emergence of 20e pulse after jha application in the end of the fourth larval instar , which consequently contributed to shortening the developmental duration of the fourth larval instar and to subsequently reducing the body weight . in addition , we noted that compared to the early stages ( 12 and 24 hat ) after jha application , a large number of genes showed more dramatic changes at the late stage of 48 hat in expression levels , indicating that these jha - modulated genes with dramatic expression changes may be just the indirect targets of jh , but the targets of 20e to a great extent . given that the body weight of the jha - induced superlarvae in the fifth larval instar was also reduced , we suggested that in the fifth larval instar , jha - induced silkworm superlarvae did not reach to critical weight as same with that in the control , so it should experience an additional larval molting , which may be the same as the fourth instar of the control . together with our observation that genes involved in the iis pathway were significantly upregulated after jha application , we therefore suggest that jh may regulate 20e signaling by mediating basic metabolic activities and the nutrient - dependent iis pathway . in this study , we profiled the genomewide alteration of gene expression in the integument of silkworm larvae after jha application and found the cues about jh regulation on metabolic homeostasis , iis pathway , and 20e signaling .

with the increase in life expectancy , there is , proportionately , a dramatic increase in age - related health conditions including , in particular , eye diseases . among all the eye pathologies , diabetic retinopathy ( dr ) is currently one of the leading causes of blindness in the working - age population in developed countries [ 13 ] . epidemiological data show that the prevalence of dr is around 30% in the diabetic population and that the annual incidence varies from 2% to 6% . every year , up to 1% of diabetic patients develop serious ocular complications leading to both poor quality of life and a socioeconomic burden . with regard to the italian setting , a study conducted in the veneto region , using clinical fundus examination , confirmed literature data with a prevalence of dr of 26.6% . a significant worldwide increase in the number of persons affected by diabetes has been estimated for the next 10 years , reaching approximately 380 million by 2025 , mostly in developing countries . in europe , compared with 2007 , there is expected to be an increase of 10 million persons with a prevalence of diabetes below 10% . as the prevalence of diabetes is expected to rise in the future , an associated increase in dr cases should also be expected . however , recently , data suggest that the prevalence of dr may decrease , due to the intensification of the screening programmes and better control of risk - factors , thus underlying their effectiveness and the significance . in this view , scholars and practitioners widely agreed that dr , at any stage of progression , requires specific management : from the screening programmes ( useful for an early diagnosis ) to the definition of pharmacological treatments needed . in recent years , the strategies for dr prevention have moved from the traditional ophthalmological examination to a faster digital retinal imaging acquisition and grading of dr . the application of these new screening programmes , followed by prompt diagnosis and a better timely management , is well known to prevent significantly the risk of diabetic blindness [ 9 , 10 ] , as well as to decrease all the costs related to the investigated pathology . in fact , from an economic point of view , the annual cost per patient affected by dr is approximately twice as high as those patients with diabetes only : thus , the implementation of effective and recognized screening programmes could represent cost - effective strategies [ 12 , 13 ] , one useful to narrow both the economic and the social burden of dr . despite the implementation of successful national screening programmes worldwide , the lack of diffuse screening activities for dr in italy is delaying the diagnosis and prompt treatment of dr . public screening programmes and prompt treatments have been shown to reduce lifetime costs related to visual disability [ 14 , 15 ] . for these reasons , vision impairments as a result of dr should be considered easy to prevent , and the development of systematic programmes of screening should represent an urgent and primary healthcare need . moving on from these premises , since dr should be one of the major priorities for the healthcare services , the present study aimed at investigating the feasibility related to the introduction of a specific and accurate screening programme in the catchment area of treviso ( defined as ulss 9 ) , in comparison with the no prevention strategy , including also the costs averted to blindness , in terms of the validity of the intervention and the direct costs absorbed ( efficiency ) by the regional healthcare service . the analysis was designed assuming the regional healthcare service ( veneto region ) point of view . in order to achieve the previously mentioned objective , a perspective study for the implementation of a screening programme devoted to dr was planned to be performed between september and december 2012 , after receiving the approval from ethic committee of the local health authority . as dr is a multiprofessional and a multidimensional eye disease , a multidisciplinary group was involved in the project , including general practitioners , diabetes experts , administrative staff , nurses , epidemiologists , and ophthalmologists . the involvement of different healthcare professionals in the screening programme could influence the resources absorption and the optimisation of the screening pathway [ 12 , 13 , 1618 ] . the screening involved all types ( type 1 and type 2 ) of diabetic patients living in the area of ponzano ( treviso ) that has 5.000 inhabitants and is part of one of the local health authorities of the veneto region : the ulss 9 of treviso . diabetic subjects were accurately identified by crossing different databases : primary and secondary diagnosis for hospitalisation , drugs and delivery systems , prescriptions , health care procedures covered by the diabetic code , and the diabetic code assigned to patients . in particular , the following patients were invited to attend the screening programme : ( i ) outpatients going to a hospital specialist visit to the diabetologists , or first visit to ophthalmologist ; ( ii ) patients going to the territorial pharmacist , in order to retrieve the specific diabetes drugs ; and ( iii ) patients referring to the general practitioners , with a diagnosis of diabetes . initially , a personal letter of invitation was sent to all the diabetic target population ( n = 498 ) to be enrolled in the screening programme . all the patients attending the screening procedure have signed a specific informed consent form , consistent with ethical aspects . three nonmydriatic , 45 field , digital retinal images were captured , in accordance with a previously validated technique . the three fundus images encompassed the following retinal fields : field 1 centred on the macula , field 2 centred on optic disc , and field 3 midperipheral superior - temporal field . all images were electronically transmitted to the reading centre and stored in an online secured database called eye knowledge network for the second step , online examination . retinal images were graded for dr at the ca ' foncello treviso hospital , where the ophthalmology unit is located , by two experts and certified readers who were members of the reading centre staff of the university of padova . dr and diabetic macular edema ( dme ) were graded in accordance with the international classification proposed by the american academy of ophthalmologists . when the quality of the images was inadequate for the clinical evaluation and when fundus images were graded as positive , the patients were referred for further ophthalmologic examination . positive findings included retinal changes that required specialist management : moderate and severe nonproliferative dr , proliferative dr , dme , and/or any other retinal abnormality . fundus images were graded as negative if no dr or nonsight threatening dr was detected . a report , with the results of the screening and the correct follow - up timetable for the negative screened population , was sent to the patient 's general practitioner within 1 month after the screening . from a methodological point of view , in order to quantify the impact of the introduction of screening programme in the clinical practice , both an activity based costing ( abc ) analysis and a budget impact analysis ( bia ) were conducted . the abc [ 21 , 22 ] is useful for the enhancement of the average costs related to each phase of the screening pathway . in particular , the main objective of the abc is the measurement of the costs and the performance activities , taking into account also the related human and materials resources for the proper development of the procedure . in this view , the economic impact of each patient was determined utilising the following components : ( i ) human resources ( i.e. , individuals involved in the different phases of the screening programme , such as administrative staff , nurses , and ophthalmologists ) ; ( ii ) materials and equipment ; ( iii ) pharmacological and/or laser treatment . the abc did not take into consideration the costs related to the delivery of the invitation letter : different technologies could be implemented in order to carry out this task ( telephonic invitation , e - mail contact , personal contact , or letter ) , differing for the related economic value . a bia allows the prediction of the potential financial impact of a new technology adoption , into a healthcare system ; influencing , in a positive or in a negative manner , the healthcare expenditure and considering both a specific point of view and a determined time horizon . in this view , two scenarios were simulated , thus comparing the so - called do nothing strategy with the implementation of a proper screening programme . in particular , in both scenarios , the occurrence and the related cost of blindness were taken into consideration as direct healthcare costs . since the analysis assumed the healthcare regional services point of view , the intangible and indirect items of expenditure were excluded from the study . drug costs were derived from the officially published nhs price list . if necessary , economic values were reported in euros , considering the 2015 inflation rate , using the consumer price index for healthcare expenditure , thus making economic measures comparable , and being based on the same year of reference . in order to ensure the robustness of the result , a sensitivity analysis was carried out . in particular , both the attendance rate to the screening programme and the percentage of patients undergoing the complete eye examination were modified , thus understanding if significant changes in the feasibility of the programme occur . literature reported that patients ' compliance with dr screening is not optimal worldwide , hence reporting attendance rates ranging from a minimum of 32% and a maximum of 92% [ 2427 ] . further analysis reported a variation in the eye examination rate . the local health authority involved declared that it could reach about the 80%85% if collaboration with community - based organisations , as well as greater information activities of dr risks among citizens , is implemented . moving on from these premises , 498 individuals were identified as being diabetic patients within the area of ponzano ( that has 5,000 inhabitants ) ; thus , it emerged that the prevalence of the diabetes in the investigated town was around 10% . out of the 498 diabetic patients originally invited to enroll in the screening programme , 340 accepted to be evaluated ( 68% ) , although it was possible to confirm a response rate equal to 80% since 57 patients did not attend the screening programme because they had already undergone a complete eye examination that showed no signs of dr development . the study population was composed predominantly of males ( 55% ) ; the average age of the sample was 68 years ( range : 2693 ) . on average , patients have been suffering from diabetes for 20 years ( range : 241 ) . the most common comorbidities developed by patients were hypertension ( 40% ) and dyslipidaemia ( 38% ) . 324 patients ( 95% ) successfully completed the procedure , though in 16 cases ( 5% ) images were noncaptured due to either systemic conditions of the patients , insufficient mydriasis , or other technical reasons related to the fundus camera . the quality of digital images was adequate for the interpretation in 260 patients ( 80% ) , although it was ungradable in 64 cases ( 20% ) due to insufficient dilation , media opacity , poor fixation , or the absence of one of the captured fields . as a result , taking into account the gradable 260 patients , 225 were classified as negative ( 87% ) and 35 as positive ( 13% ) . based on the entire screened population , 115 patients ( 34% ) were referred to an ophthalmologist . of these , 16 ( 14% ) were patients from whom it was impossible to obtain images , 64 ( 56% ) had no evaluable images , and 35 ( 30% ) were positive cases . of the previously mentioned 115 patients , 92 ( 91% ) underwent a full ophthalmological examination , giving the following results : in 67 cases ( 73% ) , dr was not detected , though 25 patients ( 27% ) presented signs of dr . in particular , 24% ( n = 6 ) had mild nonproliferative dr , 52% ( n = 13 ) had moderate nonproliferative dr , 12% ( n = 3 ) had preproliferative dr , and 12% ( n = 3 ) had proliferative dr . concomitant dme was present in 36% ( n = 9 ) of these patients . in 9 cases ( 36% ) , prompt treatment with intravitreal injections and/or laser photo - coagulation was required for proliferative and severe preproliferative retinopathy or macular edema . after these general remarks , regarding the screened population , an economic evaluation was required in order to investigate the amount of resources dedicated for this specific innovative programme . it should be noted that the veneto region 2015 tariffs were taken into account in order to investigate the value of the complete screening programme , thus also including the entire cycle of intravitreal injections . with reference to the abovementioned distribution of the patients , the economic resources absorption , with regard to the whole population screened pathway , was divided into four distinct and logical phases:340 patients attended the screening programme , involving 1 healthcare professional ( nurse ) who spent 12 minutes per patient and considering the equipment amortization.324 patients completed the procedure , after which 1 ophthalmologist interpreted their digital images , spending about 5 minutes per patient , considering also the workstation and the administrative staff costs.out of the 115 patients referred for a complete examination , only 92 underwent an ophthalmology examination , in order to obtain an in - depth analysis of the disease ( in the third phase , it is important to note that the costs of all the materials and the drugs utilised by the clinicians for the complete eye examination are included in the first visit reimbursement tariff).nine patients , who were suffering from a severe stage of pathology , received pharmacological and/or laser treatment , thus considering that both the human resources and the materials / equipment costs are included in the procedure costs , as detailed in table 2.it was first necessary to evaluate the costs related to the human resources involved in the screening programme . thus , the gross monthly salary , related to each specific professional title , was taken into consideration . in particular , its time value per minute was multiplied by the time dedicated to each procedure . 340 patients attended the screening programme , involving 1 healthcare professional ( nurse ) who spent 12 minutes per patient and considering the equipment amortization . 324 patients completed the procedure , after which 1 ophthalmologist interpreted their digital images , spending about 5 minutes per patient , considering also the workstation and the administrative staff costs . out of the 115 patients referred for a complete examination , only 92 underwent an ophthalmology examination , in order to obtain an in - depth analysis of the disease ( in the third phase , it is important to note that the costs of all the materials and the drugs utilised by the clinicians for the complete eye examination are included in the first visit nine patients , who were suffering from a severe stage of pathology , received pharmacological and/or laser treatment , thus considering that both the human resources and the materials / equipment costs are included in the procedure costs , as detailed in table 2 . table 1 shows the categories of cost impacting on each phase and the total cost per phase . table 1 shows that the fourth phase absorbed the most part of the economic resources , presenting the economic evaluation of the treatment for 9 patients . in particular , three groups of patients suffering from rd or dme were considered : ( i ) 4 patients ( 45.5% ) received a therapeutic cycle of intravitreal injections with ranibizumab alone ; ( ii ) 3 individuals ( 30% ) received laser treatment as a support of ranibizumab injection ; and ( iii ) 24.5% ( 2 patients ) received dexamethasone , thus being consistent with literature and real - life data . treatment costs were related to a period of 12 months , in which clinicians administered to the patients a cycle composed of , on average , 3.61 ( ranibizumab ) or 1.3 ( dexamethasone ) intravitreal injections , based on the observation of the clinical pathway of the nine patients under investigation in the health authority of reference and consistent with other national and international literature evidence [ 28 , 29 ] . costs for an intravitreal injection were distinguished as follows : ( a ) the cost of the specific drug ( 644.73 for ranibizumab and 951.75 for dexamethasone ) and ( b ) the cost of the procedure carried out by the health authority of reference ( 290.00 , independently of the administered drug ) . patients performing laser therapy absorbed an additional cost equal to 81.28 for every single procedure . with reference to tables 1 and 2 , the amount of all the screening costs for patients within the area of ponzano was equal to 35,899.81 , treating the 340 patients who attended the programme . after the costs of the screening programme phases had been calculated , the prevention activity was then extended to the treviso catchment area ( considering the whole ulss 9 ) , in order to estimate the feasibility of the screening procedure in a larger population . if the response rate was the same as that in the pilot study ( 80% ) , 17.600 patients would be expected to attend the screening programme within the first year . considering an average work shift of 7 hours per day , a single nurse , completely devoted to the screening activities , could be able to perform about 7,000 procedures per year . with regard to the ophthalmologist and the administrative staff , assuming an average work shift of 8 hours , it emerges that the clinician could produce on average 9,600 medical reports per year , and the administrative staff could generate a maximum amount of 19,200 documents per year . as a result , 3 members of the paramedical staff ( nurses ) , 2 ophthalmologists , and 1 administrative would also be required to treat the abovementioned attending patients . investment in the required equipment would be made to cover the need of the expected diabetic citizens ; in particular , it should be allocated to 5 pieces of equipment , thus requiring an overall investment of 72,000 . considering the incidence of blindness as a result of dr ( 0.002% per year , taking into account the whole ulss 9 that has 419,728 inhabitants ) , 8 new cases every year would be expected if the screening programme was not applied . the screening programme conducted reported an effectiveness equal to 74.59% , calculated as the difference between individuals attending the screening programme and individuals who did not participate in the prevention activities , or patients who denied to go to the ophthalmologist for the complete eye examination . considering the treviso catchment area , 4,400 individuals did not attend the screening programme whereas 1,190 did not perform the in - depth visit , thus reaching a total amount of 5,590 individuals ( 25.41% of the overall invited population ) . in this view , in the innovative scenario , only 2 patients would develop blindness . literature evidence shows that the cost of blindness is approximately $ 18,670 ( i.e. , 16,803 ) , including only medical and direct costs . in addition , therapeutic treatment should be administered to 8% of dr patients , considering both the screening pathway and the no prevention strategy . in particular , 381 patients referring to the screening pathway scenario and 609 patients ( thus considering a dr occurrence rate equal to 34.60% within the diabetic population ) for whom no prevention the distribution of the treated population , considering the administered drug ( 74.5% for ranibizumab alone or with the support of laser therapy and 25.5% for dexamethasone [ 28 , 29 ] ) , as well as the treatment frequency within a 12-month time horizon , was assumed to be the same as the conducted study previously described . with reference to these data , the total costs of the two different pathways are presented in table 3 . in this view , it emerged that in both scenarios the more significant economic resources absorption was related to the treatment phase : at 12-month time horizon , the administration of ranibizumab with the support of laser therapy , ranibizumab alone , or dexamethasone required , on average , 3,667.80 , 3,374.38 , and 1,614.28 , respectively , per patient . table 4 reports the results of the sensitivity analyses , demonstrating an overall economic advantage in all the cases in which the prevention activity is implemented , thus ensuring the robustness of the bia result . in particular , it emerged that the economic benefits of the screening programme implementation are more sensitive to the higher number of screened individuals , in comparison with the decrease of the blindness events that occurred . more investments are required from the healthcare service , if the screening programme would cover a larger number of the target population : in particular , achieving a maximum attendance rate equal to 92% , the healthcare service would equally benefit of an economic saving of 2.38% , always resulting in the preferable solution . screening for diabetic retinopathy is important because the majority of patients who develop dr show no symptoms until diabetic macular edema and/or proliferative diabetic retinopathy are present , thus confirming that , in the early stages of the investigated disease , any notable symptoms affect the patients . although the beginning of the dr has an asymptomatic nature , the attendance rate reported in the proposed study was equal to 80% , thus demonstrating the effectiveness and the validity of the screening programme . an increase in the attendance rate could be achieved with an improvement and a diversification of the communication tools to inform patients concerning the importance of the eyes examinations and controls , thus enhancing their awareness on this field . in particular , literature reported that telehealth or telemedicine programmes may facilitate early dr diagnosis and timely treatment , preserving vision . the results of the study show the importance of a screening programme , from an economic point of view , one leading to a substantial saving of 271,543.32 ( 13.71% ) in comparison with the no prevention strategy . in addition , three - field colour , 45-degree , nonmydriatic images have demonstrated a sensitivity and specificity of 82% and 92% , respectively , in the diagnosis of dr , representing an effective tool in a screening setting . the references for the correct follow - up and management are the aao and national guidelines for dr [ 33 , 34 ] . however , despite the relevance of implementing a screening programme , in the real world , these recommendations are seldom adhered to . the increasing number of diabetic patients , in particular due to the population ageing , delays access to the next ophthalmologic examination . it has been estimated that only 50% of the known diabetic patients receive the recommended regular eye examinations , something that may be considered a real concern . eye screening offers the possibility of identifying the early signs of dr , thus preventing visual loss due to dme . as previously described , new technological screening procedures based on digital mydriatic and nonmydriatic fundus images present multiple advantages . online by specifically trained ophthalmologists in a deferred time , thus sparing and optimising resources . this could lead to a significant benefit with the decreasing of waiting lists , a phenomenon perceived by citizens as a serious problem of modern healthcare systems that compromises the coverage of their health needs . in addition , because of their easy and safe use , without the administration of drugs , nonmydriatic cameras can be placed in primary care settings in order to improve the access to care . a large - scale application of this screening strategy could spare unnecessary examinations for negative patients , thus preventing irreversible loss of visual acuity for persons affected by retinopathy , due to long waiting lists for eye examination . in particular , it emerged that the screening programme presented in the study has the potential to reduce the prevalence of blindness due to dr in the veneto region . with reference to this , the nonmydriatic fundus camera is not only effective but also cost - effective in the investigated greater area and leads to significant benefits for both the regional healthcare service and for the patients . vision loss is associated not only with a large increase of costs due to the management of this condition but also with the compromise of a patient 's quality of life , thus representing a significant social burden and requiring a future in - depth analysis . in fact , with the rapid ageing of the population , and considering that older adults could stay active and also productive in this specific part of their lives , blindness should be a public healthcare priority . healthcare regulators and policy makers will benefit from the implementation of adequate screening programmes , optimising effectiveness and resources allocation within this specific target population . in this view , the present study would represent the first attempt to extent the current theories and models into the practical context of the healthcare sector , extending the results of the study , from the italian setting to other european and international contexts . the results would contribute in the advancement and establishment of organisational and management models to be applied in the prevention sector , freeing up hospital resources for more severe cases of patients and reducing the economic and social burden of waiting lists for ophthalmic procedures . the early diagnosis and the process of taking charge of the patients could be considered an effective way to offer a better healthcare delivery to the patients , a clinical pathways optimisation but , in particular , a possible economic saving for the healthcare services . a future interesting contribution for policy makers could be also the definition of the best screening programme organisational setting , maximizing both effectiveness and efficiency , involving different healthcare professionals , such as general practitioners and pharmacists . new approaches to the screening development could reduce healthcare expenditure and increase the attendance rate for patients . in this view , further researches could be addressed to the proposal of a reimbursement tariff for the diabetic screening activities , with reference to different clinical setting in which the procedure could be performed , as well as the implementation of a multidimensional assessment ( through the health technology assessment tool ) in order to understand all the possible implications ( organisational , social , and equity aspects ) of the adoption of screening programmes , measured into the clinical practice , generating significant advancement to these findings and their robustness .

aims . the study aimed to present the experience of a screening programme for early detection of diabetic retinopathy ( dr ) using a nonmydriatic fundus camera , evaluating the feasibility in terms of validity , resources absorption , and future advantages of a potential application , in an italian local health authority . methods . diabetic patients living in the town of ponzano , veneto region ( northern italy ) , were invited to be enrolled in the screening programme . the no prevention strategy with the inclusion of the estimation of blindness related costs was compared with screening costs in order to evaluate a future extensive and feasible implementation of the procedure , through a budget impact approach . results . out of 498 diabetic patients eligible , 80% was enrolled in the screening programme . 115 patients ( 34% ) were referred to an ophthalmologist and 9 cases required prompt treatment for either proliferative dr or macular edema . based on the pilot data , it emerged that an extensive use of the investigated screening programme , within the greater treviso area , could prevent 6 cases of blindness every year , resulting in a saving of 271,543.32 ( 13.71% ) . conclusions . fundus images obtained with a nonmydriatic fundus camera could be considered an effective , cost - sparing , and feasible screening tool for the early detection of dr , preventing blindness as a result of diabetes .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

among all the eye pathologies , diabetic retinopathy ( dr ) is currently one of the leading causes of blindness in the working - age population in developed countries [ 13 ] . every year , up to 1% of diabetic patients develop serious ocular complications leading to both poor quality of life and a socioeconomic burden . with regard to the italian setting , a study conducted in the veneto region , using clinical fundus examination , confirmed literature data with a prevalence of dr of 26.6% . in europe , compared with 2007 , there is expected to be an increase of 10 million persons with a prevalence of diabetes below 10% . as the prevalence of diabetes is expected to rise in the future , an associated increase in dr cases should also be expected . however , recently , data suggest that the prevalence of dr may decrease , due to the intensification of the screening programmes and better control of risk - factors , thus underlying their effectiveness and the significance . in fact , from an economic point of view , the annual cost per patient affected by dr is approximately twice as high as those patients with diabetes only : thus , the implementation of effective and recognized screening programmes could represent cost - effective strategies [ 12 , 13 ] , one useful to narrow both the economic and the social burden of dr . despite the implementation of successful national screening programmes worldwide , the lack of diffuse screening activities for dr in italy is delaying the diagnosis and prompt treatment of dr . for these reasons , vision impairments as a result of dr should be considered easy to prevent , and the development of systematic programmes of screening should represent an urgent and primary healthcare need . moving on from these premises , since dr should be one of the major priorities for the healthcare services , the present study aimed at investigating the feasibility related to the introduction of a specific and accurate screening programme in the catchment area of treviso ( defined as ulss 9 ) , in comparison with the no prevention strategy , including also the costs averted to blindness , in terms of the validity of the intervention and the direct costs absorbed ( efficiency ) by the regional healthcare service . in order to achieve the previously mentioned objective , a perspective study for the implementation of a screening programme devoted to dr was planned to be performed between september and december 2012 , after receiving the approval from ethic committee of the local health authority . the involvement of different healthcare professionals in the screening programme could influence the resources absorption and the optimisation of the screening pathway [ 12 , 13 , 1618 ] . the screening involved all types ( type 1 and type 2 ) of diabetic patients living in the area of ponzano ( treviso ) that has 5.000 inhabitants and is part of one of the local health authorities of the veneto region : the ulss 9 of treviso . in particular , the following patients were invited to attend the screening programme : ( i ) outpatients going to a hospital specialist visit to the diabetologists , or first visit to ophthalmologist ; ( ii ) patients going to the territorial pharmacist , in order to retrieve the specific diabetes drugs ; and ( iii ) patients referring to the general practitioners , with a diagnosis of diabetes . initially , a personal letter of invitation was sent to all the diabetic target population ( n = 498 ) to be enrolled in the screening programme . the three fundus images encompassed the following retinal fields : field 1 centred on the macula , field 2 centred on optic disc , and field 3 midperipheral superior - temporal field . all images were electronically transmitted to the reading centre and stored in an online secured database called eye knowledge network for the second step , online examination . dr and diabetic macular edema ( dme ) were graded in accordance with the international classification proposed by the american academy of ophthalmologists . when the quality of the images was inadequate for the clinical evaluation and when fundus images were graded as positive , the patients were referred for further ophthalmologic examination . fundus images were graded as negative if no dr or nonsight threatening dr was detected . a report , with the results of the screening and the correct follow - up timetable for the negative screened population , was sent to the patient 's general practitioner within 1 month after the screening . from a methodological point of view , in order to quantify the impact of the introduction of screening programme in the clinical practice , both an activity based costing ( abc ) analysis and a budget impact analysis ( bia ) were conducted . the abc [ 21 , 22 ] is useful for the enhancement of the average costs related to each phase of the screening pathway . in particular , the main objective of the abc is the measurement of the costs and the performance activities , taking into account also the related human and materials resources for the proper development of the procedure . , individuals involved in the different phases of the screening programme , such as administrative staff , nurses , and ophthalmologists ) ; ( ii ) materials and equipment ; ( iii ) pharmacological and/or laser treatment . the abc did not take into consideration the costs related to the delivery of the invitation letter : different technologies could be implemented in order to carry out this task ( telephonic invitation , e - mail contact , personal contact , or letter ) , differing for the related economic value . a bia allows the prediction of the potential financial impact of a new technology adoption , into a healthcare system ; influencing , in a positive or in a negative manner , the healthcare expenditure and considering both a specific point of view and a determined time horizon . in this view , two scenarios were simulated , thus comparing the so - called do nothing strategy with the implementation of a proper screening programme . in particular , in both scenarios , the occurrence and the related cost of blindness were taken into consideration as direct healthcare costs . if necessary , economic values were reported in euros , considering the 2015 inflation rate , using the consumer price index for healthcare expenditure , thus making economic measures comparable , and being based on the same year of reference . in order to ensure the robustness of the result , a sensitivity analysis was carried out . in particular , both the attendance rate to the screening programme and the percentage of patients undergoing the complete eye examination were modified , thus understanding if significant changes in the feasibility of the programme occur . the local health authority involved declared that it could reach about the 80%85% if collaboration with community - based organisations , as well as greater information activities of dr risks among citizens , is implemented . moving on from these premises , 498 individuals were identified as being diabetic patients within the area of ponzano ( that has 5,000 inhabitants ) ; thus , it emerged that the prevalence of the diabetes in the investigated town was around 10% . out of the 498 diabetic patients originally invited to enroll in the screening programme , 340 accepted to be evaluated ( 68% ) , although it was possible to confirm a response rate equal to 80% since 57 patients did not attend the screening programme because they had already undergone a complete eye examination that showed no signs of dr development . 324 patients ( 95% ) successfully completed the procedure , though in 16 cases ( 5% ) images were noncaptured due to either systemic conditions of the patients , insufficient mydriasis , or other technical reasons related to the fundus camera . the quality of digital images was adequate for the interpretation in 260 patients ( 80% ) , although it was ungradable in 64 cases ( 20% ) due to insufficient dilation , media opacity , poor fixation , or the absence of one of the captured fields . based on the entire screened population , 115 patients ( 34% ) were referred to an ophthalmologist . of these , 16 ( 14% ) were patients from whom it was impossible to obtain images , 64 ( 56% ) had no evaluable images , and 35 ( 30% ) were positive cases . of the previously mentioned 115 patients , 92 ( 91% ) underwent a full ophthalmological examination , giving the following results : in 67 cases ( 73% ) , dr was not detected , though 25 patients ( 27% ) presented signs of dr . in particular , 24% ( n = 6 ) had mild nonproliferative dr , 52% ( n = 13 ) had moderate nonproliferative dr , 12% ( n = 3 ) had preproliferative dr , and 12% ( n = 3 ) had proliferative dr . in 9 cases ( 36% ) , prompt treatment with intravitreal injections and/or laser photo - coagulation was required for proliferative and severe preproliferative retinopathy or macular edema . it should be noted that the veneto region 2015 tariffs were taken into account in order to investigate the value of the complete screening programme , thus also including the entire cycle of intravitreal injections . with reference to the abovementioned distribution of the patients , the economic resources absorption , with regard to the whole population screened pathway , was divided into four distinct and logical phases:340 patients attended the screening programme , involving 1 healthcare professional ( nurse ) who spent 12 minutes per patient and considering the equipment amortization.324 patients completed the procedure , after which 1 ophthalmologist interpreted their digital images , spending about 5 minutes per patient , considering also the workstation and the administrative staff costs.out of the 115 patients referred for a complete examination , only 92 underwent an ophthalmology examination , in order to obtain an in - depth analysis of the disease ( in the third phase , it is important to note that the costs of all the materials and the drugs utilised by the clinicians for the complete eye examination are included in the first visit reimbursement tariff).nine patients , who were suffering from a severe stage of pathology , received pharmacological and/or laser treatment , thus considering that both the human resources and the materials / equipment costs are included in the procedure costs , as detailed in table 2.it was first necessary to evaluate the costs related to the human resources involved in the screening programme . 340 patients attended the screening programme , involving 1 healthcare professional ( nurse ) who spent 12 minutes per patient and considering the equipment amortization . out of the 115 patients referred for a complete examination , only 92 underwent an ophthalmology examination , in order to obtain an in - depth analysis of the disease ( in the third phase , it is important to note that the costs of all the materials and the drugs utilised by the clinicians for the complete eye examination are included in the first visit nine patients , who were suffering from a severe stage of pathology , received pharmacological and/or laser treatment , thus considering that both the human resources and the materials / equipment costs are included in the procedure costs , as detailed in table 2 . table 1 shows that the fourth phase absorbed the most part of the economic resources , presenting the economic evaluation of the treatment for 9 patients . in particular , three groups of patients suffering from rd or dme were considered : ( i ) 4 patients ( 45.5% ) received a therapeutic cycle of intravitreal injections with ranibizumab alone ; ( ii ) 3 individuals ( 30% ) received laser treatment as a support of ranibizumab injection ; and ( iii ) 24.5% ( 2 patients ) received dexamethasone , thus being consistent with literature and real - life data . treatment costs were related to a period of 12 months , in which clinicians administered to the patients a cycle composed of , on average , 3.61 ( ranibizumab ) or 1.3 ( dexamethasone ) intravitreal injections , based on the observation of the clinical pathway of the nine patients under investigation in the health authority of reference and consistent with other national and international literature evidence [ 28 , 29 ] . costs for an intravitreal injection were distinguished as follows : ( a ) the cost of the specific drug ( 644.73 for ranibizumab and 951.75 for dexamethasone ) and ( b ) the cost of the procedure carried out by the health authority of reference ( 290.00 , independently of the administered drug ) . with reference to tables 1 and 2 , the amount of all the screening costs for patients within the area of ponzano was equal to 35,899.81 , treating the 340 patients who attended the programme . after the costs of the screening programme phases had been calculated , the prevention activity was then extended to the treviso catchment area ( considering the whole ulss 9 ) , in order to estimate the feasibility of the screening procedure in a larger population . if the response rate was the same as that in the pilot study ( 80% ) , 17.600 patients would be expected to attend the screening programme within the first year . considering an average work shift of 7 hours per day , a single nurse , completely devoted to the screening activities , could be able to perform about 7,000 procedures per year . with regard to the ophthalmologist and the administrative staff , assuming an average work shift of 8 hours , it emerges that the clinician could produce on average 9,600 medical reports per year , and the administrative staff could generate a maximum amount of 19,200 documents per year . as a result , 3 members of the paramedical staff ( nurses ) , 2 ophthalmologists , and 1 administrative would also be required to treat the abovementioned attending patients . investment in the required equipment would be made to cover the need of the expected diabetic citizens ; in particular , it should be allocated to 5 pieces of equipment , thus requiring an overall investment of 72,000 . considering the incidence of blindness as a result of dr ( 0.002% per year , taking into account the whole ulss 9 that has 419,728 inhabitants ) , 8 new cases every year would be expected if the screening programme was not applied . the screening programme conducted reported an effectiveness equal to 74.59% , calculated as the difference between individuals attending the screening programme and individuals who did not participate in the prevention activities , or patients who denied to go to the ophthalmologist for the complete eye examination . considering the treviso catchment area , 4,400 individuals did not attend the screening programme whereas 1,190 did not perform the in - depth visit , thus reaching a total amount of 5,590 individuals ( 25.41% of the overall invited population ) . in addition , therapeutic treatment should be administered to 8% of dr patients , considering both the screening pathway and the no prevention strategy . in particular , 381 patients referring to the screening pathway scenario and 609 patients ( thus considering a dr occurrence rate equal to 34.60% within the diabetic population ) for whom no prevention the distribution of the treated population , considering the administered drug ( 74.5% for ranibizumab alone or with the support of laser therapy and 25.5% for dexamethasone [ 28 , 29 ] ) , as well as the treatment frequency within a 12-month time horizon , was assumed to be the same as the conducted study previously described . in this view , it emerged that in both scenarios the more significant economic resources absorption was related to the treatment phase : at 12-month time horizon , the administration of ranibizumab with the support of laser therapy , ranibizumab alone , or dexamethasone required , on average , 3,667.80 , 3,374.38 , and 1,614.28 , respectively , per patient . in particular , it emerged that the economic benefits of the screening programme implementation are more sensitive to the higher number of screened individuals , in comparison with the decrease of the blindness events that occurred . more investments are required from the healthcare service , if the screening programme would cover a larger number of the target population : in particular , achieving a maximum attendance rate equal to 92% , the healthcare service would equally benefit of an economic saving of 2.38% , always resulting in the preferable solution . screening for diabetic retinopathy is important because the majority of patients who develop dr show no symptoms until diabetic macular edema and/or proliferative diabetic retinopathy are present , thus confirming that , in the early stages of the investigated disease , any notable symptoms affect the patients . although the beginning of the dr has an asymptomatic nature , the attendance rate reported in the proposed study was equal to 80% , thus demonstrating the effectiveness and the validity of the screening programme . an increase in the attendance rate could be achieved with an improvement and a diversification of the communication tools to inform patients concerning the importance of the eyes examinations and controls , thus enhancing their awareness on this field . the results of the study show the importance of a screening programme , from an economic point of view , one leading to a substantial saving of 271,543.32 ( 13.71% ) in comparison with the no prevention strategy . in addition , three - field colour , 45-degree , nonmydriatic images have demonstrated a sensitivity and specificity of 82% and 92% , respectively , in the diagnosis of dr , representing an effective tool in a screening setting . however , despite the relevance of implementing a screening programme , in the real world , these recommendations are seldom adhered to . the increasing number of diabetic patients , in particular due to the population ageing , delays access to the next ophthalmologic examination . it has been estimated that only 50% of the known diabetic patients receive the recommended regular eye examinations , something that may be considered a real concern . eye screening offers the possibility of identifying the early signs of dr , thus preventing visual loss due to dme . as previously described , new technological screening procedures based on digital mydriatic and nonmydriatic fundus images present multiple advantages . in addition , because of their easy and safe use , without the administration of drugs , nonmydriatic cameras can be placed in primary care settings in order to improve the access to care . in particular , it emerged that the screening programme presented in the study has the potential to reduce the prevalence of blindness due to dr in the veneto region . with reference to this , the nonmydriatic fundus camera is not only effective but also cost - effective in the investigated greater area and leads to significant benefits for both the regional healthcare service and for the patients . vision loss is associated not only with a large increase of costs due to the management of this condition but also with the compromise of a patient 's quality of life , thus representing a significant social burden and requiring a future in - depth analysis . in fact , with the rapid ageing of the population , and considering that older adults could stay active and also productive in this specific part of their lives , blindness should be a public healthcare priority . in this view , the present study would represent the first attempt to extent the current theories and models into the practical context of the healthcare sector , extending the results of the study , from the italian setting to other european and international contexts . the results would contribute in the advancement and establishment of organisational and management models to be applied in the prevention sector , freeing up hospital resources for more severe cases of patients and reducing the economic and social burden of waiting lists for ophthalmic procedures . the early diagnosis and the process of taking charge of the patients could be considered an effective way to offer a better healthcare delivery to the patients , a clinical pathways optimisation but , in particular , a possible economic saving for the healthcare services . a future interesting contribution for policy makers could be also the definition of the best screening programme organisational setting , maximizing both effectiveness and efficiency , involving different healthcare professionals , such as general practitioners and pharmacists . in this view , further researches could be addressed to the proposal of a reimbursement tariff for the diabetic screening activities , with reference to different clinical setting in which the procedure could be performed , as well as the implementation of a multidimensional assessment ( through the health technology assessment tool ) in order to understand all the possible implications ( organisational , social , and equity aspects ) of the adoption of screening programmes , measured into the clinical practice , generating significant advancement to these findings and their robustness .

according to the world health organization , there is no health without mental health .1 mental disorders make up an important independent contribution to the burden of non - communicable diseases worldwide . in fact , neuropsychiatric conditions account for up to one quarter of all disability - adjusted life years ( ie , the sum of years lived with disability and years of life lost).1 a psychiatrist s main responsibility is to render accurate diagnoses that guide the selection of appropriate medication for severely mentally ill patients.2 a crucial problem in psychiatry , which affects both research and clinical work , is that in practice it is difficult to strictly apply the scientific method to the investigation of human mental illness , since the evaluations should ( at least ) include analyses of both the mind and the brain when assessing normal and aberrant behaviors.3 a consequence of this complexity is that , even if psychopathological states can effectively be alleviated by a combination of psychotherapy and neuropharmacological interventions , the relapse rate for several psychiatric disorders is tremendously high.4 for example , the relapse rate for manic episodes in bipolar disorders was found to be 50% at 1 year , and 70% at 5 years.5 similarly , the relapse rate following a first episode of schizophrenia was reported to be 35% at 18 months , and 74% at 5 years.6 in other words , there is still much to learn in order to fill this existing gap between a partial recovery , which allows patients to return home , and the achievement of a complete recovery.7 the complexity surrounding the treatment of mental illness is also illustrated by alcohol dependence , which constitutes one of the most severe and widespread public health problems . indeed , it is estimated that 3%8% of all deaths and 4% of all disability - adjusted life years worldwide are attributable to alcohol.1 without intervention , detoxification alone does little to prevent an alcoholic s subsequent relapse . during clinical trials , placebo control groups showed a relapse rate of up to 85% , even if hospitalized until complete remission of the physical withdrawal symptoms.8 similarly , there is little evidence supporting that antidepressants alter in any way the risk factors that lead to relapse and recurrence . consequently , most patients with chronic or recurrent depression are encouraged to stay on medication indefinitely.9 in addition , while it has been well established that maintenance treatment with antipsychotic medication decreases relapse rates , a substantial proportion of patients suffering from schizophrenia either relapse despite taking medication or have trouble adhering to antipsychotic treatment.10 all of the above fuels the current strong interest for the development of alternatives to psychotherapy and medication.11 in recent years , we have seen the emergence of several intervention strategies aimed at improving psychiatric treatment , such as multisystemic,12 cognitive behavioral,13 or mindfulness14 therapies . interventions is evidently not to discredit existing treatment methods , but to provide a complementary set of tools to be used by clinicians to improve current patient assessment . therefore , the purpose of this paper is to present four key arguments that support the use of event - related potential ( erp)-based cognitive assessment in combination with psychotherapy and medication to develop more efficient psychiatric therapies . it is now largely accepted that psychiatric disorders are brain disorders,15 and brain - related tools , such as electroencephalography ( eeg)16 or repetitive transcranial magnetic stimulation ( rtms),17 have already proven their value for the management of psychiatric diseases . for instance , the combined use of rtms and eeg enables the noninvasive evaluation of functional connections between brain areas , and provides a tool to investigate cortical activity.18 thus , a non - invasive input ( tms ) known spatial and temporal characteristics with directional and precise chronometric data can be applied to the study of the brain s local reactivity and the interactions between different brain regions.19 this emerging technique allows for instance to observe that , during an 8 to 10 minute long eeg session with single - pulse tms over the right premotor cortex , patients suffering from schizophrenia present reduced evoked gamma oscillations in the frontal cortex , reinforcing the hypothesis of an intrinsic dysfunction of the frontal thalamo - cortical circuit.20 in this paper , we focus on erps , since by allowing the evaluation of the entire information processing stream , they can help pinpoint the specific neurocognitive functions that should be rehabilitated in each patient through specific and individualized cognitive remediation procedures . the aim of this paper is not to provide an exhaustive review of the literature pertaining to erps and psychiatric disorders , but to systematically illustrate four reasons why we think that it is now time to incorporate cognitive erps as a requisite step in clinical evaluations and use them to direct the development of an individualized therapy for mentally ill patients . this approach should also include a cognitive neuropsychological rehabilitation step , as an integrative part of the whole process that is classically only based on psychotherapy and medication . we will discuss the advantages , limitations , and perspectives associated with the clinical use of erps and we will propose new ways in which this technique can be effectively incorporated into patient care . cognitive deficits have gained considerable importance in the field as critical features of mental illness , and it is now believed that they might represent valid therapeutic targets.21 indeed , several studies have provided consistent evidence that mental illness involves significant cognitive impairment . for instance , virtually all areas of cognition , such as perception , attention , and memory , are known to be impaired in the schizophrenic individual,22 as well as in patients of other psychiatric afflictions , such as euthymic bipolar disorder,23 alcohol - dependence,24 or attention - deficit / hyperactivity disorder ( adhd).25 in schizophrenic patients , literature data reports a substantial decrease in cognitive function of about 1.01.75 standard deviations below the normal mean.26 moreover , some variability in the cognitive domain was also reported in euthymic bipolar patients ( large effect sizes [ d0.8 ] for executive functions and small effect sizes [ 0.2db0.5 ] for sustained attention).23 however , a proven connection between the onset of a mental illness and the presence of cognitive impairment would be needed in order to establish the requirement for cognitive therapeutics.27 additionally , while there is overwhelming evidence that medication can reduce the severity of clinical symptoms , cognitive gains are either poor or undetectable.21 thus , it is currently accepted that cognitive impairment and symptomatic manifestations of mental illness require separate therapeutic approaches.28 cognitive retraining procedures ( crps ) have brought new applications to psychiatric rehabilitation.29 cognitive remediation is a behavioral intervention designed to improve cognition in patients who have suffered a cognitive decline.30 the major elements of psychiatric rehabilitation focus on highlighting the patient s strengths , improving vocational / social competencies , working with environmental facts of an individual s life , and instilling a sense of mastery and hope.31 from this perspective , cognitive retraining techniques range from assistive or prosthetic devices ( including computers , diaries , and/or lists ) to approaches that focus on enhancing the patient s efficiency , ( eg , mnemonic strategies).32 many studies have demonstrated that the use of crps can enhance cognitive performance in diverse psychiatric diseases,33 and that these improvements are stable up to at least 6 months after the treatment is discontinued.34 additionally , crp - related cognitive gains have been proved to translate to improvements in real world activities,35 with greater impact than any drug treatment.21 for example , in a single case study involving a schizophrenic patient,35 levaux et al demonstrated that the usage of an attention training technique leads to the following beneficial effects : i ) at the cognitive level , a specific improvement in selective attention , attention switching , and resistance to distractive interference ; and ii ) at the clinical level , a reduction in intrusive thoughts in daily life and a general decrease in symptoms stable up to at least 6 months . overall , cognitive remediation is a therapy that engages patients in learning activities that enhance the neurocognitive skills relevant to their chosen recovery goals.36 a number of approaches to remediating cognition in schizophrenia have been developed and studied over the last 20 years . at the time of writing five of these , while differing in focus , determined that it has moderate to large effect sizes;3741 the remaining one didnt.42 moreover , with the spread of crp s application to patients suffering from schizophrenia , positive outcomes with crp for other psychiatric disorders , such as unipolar depression,43 social phobia,44 adhd,45 and alcohol dependence46 have begun to appear in the literature . overall , these data are consistent with the general idea that recognizing the nature of the cognitive impairment involved in a specific mental illness will be very useful for the development of treatment and rehabilitation strategies.29 although the usefulness of crps for the treatment of psychiatric disorders is gradually becoming more accepted , several questions regarding how to use them in clinical practice still remain to be resolved . among other concerns , it is unknown whether cognitive impairments will be detected in all patients or just on a case - by - case basis.27 also , the assignment of impairment requires the comparison of patients results to those of a standard cognitive control ; however , normal values may be difficult to define , mainly because of the healthy individuals variability in cognitive function . in fact , it appears that , for instance , around 20% of schizophrenics generally perform within the normal range , only showing subtle evidence of impairment in specific cognitive domains.47 indeed , the human mind can be defined by several cognitive domains , such as attention , memory , language , or executive functions , any of which may prove to be altered.48 this requires rehabilitation specialists ( usually neuropsychologists ) to thoroughly assess the cognitive status of each domain in order to decide the appropriate rehabilitative treatment . this process may be complicated by two factors : i ) the fractionation of each cognitive domain implies the involvement of various neural networks ( eg , executive function refers to a wide range of cognitive processes [ including problem - solving , flexibility , inhibition , planning , and decision - making ] , and a patient s performance in one executive function may have little or no predictive value for how the patient will perform in another);49 and ii ) time pressure is an important feature of the clinical examination , because cognitive screening needs to identify genuine impairment in as little time as possible , using an easily administered instrument.50 in the following arguments , we will describe how and why we believe that cognitive erps can aid in the cognitive screening process . a general screening of main cognitive functions can be achieved by performing rapid tasks , which are easily implementable in clinical practice and have the potential to index the cognitive areas that require further assessment and/or rehabilitation by neuropsychologists . the main goal of cognitive psychology has been to define the different stages that are required to achieve a final performance for each human cognitive function.51 indeed , every cognitive function ( eg , recognizing an emotion on a face , recalling an event , reading a text , or making a decision ) is associated with various cognitive stages . in fact , advances in brain imaging techniques ( namely positron emission tomography [ pet ] and functional resonance magnetic imaging [ fmri ] ) have revealed that each of the serial cognitive stages is implemented within separate neural processes in order to achieve normal function.52 the fact that healthy cognitive functions are implemented within distributed neural networks has important implications for the understanding of mental disorders . firstly , the use of advanced imaging methods enabled the study of the neurobiology of mental disorders , confirming the fact that mental disorders have a neurobiological component ( ie , they are not only functional or psychogenic conditions).53 secondly , imaging technology contributed insight into the pathological mechanisms of mental disorders , demonstrating that a functional alteration may result from an isolated brain lesion ( eg , after a stroke ) or deficient interactions between different brain regions ( functional connectivity).54 in this regard , imaging analysis can also provide a better understanding of the mechanisms of action of psychopharmaceuticals , potentially aiding in the search for novel therapeutic targets.53 the study of blood flow ( fmri ) or glucose metabolism ( pet ) in certain areas of the brain during activation tasks enables the identification of the areas of the brain that are activated or deactivated during specific cognitive tasks . therefore , by comparing patients to controls , we can observe which brain activities are deficient . however , from a purely cognitive perspective , these data have an important limitation , residing on the fact that brain images are averaged on seconds . thus , it is impossible to relate specific brain activations to the different cognitive stages involved in the task.55 in other words , the excellent anatomical resolution of brain imaging techniques ( 34 mm for fmri ) might allow the visualization of the brain network involved in a certain task , but its coarse temporal resolution ( 12 seconds ) impedes the determination of the activation sequence . for this purpose , we should recur to electrophysiological tools , and more precisely , to cognitive erps.56 erps , which are derived from electroencephalography , are highly sensitive and have the potential to monitor brain lectrical activity with a fine temporal resolution ( on the order of milliseconds ) . thus , when healthy individuals perform a cognitive task , it is possible to observe the various electrophysiological components representing the cognitive stages utilized to achieve performance.56 figure 1 illustrates the different electrophysiological components representing the various cognitive stages needed to perform a simple task . another highly valuable aspect of cognitive erps is that they can permit the identification / detection of the electrophysiological component(s ) consistent with the onset of a dysfunction , allowing the inference of impaired cognitive stages.56 in other words , by using a well characterized task and analyzing which erps show decreased amplitude and/or delayed latency compared to normal values , it is possible to deduce which erp component is responsible for the cognitive deficit.56 figure 2 illustrates the well - known p300 component , a late , long - lasting parietal positive wave , which is decreased and delayed in alcoholic patients compared to matched controls . however , while p300 deficit is common in many psychiatric afflictions , there is also an abundance of literature reporting ; for instance , many earlier erp components , such as the mismatch negativity or the sensory gating p50 , but also later ones , such as the n400 indexing language semantic processes , are affected in alcoholic,57 as well as in schizophrenic58 patients . this suggests that during illness , various cognitive stages of the information - processing stream may be affected , and that the recorded erp components may be considered as biological markers of the disease , indexing specific pathophysiological mechanisms that may or may not recover with the illness remission.59 obviously , erps also suffer from some limitations . their spatial resolution is very weak , so that inferences can only be made on the neural generators of the observed components , and parameters of erps ( latency and amplitude ) are subject to large inter - individual differences , so that the collection of normative data may prove fastidious.56 nevertheless , cognitive erps are advantageous , because they allow the assessment of the different stages used in the information - processing stream during performance of a task . therefore , how and why could this technique be useful to psychiatrists in their daily clinical practice ? the most widely used and studied erp component is the p300 component.60 this component is of particular interest because it is functionally related to various complex cognitive processes , such as decision - making and cognitive closure phenomena , which are associated with different types of response - related cognitive activities.61 many studies investigating p300 in dementia , schizophrenia , depression , alcoholism , drug addiction , anxiety , or personality disorders have described disturbances in recorded latency and amplitude values of this component compared to healthy individuals . these differences have been associated with deficits in behavioral performance and can indicate the severity of a clinical state , as well as its possible evolution.62 for example , if we consider the literature on the ability to recognize emotional facial expressions ( efe ) in psychiatric populations , two main points emerge : i ) at a behavioral level , many patients with psychiatric diseases display a deficit in emotional recognition , which can be indexed by decreased performance rates and longer response latencies compared to controls;63 and ii ) at an electrophysiological level , this behavioral deficit is associated with a decreased and delayed p300 component.64 however , an important question in the field has revolved around the events occurring in the information - processing stream prior to the p300 component ( ie , before the response - related stages ) . by using an emotional oddball task,64,65 we demonstrated that even if different psychopathological populations displayed a similar behavioral deficit in efe recognition , this deficit originated at different cognitive levels , as indexed by cognitive erps . indeed , while patients suffering from alcohol dependence or schizophrenia displayed altered erp components affecting all information - processing streams ( from perceptive - visual p100 , to attention n2b and response - related p300 components),66,67 depressed people exhibited only a disturbed p300 component ( with intact earlier components).68 on the other hand , ecstasy users presented differences starting at the level of attention ( n2b).69 in other words , similar patterns at the behavioral level can result from different disturbances in cognitive processes from one population to another . this point has great clinical relevance , because it suggests that a similar pattern of deficit may be attributable to different neurocognitive disturbances , supporting the notion that similar behavioral deficits should be differently rehabilitated . from a clinical point - of - view , it might not be a surprise that there are neurocognitive differences linked to different neuropsychiatric afflictions , especially considering the specificity of the pathophysiological mechanisms associated with distinct mental illnesses . however , interestingly , we also observe that such neurocognitive differences can be seen between individuals within a specific population that have a certain type of behavioral deficit . for instance , among anxious patients , rossignol et al showed that those suffering from generalized anxiety disorder only displayed p300 differences when confronted with emotional oddball task,70 whereas people with social anxiety showed earlier differences starting from the perceptive p100 component.71 this suggests that social anxiety and trait anxiety should be considered separately and treated independently.72 this realization is of great relevance , because it suggests that patients who display a similar group of symptoms and belong to a closed psychiatric category might have deficits that originate from different levels of the cognitive process . overall , the monitoring of erps is useful for identifying the cognitive origin of a behavioral deficit . by temporally analyzing the information - processing stream , the root causes of behavioral problems current data suggest that such differences not only exist between the various neuropsychiatric afflictions , but also among different patients categorized in the same psychiatric category . therefore , the next important question is : how can such information be useful for psychiatrists ? thanks to an optimal temporal resolution , erps allow the assignment of a behavioral deficit to its cognitive origin . this feature of erp analysis is of great clinical relevance , since it suggests that each patient s behavioral deficit can be specifically treated . however , even if the importance of indexing cognitive disturbances is well - established in psychiatric clinical settings , the manner with which to efficiently perform cognitive analyses is a matter of debate . this is mainly due to cognition fractionation , which means that a global assessment of all cognitive functions may take several hours to perform , and this is incompatible with both patients statuses and consultation pressures . nevertheless , erp analysis is non - invasive , painless , and cheap . in addition , erps provide the most informative profile for psychiatrists regarding the cognitive deficits presented by their patients , especially when examining erp components that are well - characterized in terms of eliciting stimuli , technical recording methods , and quantification . these defined erps are easily implementable in clinical practice and are operationally related to the neurocognitive processes they reflect.73 we would especially like to emphasize that the most useful components span different stages of the information processing stream , including p50 , mismatch negativity ( mmn ) , p300 , nogo p300 , and error - related negativity ( ern ) . the p50 component is the earliest to be observed ( around 50 ms ) and the smallest in amplitude of the auditory erps . the p50 sensory gating effect refers to an amplitude reduction in p50 upon exposure to a second stimulus in a pair of identical stimuli presented with a short inter - stimulus interval . p50 gating is one of the earliest , measurable brain sensory processing steps that are linked to screening - out and attention filtering mechanisms of redundant incoming information.74 mmn ( also called n2a ) is an erp component with peak latency around 150 ms after stimulus and maximal amplitude at the frontal scalp locations . mmn is usually evoked by a physically deviant auditory stimulus that occurs in a series of frequent standard stimuli . it is believed to reflect cortical information processing at the earliest level of the sensory cortex ; however , recent findings suggest that the transient auditory sensory memory representation underlying the mmn is facilitated by a long - term memory representation of the corresponding stimulus.75 the p300 component ( or p3 ) is a long - lasting positive component that occurs between 300 and 700 ms after stimulation . p3 is thought to reflect premotor processes , such as short - term memory updating or cognitive closure mechanisms.58 the nogo p300 has been identified as one of the markers for response inhibition,76 which involves the activation of the executive system of the frontal lobes.77 the neural basis for this executive system is believed to be a distributed circuitry , which involves the following : the prefrontal areas and anterior cingulated gyrus;78 the orbitofrontal cortex;79 the ventral frontal regions;80 the parietal , dorsal , and ventral prefrontal regions;81 and the premotor and supplementary motor areas . finally , ern represents a neural measure of error processing that peaks 50 ms after subjects make mistakes . this component is linked to the subject s ability to evaluate his actions as worse than expected , and constitutes a learning signal that can subsequently be used to adjust behavior.82 it has been suggested that the analysis of a weighted combination of these electrophysiological features may provide greater diagnostic power than any single endophenotype alone.83 for instance , price et al84 compared and contrasted four electrophysiological components , analyzing their covariance on a single cohort of schizophrenic patients , family members , and controls . their findings revealed that the use of the electrophysiological battery provided novel information on the characteristics of the different groups . in particular , they highlighted the heterogeneity of electrophysiological features among the groups and demonstrated that the combined analysis could serve to minimize the impact of such heterogeneity.84 we propose the use of p50 , mmn , p300 , nogo p300 , and ern to facilitate the indexing of cognitive stages affected in psychiatric patients by assessing , respectively , the attention - filtering , early perceptive discriminative , response - related decisional , inhibitory , and self - action evaluation processes . indeed , neurocognitive processes ( indexing attention , executive functions , and meta - cognition ) have been linked to psychopathology.85 once cognitive disturbances are characterized through erp screening for individual patients , psychiatrists will be able to orient the cognitive treatment ( individually or in groups that present homogeneous patterns of cognitive deficits ) . more precisely , specific cognitive retraining procedures could be used to target deficits and increase cognitive efficiency , which has already been shown to reduce clinical symptoms.86,87 in this regard , there are currently many crps available , including those directed at attention mechanisms ( increasing or decreasing the attention resources devoted to a specific cue),88 for which compelling evidence has already been gathered in anxiety disorders . indeed , behavioral and erp studies have shown that attention training can alter threat bias , influence vulnerability to stress , and reduce clinical anxiety symptoms by modulating top - down processes of attention control rather than processes of early attention orienting.89 in the same vein , there are also available crps increasing organization , goal achievement,90 cognitive control ( ie , inhibitory skills),46 and even self - monitoring.91 this strategy is certainly useful for some patients , but the relapse rate remains tremendously high for several psychiatric disorders . thus , in psychiatry there is currently a recognized need for alternatives to psychotherapy and medication.11 cognitive deficits have gained considerable importance in the field as critical features of mental illness.21 therefore , in the present paper , we suggest that a possible way to increase the efficiency of psychiatric treatment is to include erps and crps in clinical practice , combining these techniques with psychotherapy and medication . however , this would require a significant collaboration between psychiatry departments , clinical neurophysiology laboratories , and neuropsychological rehabilitation centers . complete erp screening to his / her patients , which is easy to perform and will not be time - consuming if only the main cognitive domains are analyzed ( attention , execution , and self - monitoring ) . the basic purpose of these erp screening tests will be to indicate the likelihood of genuine cognitive impairment , which can be inferred through the comparison of the patient s results with reference controls . thus , a borderline score or a very impaired score ( along with supporting history ) might lead the psychiatrist to order a more specialized assessment of cognition ( eg , by neuropsychologists ) , and thus thoroughly identify which cognitive domain(s ) to rehabilitate . indeed , crps have been proven to have a positive impact on daily quality of life92 and on clinical symptoms.86,87 hence , psychiatric treatment data from neurophysiologists ( erps ) could help neuropsychologists decide which cognitive processes to rehabilitate through crps in order to enhance the daily quality of life and soften the clinical symptoms of individual patients . ultimately , based on the fact that the long - lasting , positive effects of crps on clinical symptoms have already been demonstrated,35,87 we are convinced that the combination of this cognitive procedure ( involving erps and crps ) with psychotherapy and medication could lead to decreased relapse rates . although the rationale of this proposal is highly reliable and theoretically grounded , we are entirely aware that it is currently impossible to implement this proposal in clinical practice . indeed , the principal concern relates to the erp screening procedure . to date , the majority of erp related studies comparing psychiatric populations to matched controls used grand - averaged data ( ie , mean data comparing a group of healthy individuals to a group of patients ) . however , in daily clinical practice , in order to be able to decide whether an individual patient presents a genuine cognitive impairment , we need to develop normal and impaired ranges for erp parameters ( amplitude and/or latency ) . in other words , it is necessary to accumulate unambiguous referenced normative data in order to determine whether a patient requires cognitive rehabilitation . in this regard , there already exist guidelines for some erp components ( mmn and p300 ) , which could be clinically implemented.93 for other components , normative data remains to be gathered ( p50 , nogo p300 , and ern ) . therefore , there is an urgent need for further research to develop multisite guidelines that can be compared and used across studies by recording a battery of these five electrophysiological measures ( p50 , mmn , p300 , nogo p300 , and ern ) . we propose that the time needed for erp screening could be greatly reduced by focusing on the five erp components described above , while other components ( eg , contingent negative variation , p100 , n300 , and n400 ) and other related cognitive processes ( eg , anticipation effects , visual , emotional , or language processes ) may be of great interest . here , we have focused on the components related to attention ( through mmn , p50 ) , execution ( nogo p300 ) , memory and decision - making capacity ( p300 ) , and self - monitoring ( ern ) , which are the ones that have been primarily screened in psychopathological states.85 nevertheless , if our approach proves to be efficient , other batteries using different components could also be developed for future clinical use . to conclude , there is a general agreement that a multi - disciplinary approach is required for the successful treatment of mental illness . in this proposal , we have suggested that psychiatrists should continue to maintain their classic collaboration with psychologists , who are trained to provide psychotherapy . however , we now suggest that neurophysiologists and neuropsychologists may be crucial to aid psychiatrists in the identification of cognitive processes that should be rehabilitated on a patient - by - patient basis . the resulting combined approach ( ie , medication , psychotherapy , and erp - oriented cognitive rehabilitation ) should reduce relapse rates for many psychiatric afflictions , because individual cognitive interventions would be specifically targeted based on each patient s needs , thus providing an individualized or personalized medicine . therefore , the challenge of future studies will be to highlight whether this procedure is efficient enough to be incorporated into a psychiatrist s current treatment method toolbox . this strategy is certainly useful for some patients , but the relapse rate remains tremendously high for several psychiatric disorders . thus , in psychiatry there is currently a recognized need for alternatives to psychotherapy and medication.11 cognitive deficits have gained considerable importance in the field as critical features of mental illness.21 therefore , in the present paper , we suggest that a possible way to increase the efficiency of psychiatric treatment is to include erps and crps in clinical practice , combining these techniques with psychotherapy and medication . however , this would require a significant collaboration between psychiatry departments , clinical neurophysiology laboratories , and neuropsychological rehabilitation centers . complete erp screening to his / her patients , which is easy to perform and will not be time - consuming if only the main cognitive domains are analyzed ( attention , execution , and self - monitoring ) . the basic purpose of these erp screening tests will be to indicate the likelihood of genuine cognitive impairment , which can be inferred through the comparison of the patient s results with reference controls . thus , a borderline score or a very impaired score ( along with supporting history ) might lead the psychiatrist to order a more specialized assessment of cognition ( eg , by neuropsychologists ) , and thus thoroughly identify which cognitive domain(s ) to rehabilitate . indeed , crps have been proven to have a positive impact on daily quality of life92 and on clinical symptoms.86,87 hence , psychiatric treatment data from neurophysiologists ( erps ) could help neuropsychologists decide which cognitive processes to rehabilitate through crps in order to enhance the daily quality of life and soften the clinical symptoms of individual patients . ultimately , based on the fact that the long - lasting , positive effects of crps on clinical symptoms have already been demonstrated,35,87 we are convinced that the combination of this cognitive procedure ( involving erps and crps ) with psychotherapy and medication could lead to decreased relapse rates . although the rationale of this proposal is highly reliable and theoretically grounded , we are entirely aware that it is currently impossible to implement this proposal in clinical practice . indeed , the principal concern relates to the erp screening procedure . to date , the majority of erp related studies comparing psychiatric populations to matched controls used grand - averaged data ( ie , mean data comparing a group of healthy individuals to a group of patients ) . however , in daily clinical practice , in order to be able to decide whether an individual patient presents a genuine cognitive impairment , we need to develop normal and impaired ranges for erp parameters ( amplitude and/or latency ) . in other words , it is necessary to accumulate unambiguous referenced normative data in order to determine whether a patient requires cognitive rehabilitation . in this regard , there already exist guidelines for some erp components ( mmn and p300 ) , which could be clinically implemented.93 for other components , normative data remains to be gathered ( p50 , nogo p300 , and ern ) . therefore , there is an urgent need for further research to develop multisite guidelines that can be compared and used across studies by recording a battery of these five electrophysiological measures ( p50 , mmn , p300 , nogo p300 , and ern ) . we propose that the time needed for erp screening could be greatly reduced by focusing on the five erp components described above , while other components ( eg , contingent negative variation , p100 , n300 , and n400 ) and other related cognitive processes ( eg , anticipation effects , visual , emotional , or language processes ) may be of great interest . here , we have focused on the components related to attention ( through mmn , p50 ) , execution ( nogo p300 ) , memory and decision - making capacity ( p300 ) , and self - monitoring ( ern ) , which are the ones that have been primarily screened in psychopathological states.85 nevertheless , if our approach proves to be efficient , other batteries using different components could also be developed for future clinical use . to conclude , there is a general agreement that a multi - disciplinary approach is required for the successful treatment of mental illness . in this proposal , we have suggested that psychiatrists should continue to maintain their classic collaboration with psychologists , who are trained to provide psychotherapy . however , we now suggest that neurophysiologists and neuropsychologists may be crucial to aid psychiatrists in the identification of cognitive processes that should be rehabilitated on a patient - by - patient basis . the resulting combined approach ( ie , medication , psychotherapy , and erp - oriented cognitive rehabilitation ) should reduce relapse rates for many psychiatric afflictions , because individual cognitive interventions would be specifically targeted based on each patient s needs , thus providing an individualized or personalized medicine . therefore , the challenge of future studies will be to highlight whether this procedure is efficient enough to be incorporated into a psychiatrist s current treatment method toolbox .

the relapse rate for many psychiatric disorders is staggeringly high , indicating that treatment methods combining psychotherapy with neuropharmacological interventions are not entirely effective . therefore , in psychiatry , there is a current push to develop alternatives to psychotherapy and medication - based approaches . cognitive deficits have gained considerable importance in the field as critical features of mental illness , and it is now believed that they might represent valid therapeutic targets . indeed , an increase in cognitive skills has been shown to have a long - lasting , positive impact on the patients quality of life and their clinical symptoms . we hereby present four principal arguments supporting the use of event - related potentials ( erp ) that are derived from electroencephalography , which allow the identification of specific neurocognitive deficiencies in patients . these arguments could assist psychiatrists in the development of individualized , targeted therapy , as well as a follow - up and rehabilitation plan specific to each patient s deficit . furthermore , they can be used as a tool to assess the possible benefits of combination therapy , consisting of medication , psychotherapy , and erp - oriented cognitive rehabilitation . using this strategy , specific cognitive interventions could be planned based on each patient s needs , for an individualized or personalized therapy , which may have the potential to reduce relapse rates for many psychiatric disorders . the implementation of such a combined approach would require intense collaboration between psychiatry departments , clinical neurophysiology laboratories , and neuropsychological rehabilitation centers .

Introduction Argument 1: the cognitive process is a key element of psychopathological states Argument 2: the ERP method, a highly adapted tool to investigate the sequence of cognitive processes Argument 3: a similar behavioral deficit may originate from different cognitive levels Argument 4: once specific cognitive deficiencies are defined, directed cognitive retraining procedures may help to alleviate symptoms in homogeneous subgroups of patients Conclusion Creating a multi-disciplinary approach to mental illness, including neurophysiologists and neuropsychologists There is still a long way to go before this strategy can be implemented into daily clinical practice

in fact , neuropsychiatric conditions account for up to one quarter of all disability - adjusted life years ( ie , the sum of years lived with disability and years of life lost).1 a psychiatrist s main responsibility is to render accurate diagnoses that guide the selection of appropriate medication for severely mentally ill patients.2 a crucial problem in psychiatry , which affects both research and clinical work , is that in practice it is difficult to strictly apply the scientific method to the investigation of human mental illness , since the evaluations should ( at least ) include analyses of both the mind and the brain when assessing normal and aberrant behaviors.3 a consequence of this complexity is that , even if psychopathological states can effectively be alleviated by a combination of psychotherapy and neuropharmacological interventions , the relapse rate for several psychiatric disorders is tremendously high.4 for example , the relapse rate for manic episodes in bipolar disorders was found to be 50% at 1 year , and 70% at 5 years.5 similarly , the relapse rate following a first episode of schizophrenia was reported to be 35% at 18 months , and 74% at 5 years.6 in other words , there is still much to learn in order to fill this existing gap between a partial recovery , which allows patients to return home , and the achievement of a complete recovery.7 the complexity surrounding the treatment of mental illness is also illustrated by alcohol dependence , which constitutes one of the most severe and widespread public health problems . during clinical trials , placebo control groups showed a relapse rate of up to 85% , even if hospitalized until complete remission of the physical withdrawal symptoms.8 similarly , there is little evidence supporting that antidepressants alter in any way the risk factors that lead to relapse and recurrence . consequently , most patients with chronic or recurrent depression are encouraged to stay on medication indefinitely.9 in addition , while it has been well established that maintenance treatment with antipsychotic medication decreases relapse rates , a substantial proportion of patients suffering from schizophrenia either relapse despite taking medication or have trouble adhering to antipsychotic treatment.10 all of the above fuels the current strong interest for the development of alternatives to psychotherapy and medication.11 in recent years , we have seen the emergence of several intervention strategies aimed at improving psychiatric treatment , such as multisystemic,12 cognitive behavioral,13 or mindfulness14 therapies . therefore , the purpose of this paper is to present four key arguments that support the use of event - related potential ( erp)-based cognitive assessment in combination with psychotherapy and medication to develop more efficient psychiatric therapies . it is now largely accepted that psychiatric disorders are brain disorders,15 and brain - related tools , such as electroencephalography ( eeg)16 or repetitive transcranial magnetic stimulation ( rtms),17 have already proven their value for the management of psychiatric diseases . for instance , the combined use of rtms and eeg enables the noninvasive evaluation of functional connections between brain areas , and provides a tool to investigate cortical activity.18 thus , a non - invasive input ( tms ) known spatial and temporal characteristics with directional and precise chronometric data can be applied to the study of the brain s local reactivity and the interactions between different brain regions.19 this emerging technique allows for instance to observe that , during an 8 to 10 minute long eeg session with single - pulse tms over the right premotor cortex , patients suffering from schizophrenia present reduced evoked gamma oscillations in the frontal cortex , reinforcing the hypothesis of an intrinsic dysfunction of the frontal thalamo - cortical circuit.20 in this paper , we focus on erps , since by allowing the evaluation of the entire information processing stream , they can help pinpoint the specific neurocognitive functions that should be rehabilitated in each patient through specific and individualized cognitive remediation procedures . the aim of this paper is not to provide an exhaustive review of the literature pertaining to erps and psychiatric disorders , but to systematically illustrate four reasons why we think that it is now time to incorporate cognitive erps as a requisite step in clinical evaluations and use them to direct the development of an individualized therapy for mentally ill patients . this approach should also include a cognitive neuropsychological rehabilitation step , as an integrative part of the whole process that is classically only based on psychotherapy and medication . cognitive deficits have gained considerable importance in the field as critical features of mental illness , and it is now believed that they might represent valid therapeutic targets.21 indeed , several studies have provided consistent evidence that mental illness involves significant cognitive impairment . for instance , virtually all areas of cognition , such as perception , attention , and memory , are known to be impaired in the schizophrenic individual,22 as well as in patients of other psychiatric afflictions , such as euthymic bipolar disorder,23 alcohol - dependence,24 or attention - deficit / hyperactivity disorder ( adhd).25 in schizophrenic patients , literature data reports a substantial decrease in cognitive function of about 1.01.75 standard deviations below the normal mean.26 moreover , some variability in the cognitive domain was also reported in euthymic bipolar patients ( large effect sizes [ d0.8 ] for executive functions and small effect sizes [ 0.2db0.5 ] for sustained attention).23 however , a proven connection between the onset of a mental illness and the presence of cognitive impairment would be needed in order to establish the requirement for cognitive therapeutics.27 additionally , while there is overwhelming evidence that medication can reduce the severity of clinical symptoms , cognitive gains are either poor or undetectable.21 thus , it is currently accepted that cognitive impairment and symptomatic manifestations of mental illness require separate therapeutic approaches.28 cognitive retraining procedures ( crps ) have brought new applications to psychiatric rehabilitation.29 cognitive remediation is a behavioral intervention designed to improve cognition in patients who have suffered a cognitive decline.30 the major elements of psychiatric rehabilitation focus on highlighting the patient s strengths , improving vocational / social competencies , working with environmental facts of an individual s life , and instilling a sense of mastery and hope.31 from this perspective , cognitive retraining techniques range from assistive or prosthetic devices ( including computers , diaries , and/or lists ) to approaches that focus on enhancing the patient s efficiency , ( eg , mnemonic strategies).32 many studies have demonstrated that the use of crps can enhance cognitive performance in diverse psychiatric diseases,33 and that these improvements are stable up to at least 6 months after the treatment is discontinued.34 additionally , crp - related cognitive gains have been proved to translate to improvements in real world activities,35 with greater impact than any drug treatment.21 for example , in a single case study involving a schizophrenic patient,35 levaux et al demonstrated that the usage of an attention training technique leads to the following beneficial effects : i ) at the cognitive level , a specific improvement in selective attention , attention switching , and resistance to distractive interference ; and ii ) at the clinical level , a reduction in intrusive thoughts in daily life and a general decrease in symptoms stable up to at least 6 months . at the time of writing five of these , while differing in focus , determined that it has moderate to large effect sizes;3741 the remaining one didnt.42 moreover , with the spread of crp s application to patients suffering from schizophrenia , positive outcomes with crp for other psychiatric disorders , such as unipolar depression,43 social phobia,44 adhd,45 and alcohol dependence46 have begun to appear in the literature . overall , these data are consistent with the general idea that recognizing the nature of the cognitive impairment involved in a specific mental illness will be very useful for the development of treatment and rehabilitation strategies.29 although the usefulness of crps for the treatment of psychiatric disorders is gradually becoming more accepted , several questions regarding how to use them in clinical practice still remain to be resolved . in fact , it appears that , for instance , around 20% of schizophrenics generally perform within the normal range , only showing subtle evidence of impairment in specific cognitive domains.47 indeed , the human mind can be defined by several cognitive domains , such as attention , memory , language , or executive functions , any of which may prove to be altered.48 this requires rehabilitation specialists ( usually neuropsychologists ) to thoroughly assess the cognitive status of each domain in order to decide the appropriate rehabilitative treatment . this process may be complicated by two factors : i ) the fractionation of each cognitive domain implies the involvement of various neural networks ( eg , executive function refers to a wide range of cognitive processes [ including problem - solving , flexibility , inhibition , planning , and decision - making ] , and a patient s performance in one executive function may have little or no predictive value for how the patient will perform in another);49 and ii ) time pressure is an important feature of the clinical examination , because cognitive screening needs to identify genuine impairment in as little time as possible , using an easily administered instrument.50 in the following arguments , we will describe how and why we believe that cognitive erps can aid in the cognitive screening process . a general screening of main cognitive functions can be achieved by performing rapid tasks , which are easily implementable in clinical practice and have the potential to index the cognitive areas that require further assessment and/or rehabilitation by neuropsychologists . firstly , the use of advanced imaging methods enabled the study of the neurobiology of mental disorders , confirming the fact that mental disorders have a neurobiological component ( ie , they are not only functional or psychogenic conditions).53 secondly , imaging technology contributed insight into the pathological mechanisms of mental disorders , demonstrating that a functional alteration may result from an isolated brain lesion ( eg , after a stroke ) or deficient interactions between different brain regions ( functional connectivity).54 in this regard , imaging analysis can also provide a better understanding of the mechanisms of action of psychopharmaceuticals , potentially aiding in the search for novel therapeutic targets.53 the study of blood flow ( fmri ) or glucose metabolism ( pet ) in certain areas of the brain during activation tasks enables the identification of the areas of the brain that are activated or deactivated during specific cognitive tasks . for this purpose , we should recur to electrophysiological tools , and more precisely , to cognitive erps.56 erps , which are derived from electroencephalography , are highly sensitive and have the potential to monitor brain lectrical activity with a fine temporal resolution ( on the order of milliseconds ) . another highly valuable aspect of cognitive erps is that they can permit the identification / detection of the electrophysiological component(s ) consistent with the onset of a dysfunction , allowing the inference of impaired cognitive stages.56 in other words , by using a well characterized task and analyzing which erps show decreased amplitude and/or delayed latency compared to normal values , it is possible to deduce which erp component is responsible for the cognitive deficit.56 figure 2 illustrates the well - known p300 component , a late , long - lasting parietal positive wave , which is decreased and delayed in alcoholic patients compared to matched controls . however , while p300 deficit is common in many psychiatric afflictions , there is also an abundance of literature reporting ; for instance , many earlier erp components , such as the mismatch negativity or the sensory gating p50 , but also later ones , such as the n400 indexing language semantic processes , are affected in alcoholic,57 as well as in schizophrenic58 patients . their spatial resolution is very weak , so that inferences can only be made on the neural generators of the observed components , and parameters of erps ( latency and amplitude ) are subject to large inter - individual differences , so that the collection of normative data may prove fastidious.56 nevertheless , cognitive erps are advantageous , because they allow the assessment of the different stages used in the information - processing stream during performance of a task . these differences have been associated with deficits in behavioral performance and can indicate the severity of a clinical state , as well as its possible evolution.62 for example , if we consider the literature on the ability to recognize emotional facial expressions ( efe ) in psychiatric populations , two main points emerge : i ) at a behavioral level , many patients with psychiatric diseases display a deficit in emotional recognition , which can be indexed by decreased performance rates and longer response latencies compared to controls;63 and ii ) at an electrophysiological level , this behavioral deficit is associated with a decreased and delayed p300 component.64 however , an important question in the field has revolved around the events occurring in the information - processing stream prior to the p300 component ( ie , before the response - related stages ) . indeed , while patients suffering from alcohol dependence or schizophrenia displayed altered erp components affecting all information - processing streams ( from perceptive - visual p100 , to attention n2b and response - related p300 components),66,67 depressed people exhibited only a disturbed p300 component ( with intact earlier components).68 on the other hand , ecstasy users presented differences starting at the level of attention ( n2b).69 in other words , similar patterns at the behavioral level can result from different disturbances in cognitive processes from one population to another . it is believed to reflect cortical information processing at the earliest level of the sensory cortex ; however , recent findings suggest that the transient auditory sensory memory representation underlying the mmn is facilitated by a long - term memory representation of the corresponding stimulus.75 the p300 component ( or p3 ) is a long - lasting positive component that occurs between 300 and 700 ms after stimulation . their findings revealed that the use of the electrophysiological battery provided novel information on the characteristics of the different groups . in particular , they highlighted the heterogeneity of electrophysiological features among the groups and demonstrated that the combined analysis could serve to minimize the impact of such heterogeneity.84 we propose the use of p50 , mmn , p300 , nogo p300 , and ern to facilitate the indexing of cognitive stages affected in psychiatric patients by assessing , respectively , the attention - filtering , early perceptive discriminative , response - related decisional , inhibitory , and self - action evaluation processes . more precisely , specific cognitive retraining procedures could be used to target deficits and increase cognitive efficiency , which has already been shown to reduce clinical symptoms.86,87 in this regard , there are currently many crps available , including those directed at attention mechanisms ( increasing or decreasing the attention resources devoted to a specific cue),88 for which compelling evidence has already been gathered in anxiety disorders . indeed , behavioral and erp studies have shown that attention training can alter threat bias , influence vulnerability to stress , and reduce clinical anxiety symptoms by modulating top - down processes of attention control rather than processes of early attention orienting.89 in the same vein , there are also available crps increasing organization , goal achievement,90 cognitive control ( ie , inhibitory skills),46 and even self - monitoring.91 this strategy is certainly useful for some patients , but the relapse rate remains tremendously high for several psychiatric disorders . thus , in psychiatry there is currently a recognized need for alternatives to psychotherapy and medication.11 cognitive deficits have gained considerable importance in the field as critical features of mental illness.21 therefore , in the present paper , we suggest that a possible way to increase the efficiency of psychiatric treatment is to include erps and crps in clinical practice , combining these techniques with psychotherapy and medication . however , this would require a significant collaboration between psychiatry departments , clinical neurophysiology laboratories , and neuropsychological rehabilitation centers . indeed , crps have been proven to have a positive impact on daily quality of life92 and on clinical symptoms.86,87 hence , psychiatric treatment data from neurophysiologists ( erps ) could help neuropsychologists decide which cognitive processes to rehabilitate through crps in order to enhance the daily quality of life and soften the clinical symptoms of individual patients . ultimately , based on the fact that the long - lasting , positive effects of crps on clinical symptoms have already been demonstrated,35,87 we are convinced that the combination of this cognitive procedure ( involving erps and crps ) with psychotherapy and medication could lead to decreased relapse rates . in this regard , there already exist guidelines for some erp components ( mmn and p300 ) , which could be clinically implemented.93 for other components , normative data remains to be gathered ( p50 , nogo p300 , and ern ) . therefore , there is an urgent need for further research to develop multisite guidelines that can be compared and used across studies by recording a battery of these five electrophysiological measures ( p50 , mmn , p300 , nogo p300 , and ern ) . here , we have focused on the components related to attention ( through mmn , p50 ) , execution ( nogo p300 ) , memory and decision - making capacity ( p300 ) , and self - monitoring ( ern ) , which are the ones that have been primarily screened in psychopathological states.85 nevertheless , if our approach proves to be efficient , other batteries using different components could also be developed for future clinical use . to conclude , there is a general agreement that a multi - disciplinary approach is required for the successful treatment of mental illness . however , we now suggest that neurophysiologists and neuropsychologists may be crucial to aid psychiatrists in the identification of cognitive processes that should be rehabilitated on a patient - by - patient basis . the resulting combined approach ( ie , medication , psychotherapy , and erp - oriented cognitive rehabilitation ) should reduce relapse rates for many psychiatric afflictions , because individual cognitive interventions would be specifically targeted based on each patient s needs , thus providing an individualized or personalized medicine . this strategy is certainly useful for some patients , but the relapse rate remains tremendously high for several psychiatric disorders . thus , in psychiatry there is currently a recognized need for alternatives to psychotherapy and medication.11 cognitive deficits have gained considerable importance in the field as critical features of mental illness.21 therefore , in the present paper , we suggest that a possible way to increase the efficiency of psychiatric treatment is to include erps and crps in clinical practice , combining these techniques with psychotherapy and medication . however , this would require a significant collaboration between psychiatry departments , clinical neurophysiology laboratories , and neuropsychological rehabilitation centers . the basic purpose of these erp screening tests will be to indicate the likelihood of genuine cognitive impairment , which can be inferred through the comparison of the patient s results with reference controls . indeed , crps have been proven to have a positive impact on daily quality of life92 and on clinical symptoms.86,87 hence , psychiatric treatment data from neurophysiologists ( erps ) could help neuropsychologists decide which cognitive processes to rehabilitate through crps in order to enhance the daily quality of life and soften the clinical symptoms of individual patients . ultimately , based on the fact that the long - lasting , positive effects of crps on clinical symptoms have already been demonstrated,35,87 we are convinced that the combination of this cognitive procedure ( involving erps and crps ) with psychotherapy and medication could lead to decreased relapse rates . in this regard , there already exist guidelines for some erp components ( mmn and p300 ) , which could be clinically implemented.93 for other components , normative data remains to be gathered ( p50 , nogo p300 , and ern ) . therefore , there is an urgent need for further research to develop multisite guidelines that can be compared and used across studies by recording a battery of these five electrophysiological measures ( p50 , mmn , p300 , nogo p300 , and ern ) . to conclude , there is a general agreement that a multi - disciplinary approach is required for the successful treatment of mental illness . however , we now suggest that neurophysiologists and neuropsychologists may be crucial to aid psychiatrists in the identification of cognitive processes that should be rehabilitated on a patient - by - patient basis . the resulting combined approach ( ie , medication , psychotherapy , and erp - oriented cognitive rehabilitation ) should reduce relapse rates for many psychiatric afflictions , because individual cognitive interventions would be specifically targeted based on each patient s needs , thus providing an individualized or personalized medicine .

the issue of accurate pairwise sequence alignment is common to many fields in bioinformatics , whether as a core tool in fields such as reference - guided genome assembly ( 16 ) or as the seed for the generation of a progressive multiple sequence alignment ( 79 ) . ideally , analysis of a pairwise alignment should proceed in the knowledge that no inherent bias exists as a result of the alignment approach used . this is particularly challenging in the era of high - throughput sequencing , where every platform produces systematic errors ( 1015 ) that should be considered in constructing an alignment . the alignment of coding dna , in particular , presents a unique challenge as it is critical that the final alignment takes into account the correct reading frame . preservation of the reading frame ensures correct calling of gene structure ( 1618 ) and snps , whether in , for example , the realignment phase of an exome sequencing pipeline ( 19 ) , single - nucleotide polymorphism ( snp ) calling from existing rna - seq data ( 20,21 ) or amplicon - based analyses such as human immunodeficiency virus ( hiv ) drug resistance genotyping ( 22 ) . when aligning coding dna generated using high - throughput sequencing platforms , it is critical that codons present in the open reading frame remain intact and that codon - sized insertions and deletions are recognized and called correctly , as distinct from both genuine frameshifts and single indels created through sequencing error . the landscape of pairwise alignment and reference mapping tools for high - throughput sequencing data is broad . tools such as bowtie 2 ( 2 ) and bwa - mem ( 3 ) , while well - suited to mapping the location of query sequence reads within a complete reference genome , lack the subtle nuances required to correctly distinguish spurious indels from genuine codon - sized mutations in coding dna . other tools , such as mosaik ( 6 ) and ssaha2 ( 5 ) , perform full mapping and realignment using a smith waterman approach , with a focus on correcting next - generation sequencing errors . however , basic smith waterman alignment , even when taking into account quality scores as in mosaik and bwa - mem , is not appropriate for reference mapping of coding dna as it fails to maintain the intactness of codons . finally , the genome analysis toolkit ( gatk ) ( 23,24 ) performs the realignment step for tools such as bwa - mem in , for example , the 1000 genomes project pipeline ( 25 ) , but does not consider the biological context ( coding or non - coding ) in its alignment . one approach to ensure codons are maintained for downstream analysis , taken by the revtrans program ( 26 ) , is to initially translate query sequences into their corresponding amino acid sequences , align them to a translated reference sequence at the amino acid level and then back - align the nucleotide sequences based on the amino acid alignment ( 26 ) . while effective in some cases , this approach is useless in the presence of indels in the open reading frame , which results in mistranslation to amino acid space . tools such as transalign ( 27 ) repetitively translate , align , back - translate and correct multiple sequence alignments , which to some extent addresses frameshift errors . this approach is , however , time - consuming , as in order to align robustly it requires a full amino acid multiple sequence alignment to create a back - translated dna profile to which low - scoring sequences ( assumed to be frameshifted ) are compared . codon spaceweighting the likelihood of each nucleotide substitution within the context of the codon to which it belongs . this , thus , facilitates the identification of indels , whether genuine frameshift mutations or the result of sequencing error . initial models of coding dna such as those used by pairwise ( 16 ) and est_genome ( 28 ) were designed specifically for gene prediction , comparing sequences against all six translated frames simultaneously using a classical dynamic programming approach to discover exons . however , this approach was found to be time - consuming and overly specific ( 29 ) . the hmmer tool ( 30 ) took a different approach , using profile hidden markov models to align and compare protein sequences . genewise and genomewise ( 29 ) use a hidden markov model approach to allow for the alignment of a nucleotide sequence , potentially including frameshifts , to an existing protein sequence , and thus represent the closest approximation to a codon codon alignment tool . however , because the alignment is to a protein sequence , it is impossible to penalize synonymous mutations . protein mismatches are all scored equally , as are single - nucleotide insertions resulting from sequencing error . this prevents the use of genewise and genomewise to correctly identify and account for homopolymer errors in next - generation sequencing platforms . here , we present a novel reference mapper ( ramics : rapid amplicon mapping in codon space ) that uses a hidden markov model approach to align large numbers of coding dna sequences to a coding region of a reference sequence in an accurate and biologically relevant manner , accounting for the inherent error biases of the sequencing platform used . ramics is developed to be executed on graphics processing units ( gpus ) and , thus , provides significant speed improvements over other , similarly complex , mapping tools . ramics is , thus , ideal for any situation where a large number of sequence reads from a known , coding location in a genome must be mapped to a coding reference sequence . the tool first discerns the optimal open reading frame of the reference sequence by selecting a frame that would minimize the number of stop codons . codon space using a hidden markov model ( 31 ) ( figure 1 and supplementary information ) . single- and double - nucleotide indels are identified and accounted for , thereby ensuring that biologically relevant complete codons are kept intact . this enables a correct and relevant comparison of a query sequence to the reference , as the concept of a codon - sized insertion that begins within one codon and ends within another ( as would be output by most conventional alignment approaches ) is structurally meaningless in a biological context ( figure 2a ) . match states are shown as circles , delete states as squares and insert states as diamonds . d are whole - codon insertions and deletions , and are the only self - transitioning states . one copy of this hmm exists for every codon in the reference sequence , and they are chained together to build a profile hmm . dashed lines indicate an unshown transition to the next set of states in the chain .. ( a ) biologically relevant alignments. sequences x and y differ from each other by a three - nucleotide insertion . conventional nucleotide - based approaches generate a statistically optimal pairwise alignment matching as many nucleotides as possible . ramics , on the other hand , considers the biological importance of codons and generates a biologically relevant alignment preserving the codon structure of the coding dna being aligned . ( b ) correctly accounting for homopolymer errors. comparing alignments of an hiv pol gene sequence containing a homopolymer error ( additional adenosine , green arrow ) to a reference sequence shows that ramics correctly identifies and accounts for this error . conventional nucleotide alignment approaches would call this codon as aaa ( flagging the g as an insertion ) which encodes for lysine ( sensitive form ) , while ramics would call the codon as aga ( arginine , the resistant form ) correctly identifying and discounting the extra adenosine introduced as a result of the sequencing error at the homopolymer region . ramics uses models of codon substitution ( 3234 ) to give synonymous codon substitutions higher transition probabilities as well as favoring more likely non - synonymous substitutions . ramics can also be set to use a simple non - coding substitution model to align non - coding dna to a non - coding reference sequence . however , ramics can not concurrently align sequence reads to coding and non - coding regions of the same reference sequence ( e.g. a reference sequence containing both introns and exons ) , and , as such , is unsuitable for use as the first - pass mapping tool in full - genome association studies . it can , however , be subsequently used for targeted realignment of coding regions as a second - pass mapping tool . ramics uses reported average error rates for given sequencing platforms ( 10,12,13,15 ) to set the transition probabilities to single- and double - nucleotide indel states in the profile hidden markov model . in addition , the model is more likely to place a single - nucleotide indel on either side of a homopolymer region when the sequencing platform is set to roche/454 ( 35 ) or ion torrent personal genome machine ( 36 ) , as homopolymer miscalls are a common sequencing error on these platforms ( 13 ) . to illustrate the necessity of considering the sequencing platform used to generate the sequence data , we take an example from hiv-1 antiretroviral drug resistance testing . the drug resistance profile of a sequence is determined by characterizing mutations at various amino acid positions throughout the polymerase gene mapped in relation to the hxb2 hiv reference sequence ( 51 ) . drug resistant mutations include the mutation of lysine ( k ) to asparagine ( n ) at codon 103 ( k103n ) , which confers drug resistance to certain non - nucleoside reverse transcriptase inhibitors ( nnrtis ) ( 22 ) . the k103n codon is located in a homopolymer region and some high - throughput sequencing platforms such as roche/454 ( 35 ) and ion torrent pgm ( 32 ) , as well as to a lesser extent the illumina platform ( 37 ) , are prone to increased error rates in regions containing homopolymers , as they can not successfully identify the correct number of nucleotides in these regions ( 12,13 ) . incorrect assignment of a homopolymer indel during the mapping step would result in a viral sequence being falsely called as susceptible when it is actually resistant ( 54 ) . wunsch alignment to the hxb2 reference sequence of a sequence containing both the k103n mutation and a spurious extra adenosine directly following homopolymer region would result in the virus in question being falsely classified as susceptible to nnrtis such as efavirenz ( figure 2b ) . mapping of the same data using ramics , however , weighs the possibility of a false tyrosine ( t ) insertion in the center of a homopolymer region against a false adenosine ( a ) insertion at the end of the region , assigns the latter a higher probability and , thus , correctly calls the presence of the k103 m mutation in spite of the hompolymer error ( figure 2b ) . thus , a major strength of ramics is in its application to situations where the correctness of alignment in a coding region is crucial , such as alignment optimization in rna - seq and exome sequencing ( 1921,38,39 ) . it can be a common case in reference mapping , particularly for amplicon - based analyses , that a set of query sequence reads will be more closely related to each other than they will to a given reference sequence this is true in reference - guided assembly ( 40 ) , drug - resistance genotyping ( 41 ) and applications in metagenomic function prediction ( 42 ) . ramics circumvents any potential bias introduced through a distantly related reference sequence by allowing the use of the reference sequence as a guide for the initial mapping and then iteratively training the underlying hidden markov model to the resulting pairwise alignments . the result is a set of emission and transition probabilities that will correctly align outlier sequences ( potentially with many sequencing errors ) in the query set , based on the substitutions in sequences more similar to the reference . this approach generates a global reference alignment that approaches a reference - seeded multiple sequence alignment , and which can be used as such . ramics produces results in various formats with the choice of output format very much driven by the end - users requirements : a global nucleotide alignment of all query sequences to the reference sequence , in fasta format.a derivation of the above global nucleotide alignment in which all single- and double - nucleotide insertions are widened to the width of a full codon using gaps ( codons.fasta ) . this does not alter the sequences , but allows the open reading frame to be read clearly in alignment viewers such as seaview ( 43).a global alignment with all single- and double - nucleotide insertions removed ( clean.fasta ) . this format does edit the sequences by assuming that all single- and double - indels are the result of sequencing / polymerase chain reaction ( pcr ) error . while this is extremely useful in some amplicon - based analyses where frameshift mutation can be safely treated as pcr / sequencing errors , it should not be used in cases where a frameshift mutation may have biological relevance.a global alignment containing only one instance of each identical sequence with a count of the number of contributing sequence reads added to the sequence descriptor.a set of sam files describing mapped reads , with soft clipping used beyond the end of the reference sequence.a file containing a set of pairwise gapped fasta format alignments of each query sequence to the reference . a global nucleotide alignment of all query sequences to the reference sequence , in fasta format . a derivation of the above global nucleotide alignment in which all single- and double - nucleotide insertions are widened to the width of a full codon using gaps ( codons.fasta ) . this does not alter the sequences , but allows the open reading frame to be read clearly in alignment viewers such as seaview ( 43 ) . a global alignment with all single- and double - nucleotide insertions removed ( clean.fasta ) . this format does edit the sequences by assuming that all single- and double - indels are the result of sequencing / polymerase chain reaction ( pcr ) error . while this is extremely useful in some amplicon - based analyses where frameshift mutation can be safely treated as pcr / sequencing errors , it should not be used in cases where a frameshift mutation may have biological relevance . a global alignment containing only one instance of each identical sequence with a count of the number of contributing sequence reads added to the sequence descriptor . a set of sam files describing mapped reads , with soft clipping used beyond the end of the reference sequence . a file containing a set of pairwise gapped fasta format alignments of each query sequence to the reference . ramics is designed to map large numbers of high - throughput sequencing reads from a coding dna region to a similar reference sequence , both quickly and accurately . ramics also has potential as an alignment refiner for the coding regions of whole - genome mapping or as the seed pairwise aligner for a profile hmm multiple sequence aligner in the style of the protein sequence aligner clustal omega ( 44 ) . both as a standalone amplicon mapper and as a component of these larger toolchains , it can align large numbers of reads quickly as well as aligning reads accurately in regions that have similar biological features but contain many synonymous or functionally similar substitutions . although ramics has the ability to remove frameshifts in reads , under the assumption that they are sequencing errors , we did not remove any indels before evaluating the alignments . we first compared the quality of alignments with ramics in non - coding mode , coding mode without training and coding mode with training , respectively . we then compared ramics for quality and speed against bowtie-2 ( 2 ) , bwa - mem ( 3 ) , bwa - mem post - processed by the indelrealigner from the gatk ( 23 ) , mosaik ( 6 ) , the basic needleman wunsch aligners emboss needle and water ( 45 ) ( needle is used where a global alignment is more appropriate , water for local alignment ) , nextgenmap ( 46 ) , ssaha-2 ( 5 ) and shrimp2 ( 1 ) . these tools represent a mix of global , local and glocal alignment techniques ( 47 ) , as for the mapping of short reads to a relatively short amplicon all methods are potentially valid . several benchmarks for the mapping of simulated next - generation sequencing data exist ( 48,49 ) . the seal benchmark ( 49 ) is not easily extensible to new alignment tools , so we first performed a simple benchmark of ramics relative ability to correctly de - align spurious indels using the rabema benchmark ( 48 ) . rabema uses the mason read simulator ( 50 ) to simulate artificial short reads containing sequencing errors from a user - provided reference sequence and to output a the alignments generated by a mapping tool are then compared to the gold standard alignments . the tool then bins reads by the number of errors found , ranging from perfect reads to highly erroneous outlier reads containing 4% or more errors . this allows us to test alignment performance not only of reads exhibiting the average error rates reported by manufacturers but also of rare outlier reads that exhibit a higher error rate than the reported average . the rabema output gives the percentage of correctly mapped reads as a function of percentage sequencing error in the read . we ran each benchmark 10 times with 10 different random seeds to ensure statistical significance . while rabema is a good benchmark of a mapping tool 's ability to detect sequencing errors such as spurious indels , it only determines whether an alignment is correct or incorrect and , thus , does not allow quantification of how incorrect the alignment is from the gold standard. further , because all of the reads generated by rabema originate from the same reference sequence , the levels of diversity of the query sequences being mapped are lower than what would be generated in many genuine sequencing experiments . thus , we developed a flexible alignment benchmark ( afab ) that could evaluate not only whether a correct alignment was found , but also how similar this alignment was to a gold standard alignment , in the case where very divergent sequences made perfect mapping impossible . gold standard alignment of protein coding dna which was the highly manually curated hiv-1 group m subtype reference alignment of the hiv envelope gene from the lanl hiv reference sequence database ( 51 ) . this alignment was chosen for two reasons : ( i ) it encompasses the broad spectrum of diversity present within the hiv-1 group m subtypes ( pairwise genetic diversity measured with the generalized time - reversible substitution model ( 52 ) ranged from 0.15 to 0.24 substitutions per site , median 0.218 ) and ( ii ) the envelope gene comprises both conserved and variable regions . this ensured that the benchmarking was performed on a complex example of real - life data to which mapping tools are applied . using one representative sequence per subtype we extracted all possible pairwise alignments resulting in 55 unique alignments . for each alignment we used art ( 48 ) to generate artificial roche/454 and illumina reads ( 1000 reads per pairwise alignment ) from the second sequence of the pair . further , art generated sam files containing the correct alignment of each simulated read to the reference sequence ( the first sequence ) , which were used as the gold standard for evaluation of the accuracy of the various mapping tools . the choice of art has the added benefit of creating greater test coverage , ensuring that ramics is not just incidentally biased to mason 's model of read simulation . for both the roche/454 and illumina data the qscore tool ( 8) was used to evaluate each of the mapping tools , giving the q score and cline shift similarity scores of each tool 's generated alignment to the gold standard . we then ranked the q score and cline shift for each tool that chose to map each read , without penalizing tools that chose not to map the read as rabema does . when multiple tools achieved the same score ( most often 1.0 in the case of trivially mappable reads ) , we assigned each tool the average score they would receive , to avoid artificially inflating the scores of tools that chose to map only trivially mappable reads . this follows the ranking scheme used in the friedman test ( 53 ) and removes the assumption of normality across easy and hard to map reads . we then report the average score obtained across all reads mapped , with a score closer to 1 indicating a consistently higher ranking of a mapping approach . we repeated each benchmark five times with five different random seeds to ensure statistical significance . we benchmarked the wall clock time of ramics , and other tools , in mapping amplicon - style data . we mapped one million roche gs junior fastq reads ( average length 237 base pairs ) from the hiv reverse transcriptase ( rt ) gene to a short , 312 base pair region of the hxb2 hiv-1 reference sequence ( 54 ) . all benchmarks were performed on an intel core i53210 m cpu @ 2.50 ghz and an nvidia geforce gt 525 m graphics card . initially we evaluated the various components of the ramics algorithm to explore whether the addition of complexity improves the quality of the resulting alignment . we mapped both the roche/454 and illumina versions of the afab benchmarking data using the simplest incarnation of ramics ( without codon alignment or training ) , ramics with codon alignment and the full ramics algorithm ( with both the codon - based alignment and training parameters invoked ) . the addition of codon - based alignment increased the number of perfectly aligned sequence reads by 7.9 and 10.5% for the roche/454 and illumina data , respectively ( figure 3 ) . further , including three rounds of training of the hmm to the query data provided marginal improvements in the number of perfectly mapped reads by 1.5% ( roche/454 ) and 0.9% ( illumina ) . the number of discarded reads remained mostly unchanged regardless of the parameters used ( figure 3 ) showing that while alignment accuracy increases with algorithmic complexity , ramics is consistently capable of distinguishing between mappable and unmappable reads . the lesser impact of the training parameter can be explained by its intended use case : the proper alignment of extreme outlier reads with large numbers of sequencing errors . thus , in most instances we suggest that ramics should be used with both the codon - based alignment and training parameters invoked . for all further benchmarking analysis we used the full ramics algorithm including both the codon - based alignment and training parameters . , we show the percentage of simulated ( a ) roche/454 and ( b ) illumina reads that were misaligned ( i.e. not discarded by the mapping tool but with an incorrect alignment when compared to the gold standard ) as well as the percentage of reads that resulted in a perfect alignment when compared with the gold standard alignment . for both of the data the use case for which ramics is designed is rather unlike that for which most other mapping tools are tailored , because of its focus on coding dna . despite this , many studies have used various mapping tools to analyze sequence reads in ways similar to that which ramics was designed for ( 5558 ) and , thus , we present comparisons of ramics to a number of commonly used mapping tools on the basis of alignment quality and speed . initially we compared ramics mapping quality against other tools using the rabema benchmark ( 39 ) for both roche/454 and illumina data . reads were simulated from a single reference sequence using the reported error rates of the sequencing platforms as encoded by the mason read simulator ( 50 ) . these resulting sequence reads are then binned by their percentage difference from their seed sequence . for example , if the seed sequence used to generate a read was 100 nucleotides long and the resulting read had three differences relative to its seed , then that read would be added into the 3% error bin . this allowed the evaluation of the mapping tools using realistic error rates for both typical and outlier sequences . as would be expected , the observed error rate for vast majority of sequence reads for the illumina data set fell around 1% with 88% of sequences exhibiting an error rate of 1% . for the 454 data , 49% of the sequence reads exhibited an error rate of 1% while the error rate of the remaining reads was in excess of 1% . the higher - than - expected number of sequences with an error rate of > 1% most likely results from the fact that the single reference sequence used to seed the simulations contained a number of homopolymer regions , which are known to increase the overall observed error rate of the roche/454 platform ( 12,13 ) . for the roche/454 benchmark , the performance of all of the tools tested was comparable for reads containing zero errors , ranging from 100% to 97.9% of reads correctly mapped for ramics and ssaha , respectively ( figure 4a ) . such high performance is to be expected , as these sequence reads are identical to the reference sequence . as the percentage of erroneous nucleotides in a read increased , the accuracy of each of the mapping tools , with the exception of ramics , reduced ( figure 4a ) . shrimp ( 1 ) was excluded from the roche/454 benchmark as it discarded over 70% of the reads containing 4% erroneous nucleotides with the recommended settings . the percentage of reads mapped correctly as a function of the percentage error in simulated sequence reads for both the roche/454 ( a ) and illumina ( b ) sequencing platforms . reads are binned together by the rabema tool according to the percentage of erroneous nucleotides in the simulated read relative to its seed sequence . the numbers in parentheses following the percentage error represent the mean percentage of simulated reads ( across the five rabema runs ) that were binned by rabema into that error category . we have excluded bwa - mem without gatk as it performed identically to bwa - mem + gatk . shrimp was excluded from the roche/454 benchmark as it performed extremely poorly on this test . a similar result was observed for the illumina benchmarking data with all methods correctly mapping 100% of reads at zero error rate and the accuracy of the various approaches , with the exception of ramics , reducing as the percentage error rate increased ( figure 4b ) . for the illumina benchmark data we observed that compared to the roche/454 data , we suggest this effect is due to the shorter lengths of illumina reads , which leave fewer correct nucleotides on either side of an error with which to anchor an alignment . ramics outperforms all of the other approaches for both the roche/454 and illumina benchmarking data sets . this is because ramics renders sequencing error in reads when aligning to a reference sequence almost irrelevant , even in outlier reads with a large number of errors , by correctly identifying likely sequencing errors . mosaik was the next best performing tool ( albeit with a reduction of 8% in accuracy at an error rate of 4% in the illumina data ) , while shrimp and ssaha2 , both of which conservatively choose not to align reads , quickly deteriorate in their mapping ability with increasing error rate ( figure 4 ) . the gatk indelrealigner ( 23 ) made no statistically significant difference to the test results , so we have removed it from figure 4 for clarity . the rabema benchmark clearly shows that ramics outperforms all other mapping approaches on the outlier sequences with a higher - than - average error rate . however , these data are not a true reflection of the type of data for which ramics has been optimized , namely mapping a diverse set of sequence reads to a related reference sequence . rabema data only differ from the reference sequence through the insertion of sequencing errors and , thus , we developed a benchmarking tool to enable the assessment of mapping tools at aligning coding sequence reads containing sequencing errors that are diverse from the reference sequence . in contrast to the rabema benchmarking tool , this approach enabled the use of two robust methods for evaluating relative alignment accuracy enabling the identification of perfectly aligned reads and misaligned reads . reads discarded during mapping were not penalized in the final ranking thereby allowing tools to discard ambiguous reads without incurring unnecessary bias in the benchmarking process . extraction of all possible pairwise alignments of subtypes a through g from the gold standard hiv alignment resulted in 55 sequence pairs , each of which were evaluated using this approach . firstly , we considered the percentage of reads that each mapping approach was capable of aligning regardless of the quality of the alignment . needle ( 45 ) does not possess the capability to discard any reads , resulting in 100% of reads being mapped for both the illumina and roche/454 data ( figure 5 ) . the percentage of reads retained by the other approaches ranges from 38% ( shrimp ) to 94% ( mosaik ) for the roche/454 data and 20% ( bwa - mem ) and 62% ( ramics ) for the illumina data ( figure 5 ) . evaluated we show the percentage of simulated ( a ) roche/454 and ( b ) illumina reads that were misaligned ( i.e. not discarded by the mapping tool but with an incorrect alignment when compared to the gold standard ) as well as the percentage of reads that resulted in a perfect alignment when compared with the gold standard alignment . we have excluded bwa - mem without gatk as it performed identically to bwa - mem + gatk . as expected , because these data contained sequencing errors and were diverse from the reference sequence , the percentage of perfectly mapped reads was lower for all approaches than was observed for the rabema benchmarking data . for the illumina data , ramics significantly outperforms all of the other mapping approaches by aligning 62% of the total number of reads perfectly , with the next best approach ( needle ) mapping 48% of all reads correctly ( figure 5 ) . for the roche/454 data ramics performed best , correctly mapping 36% of reads perfectly , while the performance of the other methods ranges between 19% ( mosaik ) and 34% ( ssaha2 ) . the gatk indelrealigner made no difference to the outcome for bwa - mem , so we have removed standalone bwa - mem from figure 5 for clarity . it is also interesting to consider the read discarding strategies employed by the various approaches . ideally , a mapping tool would discard all reads that it could not align perfectly , while still aligning as many reads as possible . considering that all reads in this benchmark data set are simulated from a gold standard alignment , bowtie and bwa - mem 's strategy of discarding almost 80% of the reads leads to a high rate of perfect alignment , but an unacceptable loss of coverage depth in situations where many variants are being aligned or deep coverage is necessary . for all of the reads that were aligned by one or more mapping tools we used two approaches for evaluating alignment quality , the q score ( 59 ) and cline shift score ( 60 ) , to rank each of the mapping tools for each of the 5500 pairs of sequences reads that were tested . the perfect mapping tool would rank first or joint first compared to all other tools for every single comparison . when two or more tools received equal ranking ties were broken according to the friedman test ( 53 ) by assigning , for example , a ranking of 1.5 to each tool if two tools were equally first , preventing a bias toward tools that aligned more reads . the average q score ranks for the methods ranged from 2.512 to 4.846 and 2.978 to 4.562 for the illumina and roche/454 data , respectively ( table 1 ) . the average cline shift ranks range from 2.522 to 4.565 and 3.039 to 4.817 for the illumina and roche/454 data , respectively . for both benchmarking data sets ramics was the highest ranked mapping approach tested , with bwa - mem performing worst over both data sets ( table 1 ) . the addition of the gatk ( 23 ) to the bwa - mem toolchain , the approach used in the 1000 genomes project ( 25 ) , had a negligible effect on the quality of alignment for this use case . table 1.the average rank over aligned reads only for the given mapping toolstoolillumina rankroche/454 rankbowtie 24.003.76bwa - mem4.134.00mosaik3.983.98needle2.973.70nextgenmap3.453.80ramics2.432.46shrimp3.713.82ssaha23.373.43for both test data sets , the highest ranked mapping approach is marked in bold . the average rank over aligned reads only for the given mapping tools for both test data sets , the highest ranked mapping approach is marked in bold . thus , for both benchmarking approaches used across both illumina and roche/454 data , ramics outperforms all other methods tested here in terms of alignment quality . we must reiterate , however , that all approaches here are being tested against data that have been simulated to test the use case for which ramics has been specifically designed . while ramics outperforms all of the mapping approaches tested here , it is context based ( focused on coding dna that is divergent from the reference sequence ) and , thus , it should not be assumed that ramics outperforms all methods in all scenarios , e.g. whole - genome mapping . while alignment accuracy for mapping tools is essential , the scale of the data sets being generated by high - throughput sequencing approaches means that mapping speed is also a critical factor for an optimal method . with this in mind we have designed ramics to harness the computing power of graphic processing units ( gpus ) for optimal speed . we have also developed a cpu - based version for researchers who do not have access to gpu hardware . here , we have compared the mapping speeds of both the cpu and gpu versions of ramics against the other mapping approaches in mapping one million roche/454 reads from the hiv rt gene to a reference sequence . we found that , on average , the speeds for all approaches scale linearly as the data set size increases ( data not shown ) and find that in most instances nextgenmap is the fastest of the tools tested while mosaik is the slowest ( figure 6 ) . not surprisingly , we find that the gpu version of ramics outperforms the cpu version with as much as a 3-fold speedup . nonetheless we find that ramics performs in line with most of the read mappers tested . comparing mapping tools in terms of both speed ( reads mapped per second ) and accuracy ( average q score of the roche/454 reads generated using the afab benchmarking tool ) . the error bars on each data point reflect the range for both the speed ( y - axis ) and accuracy ( x - axis ) . ramics speed performance remains competitive with some of the fastest mappers , while its quality performance outstrips them significantly . the majority of the other mapping tools are built to align reads quickly to a large genome using various heuristics and , thus , much of the overhead necessary to hash a large genome is redundant in these analyses . ramics , on the other hand , is designed to perform a full dynamic programming alignment to a shorter reference sequence quickly and accurately . comparing the ramics gpu version to the emboss tool needle ( 45 ) , which performs a full dynamic programming alignment , shows that ramics is roughly twice as fast ( figure 6 ) . further , the efficiency of ramics is evident in that the codon - based approach implemented means it must perform eight comparisons to needle 's three , and makes extensive use of floating point arithmetic to achieve the fine - grained distinctions required for its more complicated alignment model . wunsch style alignment but takes into account homopolymer errors , is itself twice as fast as the full ramics algorithm ( data not shown ) . there should , however , be no trade - off in quality for speed or vice versa and , thus , the optimal mapping approach is one that can map the greatest number of reads with the highest accuracy in the shortest time . by evaluating each mapping approach in terms of speed and accuracy together ( figure 6 ) , we find that the gpu - based version of the full ramics codon - based algorithm outperforms all other approaches suggesting that , for amplicon - style analyses at least , many of the other approaches exhibit such a trade - off . to our knowledge , ramics is the first mapping tool that undertakes accurate mapping of coding dna to a reference sequence at speed , while concurrently identifying and correcting sequencing platform - induced errors . ramics accuracy in generating biologically correct alignments of coding dna means that it can be applied wherever the generation of high - quality alignments for sensitive detection of snps and novel variants are required . the ramics software ( university of the western cape , copyright reserved , 2013 ) is available under license from the university of the western cape , south africa at http://hiv.sanbi.ac.za/tools#/ramics . terms of the license will include but are not limited to the following : an annual up - front licensing fee for use only will be applicable for any use of the software for commercial purposes ( to be defined ) , and a free use only license will be available to verified and approved academic institutions and public not - for - profit research organizations for non - commercial use and/or application only . south african department of science and technology [ to s.a.t . ] ; south african national research foundation daad study bursary ( 11/4/1 ) [ to i.a.w . ] ; south african medical research council [ to sanbi ] .

the challenge presented by high - throughput sequencing necessitates the development of novel tools for accurate alignment of reads to reference sequences . current approaches focus on using heuristics to map reads quickly to large genomes , rather than generating highly accurate alignments in coding regions . such approaches are , thus , unsuited for applications such as amplicon - based analysis and the realignment phase of exome sequencing and rna - seq , where accurate and biologically relevant alignment of coding regions is critical . to facilitate such analyses , we have developed a novel tool , ramics , that is tailored to mapping large numbers of sequence reads to short lengths ( < 10 000 bp ) of coding dna . ramics utilizes profile hidden markov models to discover the open reading frame of each sequence and aligns to the reference sequence in a biologically relevant manner , distinguishing between genuine codon - sized indels and frameshift mutations . this approach facilitates the generation of highly accurate alignments , accounting for the error biases of the sequencing machine used to generate reads , particularly at homopolymer regions . performance improvements are gained through the use of graphics processing units , which increase the speed of mapping through parallelization . ramics substantially outperforms all other mapping approaches tested in terms of alignment quality while maintaining highly competitive speed performance .

INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION AVAILABILITY SUPPLEMENTARY DATA FUNDING

the issue of accurate pairwise sequence alignment is common to many fields in bioinformatics , whether as a core tool in fields such as reference - guided genome assembly ( 16 ) or as the seed for the generation of a progressive multiple sequence alignment ( 79 ) . this is particularly challenging in the era of high - throughput sequencing , where every platform produces systematic errors ( 1015 ) that should be considered in constructing an alignment . the alignment of coding dna , in particular , presents a unique challenge as it is critical that the final alignment takes into account the correct reading frame . preservation of the reading frame ensures correct calling of gene structure ( 1618 ) and snps , whether in , for example , the realignment phase of an exome sequencing pipeline ( 19 ) , single - nucleotide polymorphism ( snp ) calling from existing rna - seq data ( 20,21 ) or amplicon - based analyses such as human immunodeficiency virus ( hiv ) drug resistance genotyping ( 22 ) . when aligning coding dna generated using high - throughput sequencing platforms , it is critical that codons present in the open reading frame remain intact and that codon - sized insertions and deletions are recognized and called correctly , as distinct from both genuine frameshifts and single indels created through sequencing error . the landscape of pairwise alignment and reference mapping tools for high - throughput sequencing data is broad . tools such as bowtie 2 ( 2 ) and bwa - mem ( 3 ) , while well - suited to mapping the location of query sequence reads within a complete reference genome , lack the subtle nuances required to correctly distinguish spurious indels from genuine codon - sized mutations in coding dna . while effective in some cases , this approach is useless in the presence of indels in the open reading frame , which results in mistranslation to amino acid space . tools such as transalign ( 27 ) repetitively translate , align , back - translate and correct multiple sequence alignments , which to some extent addresses frameshift errors . this , thus , facilitates the identification of indels , whether genuine frameshift mutations or the result of sequencing error . initial models of coding dna such as those used by pairwise ( 16 ) and est_genome ( 28 ) were designed specifically for gene prediction , comparing sequences against all six translated frames simultaneously using a classical dynamic programming approach to discover exons . the hmmer tool ( 30 ) took a different approach , using profile hidden markov models to align and compare protein sequences . genewise and genomewise ( 29 ) use a hidden markov model approach to allow for the alignment of a nucleotide sequence , potentially including frameshifts , to an existing protein sequence , and thus represent the closest approximation to a codon codon alignment tool . here , we present a novel reference mapper ( ramics : rapid amplicon mapping in codon space ) that uses a hidden markov model approach to align large numbers of coding dna sequences to a coding region of a reference sequence in an accurate and biologically relevant manner , accounting for the inherent error biases of the sequencing platform used . ramics is developed to be executed on graphics processing units ( gpus ) and , thus , provides significant speed improvements over other , similarly complex , mapping tools . ramics is , thus , ideal for any situation where a large number of sequence reads from a known , coding location in a genome must be mapped to a coding reference sequence . the tool first discerns the optimal open reading frame of the reference sequence by selecting a frame that would minimize the number of stop codons . this enables a correct and relevant comparison of a query sequence to the reference , as the concept of a codon - sized insertion that begins within one codon and ends within another ( as would be output by most conventional alignment approaches ) is structurally meaningless in a biological context ( figure 2a ) . ramics , on the other hand , considers the biological importance of codons and generates a biologically relevant alignment preserving the codon structure of the coding dna being aligned . however , ramics can not concurrently align sequence reads to coding and non - coding regions of the same reference sequence ( e.g. it can , however , be subsequently used for targeted realignment of coding regions as a second - pass mapping tool . to illustrate the necessity of considering the sequencing platform used to generate the sequence data , we take an example from hiv-1 antiretroviral drug resistance testing . the k103n codon is located in a homopolymer region and some high - throughput sequencing platforms such as roche/454 ( 35 ) and ion torrent pgm ( 32 ) , as well as to a lesser extent the illumina platform ( 37 ) , are prone to increased error rates in regions containing homopolymers , as they can not successfully identify the correct number of nucleotides in these regions ( 12,13 ) . wunsch alignment to the hxb2 reference sequence of a sequence containing both the k103n mutation and a spurious extra adenosine directly following homopolymer region would result in the virus in question being falsely classified as susceptible to nnrtis such as efavirenz ( figure 2b ) . mapping of the same data using ramics , however , weighs the possibility of a false tyrosine ( t ) insertion in the center of a homopolymer region against a false adenosine ( a ) insertion at the end of the region , assigns the latter a higher probability and , thus , correctly calls the presence of the k103 m mutation in spite of the hompolymer error ( figure 2b ) . thus , a major strength of ramics is in its application to situations where the correctness of alignment in a coding region is crucial , such as alignment optimization in rna - seq and exome sequencing ( 1921,38,39 ) . it can be a common case in reference mapping , particularly for amplicon - based analyses , that a set of query sequence reads will be more closely related to each other than they will to a given reference sequence this is true in reference - guided assembly ( 40 ) , drug - resistance genotyping ( 41 ) and applications in metagenomic function prediction ( 42 ) . ramics circumvents any potential bias introduced through a distantly related reference sequence by allowing the use of the reference sequence as a guide for the initial mapping and then iteratively training the underlying hidden markov model to the resulting pairwise alignments . the result is a set of emission and transition probabilities that will correctly align outlier sequences ( potentially with many sequencing errors ) in the query set , based on the substitutions in sequences more similar to the reference . ramics produces results in various formats with the choice of output format very much driven by the end - users requirements : a global nucleotide alignment of all query sequences to the reference sequence , in fasta format.a derivation of the above global nucleotide alignment in which all single- and double - nucleotide insertions are widened to the width of a full codon using gaps ( codons.fasta ) . this does not alter the sequences , but allows the open reading frame to be read clearly in alignment viewers such as seaview ( 43).a global alignment with all single- and double - nucleotide insertions removed ( clean.fasta ) . while this is extremely useful in some amplicon - based analyses where frameshift mutation can be safely treated as pcr / sequencing errors , it should not be used in cases where a frameshift mutation may have biological relevance.a global alignment containing only one instance of each identical sequence with a count of the number of contributing sequence reads added to the sequence descriptor.a set of sam files describing mapped reads , with soft clipping used beyond the end of the reference sequence.a file containing a set of pairwise gapped fasta format alignments of each query sequence to the reference . a global nucleotide alignment of all query sequences to the reference sequence , in fasta format . this does not alter the sequences , but allows the open reading frame to be read clearly in alignment viewers such as seaview ( 43 ) . a global alignment containing only one instance of each identical sequence with a count of the number of contributing sequence reads added to the sequence descriptor . a set of sam files describing mapped reads , with soft clipping used beyond the end of the reference sequence . a file containing a set of pairwise gapped fasta format alignments of each query sequence to the reference . ramics is designed to map large numbers of high - throughput sequencing reads from a coding dna region to a similar reference sequence , both quickly and accurately . ramics also has potential as an alignment refiner for the coding regions of whole - genome mapping or as the seed pairwise aligner for a profile hmm multiple sequence aligner in the style of the protein sequence aligner clustal omega ( 44 ) . both as a standalone amplicon mapper and as a component of these larger toolchains , it can align large numbers of reads quickly as well as aligning reads accurately in regions that have similar biological features but contain many synonymous or functionally similar substitutions . these tools represent a mix of global , local and glocal alignment techniques ( 47 ) , as for the mapping of short reads to a relatively short amplicon all methods are potentially valid . rabema uses the mason read simulator ( 50 ) to simulate artificial short reads containing sequencing errors from a user - provided reference sequence and to output a the alignments generated by a mapping tool are then compared to the gold standard alignments . while rabema is a good benchmark of a mapping tool 's ability to detect sequencing errors such as spurious indels , it only determines whether an alignment is correct or incorrect and , thus , does not allow quantification of how incorrect the alignment is from the gold standard. thus , we developed a flexible alignment benchmark ( afab ) that could evaluate not only whether a correct alignment was found , but also how similar this alignment was to a gold standard alignment , in the case where very divergent sequences made perfect mapping impossible . gold standard alignment of protein coding dna which was the highly manually curated hiv-1 group m subtype reference alignment of the hiv envelope gene from the lanl hiv reference sequence database ( 51 ) . further , art generated sam files containing the correct alignment of each simulated read to the reference sequence ( the first sequence ) , which were used as the gold standard for evaluation of the accuracy of the various mapping tools . for both the roche/454 and illumina data the qscore tool ( 8) was used to evaluate each of the mapping tools , giving the q score and cline shift similarity scores of each tool 's generated alignment to the gold standard . when multiple tools achieved the same score ( most often 1.0 in the case of trivially mappable reads ) , we assigned each tool the average score they would receive , to avoid artificially inflating the scores of tools that chose to map only trivially mappable reads . we mapped both the roche/454 and illumina versions of the afab benchmarking data using the simplest incarnation of ramics ( without codon alignment or training ) , ramics with codon alignment and the full ramics algorithm ( with both the codon - based alignment and training parameters invoked ) . the addition of codon - based alignment increased the number of perfectly aligned sequence reads by 7.9 and 10.5% for the roche/454 and illumina data , respectively ( figure 3 ) . the number of discarded reads remained mostly unchanged regardless of the parameters used ( figure 3 ) showing that while alignment accuracy increases with algorithmic complexity , ramics is consistently capable of distinguishing between mappable and unmappable reads . the lesser impact of the training parameter can be explained by its intended use case : the proper alignment of extreme outlier reads with large numbers of sequencing errors . thus , in most instances we suggest that ramics should be used with both the codon - based alignment and training parameters invoked . not discarded by the mapping tool but with an incorrect alignment when compared to the gold standard ) as well as the percentage of reads that resulted in a perfect alignment when compared with the gold standard alignment . for both of the data the use case for which ramics is designed is rather unlike that for which most other mapping tools are tailored , because of its focus on coding dna . despite this , many studies have used various mapping tools to analyze sequence reads in ways similar to that which ramics was designed for ( 5558 ) and , thus , we present comparisons of ramics to a number of commonly used mapping tools on the basis of alignment quality and speed . reads were simulated from a single reference sequence using the reported error rates of the sequencing platforms as encoded by the mason read simulator ( 50 ) . for example , if the seed sequence used to generate a read was 100 nucleotides long and the resulting read had three differences relative to its seed , then that read would be added into the 3% error bin . as would be expected , the observed error rate for vast majority of sequence reads for the illumina data set fell around 1% with 88% of sequences exhibiting an error rate of 1% . for the 454 data , 49% of the sequence reads exhibited an error rate of 1% while the error rate of the remaining reads was in excess of 1% . the higher - than - expected number of sequences with an error rate of > 1% most likely results from the fact that the single reference sequence used to seed the simulations contained a number of homopolymer regions , which are known to increase the overall observed error rate of the roche/454 platform ( 12,13 ) . for the roche/454 benchmark , the performance of all of the tools tested was comparable for reads containing zero errors , ranging from 100% to 97.9% of reads correctly mapped for ramics and ssaha , respectively ( figure 4a ) . such high performance is to be expected , as these sequence reads are identical to the reference sequence . as the percentage of erroneous nucleotides in a read increased , the accuracy of each of the mapping tools , with the exception of ramics , reduced ( figure 4a ) . the percentage of reads mapped correctly as a function of the percentage error in simulated sequence reads for both the roche/454 ( a ) and illumina ( b ) sequencing platforms . a similar result was observed for the illumina benchmarking data with all methods correctly mapping 100% of reads at zero error rate and the accuracy of the various approaches , with the exception of ramics , reducing as the percentage error rate increased ( figure 4b ) . for the illumina benchmark data we observed that compared to the roche/454 data , we suggest this effect is due to the shorter lengths of illumina reads , which leave fewer correct nucleotides on either side of an error with which to anchor an alignment . the rabema benchmark clearly shows that ramics outperforms all other mapping approaches on the outlier sequences with a higher - than - average error rate . however , these data are not a true reflection of the type of data for which ramics has been optimized , namely mapping a diverse set of sequence reads to a related reference sequence . rabema data only differ from the reference sequence through the insertion of sequencing errors and , thus , we developed a benchmarking tool to enable the assessment of mapping tools at aligning coding sequence reads containing sequencing errors that are diverse from the reference sequence . in contrast to the rabema benchmarking tool , this approach enabled the use of two robust methods for evaluating relative alignment accuracy enabling the identification of perfectly aligned reads and misaligned reads . firstly , we considered the percentage of reads that each mapping approach was capable of aligning regardless of the quality of the alignment . not discarded by the mapping tool but with an incorrect alignment when compared to the gold standard ) as well as the percentage of reads that resulted in a perfect alignment when compared with the gold standard alignment . as expected , because these data contained sequencing errors and were diverse from the reference sequence , the percentage of perfectly mapped reads was lower for all approaches than was observed for the rabema benchmarking data . for the illumina data , ramics significantly outperforms all of the other mapping approaches by aligning 62% of the total number of reads perfectly , with the next best approach ( needle ) mapping 48% of all reads correctly ( figure 5 ) . for the roche/454 data ramics performed best , correctly mapping 36% of reads perfectly , while the performance of the other methods ranges between 19% ( mosaik ) and 34% ( ssaha2 ) . for all of the reads that were aligned by one or more mapping tools we used two approaches for evaluating alignment quality , the q score ( 59 ) and cline shift score ( 60 ) , to rank each of the mapping tools for each of the 5500 pairs of sequences reads that were tested . the addition of the gatk ( 23 ) to the bwa - mem toolchain , the approach used in the 1000 genomes project ( 25 ) , had a negligible effect on the quality of alignment for this use case . thus , for both benchmarking approaches used across both illumina and roche/454 data , ramics outperforms all other methods tested here in terms of alignment quality . while ramics outperforms all of the mapping approaches tested here , it is context based ( focused on coding dna that is divergent from the reference sequence ) and , thus , it should not be assumed that ramics outperforms all methods in all scenarios , e.g. while alignment accuracy for mapping tools is essential , the scale of the data sets being generated by high - throughput sequencing approaches means that mapping speed is also a critical factor for an optimal method . we have also developed a cpu - based version for researchers who do not have access to gpu hardware . here , we have compared the mapping speeds of both the cpu and gpu versions of ramics against the other mapping approaches in mapping one million roche/454 reads from the hiv rt gene to a reference sequence . comparing mapping tools in terms of both speed ( reads mapped per second ) and accuracy ( average q score of the roche/454 reads generated using the afab benchmarking tool ) . the error bars on each data point reflect the range for both the speed ( y - axis ) and accuracy ( x - axis ) . the majority of the other mapping tools are built to align reads quickly to a large genome using various heuristics and , thus , much of the overhead necessary to hash a large genome is redundant in these analyses . further , the efficiency of ramics is evident in that the codon - based approach implemented means it must perform eight comparisons to needle 's three , and makes extensive use of floating point arithmetic to achieve the fine - grained distinctions required for its more complicated alignment model . there should , however , be no trade - off in quality for speed or vice versa and , thus , the optimal mapping approach is one that can map the greatest number of reads with the highest accuracy in the shortest time . by evaluating each mapping approach in terms of speed and accuracy together ( figure 6 ) , we find that the gpu - based version of the full ramics codon - based algorithm outperforms all other approaches suggesting that , for amplicon - style analyses at least , many of the other approaches exhibit such a trade - off . to our knowledge , ramics is the first mapping tool that undertakes accurate mapping of coding dna to a reference sequence at speed , while concurrently identifying and correcting sequencing platform - induced errors . ramics accuracy in generating biologically correct alignments of coding dna means that it can be applied wherever the generation of high - quality alignments for sensitive detection of snps and novel variants are required . terms of the license will include but are not limited to the following : an annual up - front licensing fee for use only will be applicable for any use of the software for commercial purposes ( to be defined ) , and a free use only license will be available to verified and approved academic institutions and public not - for - profit research organizations for non - commercial use and/or application only .

so - called scent glands ( syn . defensive glands ) are not only considered an important synapomorphic character of all harvestmen ( opiliones ) , but also represent by far the largest exocrine system in this arachnid order . generally , these glands constitute paired sacs in the opilionid prosoma , opening onto the body surface via one large orifice ( ozopore ) on each of the lateral margins of the carapax dorsal to the coxae of legs i or ii . their main biological role is thought to be predator defense ( e.g. , martens , 1978 ) , but scent glands also are sites of production of antimicrobial agents ( e.g. , fieser and ardao , 1956 ) or , in certain species , may emit pheromones ( holmberg , 1986 ; machado et al . , 2002 ) . moreover , opilionid scent glands are the sources of a diversity of rare natural products , and the chemistry of their secretions is characteristic for specific opilionid groups . for example , the secretions of cyphophthalmi are complex and contain specific blends of naphthoquinones and methyl ketones ( raspotnig et al . hitherto known secretions of the palpatores include acyclic ketones , alcohols , and aldehydes , present in some sclerosomatid eupnoi ( blum and edgar , 1971 ; meinwald et al . , 1971 ; jones et al . , 1976 , 1977 ; ekpa et al . , 1985 ) , and naphthoquinones reported from one species of phalangiid eupnoi ( wiemer et al . , 1978 only one study is available on scent gland secretions of the dyspnoi , demonstrating the presence of naphthoquinones , methoxy - naphthoquinones , and anthraquinones in the scent glands of a nemastomatid species ( raspotnig et al . , 2010 ) . however , with respect to the number of species investigated , secretions of the mostly tropical laniatores appear to be best studied . laniatorean secretions are chemically distinct from the secretions of cyphophthalmids and palpatoreans , and specific patterns of phenols and benzoquinones have been demonstrated from a growing number of species since the 1950s ( e.g. , estable et al . , 1955 ; eisner et al . , 1971 , 1977 , 2004 ; roach et al . , 1980 ; hara et al . , 2005 ; review by gnaspini and hara , 2007 ; shear et al . , 2010a , b ; pomini et al . , 2010 ; fttinger et al . , essentially , nearly all published studies deal with representatives of groups of more - derivative laniatores . thus , the current knowledge on laniatorean secretions is strongly biased in favor of the grassatores . a second , more basal laniatorean group is almost unknown , but is crucial for a consistent picture of opilionid scent gland chemistry . only one example of insidiatorean chemistry has been published so far ( ekpa et al . , 1984 ) , revealing a series of completely aberrant components from an american travunioid species , sclerobunus robustus . these compounds include terpenes , such as bornyl esters , camphene , and limonene , and two alkaloids , namely n , n - dimethyl - - phenylethylamine and the tobacco alkaloid nicotine . one important and well recognized taxon within the insidiatores is the cladonychiidae ( superfamily travunioidea ) , including about 15 species in 5 extant genera ( a 6th genus was described from baltic amber by ubick and dunlop in 2005 ) . while genera cryptomaster , erebomaster , speleomaster , and theromaster are restricted to the northern united states , an exclusively european clade is represented by eight species of holoscotolemon , occurring from western europe to romania and southwards to serbia and montenegro . as a first contribution to scent gland research in european insidiatores , we here report on the scent gland chemistry of four species of holoscotolemon , covering a representative part of european cladonychiidae . collection of species specimens of holoscotolemon jaqueti ( corti , 1905 ) , h. lessiniense martens , 1978 , h. oreophilum martens , 1978 , and h. unicolor roewer , 1915 , were collected by hand under stones and litter , and from samples of leaf litter by means of a soil sifter . while h. jaqueti is a species with a disjunct south - east european and carpathian distribution ( hungary , slovakia , romania , ukraine , bosnia and hercegovina , serbia ) , h. unicolor is restricted to the eastern alps ( austria , slovenia , north eastern italy ) , and both h. lessiniense and h. oreophilum are local endemics of the southwestern alps ( italy ) . holoscotolemon oreophilum can be found in a small area in the sea alps / alpes maritimes and ligurian alps in italy , and h. lessiniense inhabits the monti lessini close to the lake garda in the southern alps . the investigated material was collected in serbia ( h. jaqueti ) , italy ( h. oreophilum , h. lessiniense ) , and austria ( h. unicolor ) ( table 1 ) . for determination of the holoscotolemon species , we used descriptions of martens ( 1978 ) . komposch ( graz ) , g. raspotnig ( graz ) , and i. karaman ( novi sad ) . table 1collection of holoscotolemon - species and preparation of extractsspecieslocation ( co - ordinates , altitude)dateextracts no.(individuals)h . jaquetiserbia : ovar - kablar gorge , above ovar banja ( 4353n , 2011e , 375 m)20 mai 20091754 ( 1 juv ) , 1755 ( 1 ) , m430 ( 1 juv)h . oreophilumitaly : alpi marittime , coli de tende s limonette ( 4409 n , 732 e , 1,475 m)29 july 20102447 ( 1 ) , 2448 ( 1 )h . lessini , monte pasubio ( 4546 n , 1107 e , 760 m)17 july 2009m353 ( 1 ) , m354 ( 1 ) , m355 ( 1 ) , m356 ( 1)italy : mt . lessini , nne verona , s velo veronese ( 4535n , 1104 e , 920 m)31 july 20102449 ( 1 )h . unicoloraustria : carinthia , villach , eichholzgraben ( 4638 n , 1350 e , 590 m)5 april 20101935 ( 1 ) , 1936 ( 1)austria : styria , graz , maria trost ( 47 06 n , 15 29 e , 445 m)20 april 20101973 ( 1 juv ) , 1983 ( 3 juv)25 april 20101995 ( 3 juv)10 may 20102000 ( 2 juv ) , 2001 ( 2 juv)austria : styria , aibl ( 46 41 n , 15 13 e , 400 m)27 june 20102127 ( 1 juv)austria : carinthia , soboth , road between krumbach and rothwein ( 4681 n , 1507 e , 1,000 m)23 june 20102243 ( 1 ) , 2244 ( 1 ) , 2245 ( 1)refers to intern labelling of extracts collection of holoscotolemon - species and preparation of extracts refers to intern labelling of extracts extraction and analysis of secretions scent gland secretion was obtained by whole body extraction of individuals in 150 l of hexane or ethyl acetate for about 30 min as previously described and standardized for other opilionids ( raspotnig et al . 21 extracts were prepared , mostly extracts of single individuals ( adults ) , but in some cases also pooled extracts ( juveniles ) , as summarized in table 1 . aliquots of extracts ( 2 l ) were subject to gas chromatographic - mass spectrometric ( gc - ms ) analysis , using a trace gc2000 coupled to a voyager ms , both from thermo ( vienna , austria ) . the gc was equipped with a zb-5ms fused silica capillary column ( 30 m 0.25 mm i.d . injection was splitless with helium ( at 1.5 ml / min ) as carrier gas . the column temperature was programmed from 50c ( held for 1 min ) to 200c at 10c / min , and then to 300c at 15c / min . the ion source of the ms and the transfer line were kept at 170c and 310c , respectively . electron impact ( ei ) reference compounds authentic nicotine ( mixture of ( r)- and ( s)-nicotine ) for comparison of gc - ms data was purchased from sigma ( vienna , austria ) . as a reference for authentic anabaseine , we used anabasine ( from sigma , vienna , austria ) , which also contained a small amount of anabaseine ( about 1% ) . quantification of secretion components determination of absolute amounts of nicotine in secretions are based on the integration of peak areas in the chromatograms , and comparisons to a calibration curve established for authentic nicotine . scanning electron microscopy for scanning electron microscopy ( sem ) , specimens were fixed in bouin , washed , dehydrated , air - dried , and mounted on aluminum stubs prior to sputter coating with gold ( agar sputter coater , grpl , tulln , austria ) . micrographs ( sem ) were taken with a philips xl30 esem ( philips / fei , vienna , austria ) at high vacuum mode and 20 kv accelerating voltage . chemical identification of extract components gc - ms analyses of extracts of adult holoscotolemon jaqueti , h. oreophilum , and h. lessiniense revealed two major ( components a and e ) , and three minor compounds ( components b , c , and d ) ( fig . 1 ) . all components were identified as pyridine alkaloids as outlined in the following and as summarized in table 2 . compound a [ nicotine ] , compound b [ 3-(1-methyl-2-piperidinyl)-pyridine ] , compound c [ anabasine ] , compound d [ 2,3-bipyridyl ] , compound e [ anabaseine ] . * compounds at rt = 13.56 min and rt = 14.55 min were found inconsistently in extracts of holoscotolemon lessiniense , and possibly represent isomeric , not fully characterized pyridines with a molecular weight of m = 174table 2gas chromatographic and mass spectral data to components from extracts of holoscotolemon lessiniense , h. oreophilum , and h. jaquetipeakretention time ( min)ei - fragmentation ( m / z)identified asa10.54162 ( m , 49 ) , 161 ( 46 ) , 133 ( 79 ) , 119 ( 24 ) , 118 ( 16 ) , 92 ( 23 ) , 84 ( 100 ) , 65 ( 21 ) , 51 ( 22 ) , 42 ( 61)nicotine = ( s)-3-(1-methyl-2-pyrrolidinyl)-pyridineb11.85176 ( m , 12 ) , 175 ( 7 ) , 147 ( 7 ) , 133 ( 9 ) , 119 ( 24 ) , 98 ( 100 ) , 42 ( 28)3-(1-methyl-2-piperidinyl)-pyridinec12.29162 ( m , 26 ) , 161 ( 23 ) , 133 ( 45 ) , 119 ( 45 ) , 106 ( 49 ) , 105 ( 60 ) , 92 ( 19 ) , 84 ( 100 ) , 80 ( 22 ) , 78 ( 20 ) , 57 ( 29 ) , 51 ( 28 ) , 41 ( 38)anabasine = 3-(2-piperidinyl)-pyridined12.89157 ( 12 ) , 156 ( m , 100 ) , 155 ( 71 ) , 130 ( 20 ) , 128 ( 12 ) , 104 ( 7 ) , 79 ( 13 ) , 78 ( 22 ) , 51 ( 13)2,3-bipyridinyl = 2-(3-pyridinyl)-pyridinee13.35161 ( 12 ) , 160 ( m , 97 ) , 159 ( 100 ) , 145 ( 46 ) , 132 ( 14 ) , 131 ( 79 ) , 118 ( 8) , 105 ( 44 ) , 104 ( 76 ) , 78 ( 27 ) , 77 ( 29 ) , 51 ( 36 ) , 41 ( 29)anabaseine = 3,4,4,6-tetrahydro-2,3-bipyridinetentatively identified on the basis of mass spectral data chemical profiles of scent gland secretions in three species of holoscotolemon . compound a [ nicotine ] , compound b [ 3-(1-methyl-2-piperidinyl)-pyridine ] , compound c [ anabasine ] , compound d [ 2,3-bipyridyl ] , compound e [ anabaseine ] . * compounds at rt = 13.56 min and rt = 14.55 min were found inconsistently in extracts of holoscotolemon lessiniense , and possibly represent isomeric , not fully characterized pyridines with a molecular weight of m = 174 gas chromatographic and mass spectral data to components from extracts of holoscotolemon lessiniense , h. oreophilum , and h. jaqueti tentatively identified on the basis of mass spectral data major components component a showed an ei - mass spectrum consistent with that of nicotine [ 3-(1-methyl-2-pyrrolidinyl)-pyridine ] , exhibiting a molecular ion ( m ) at a mass charge / ratio ( m / z ) of 162 , loss of a hydrogen atom ( m / z 161 ) , and a base ion at m / z 84 . the base ion corresponded to loss of the pyridyl moiety ( m 78 ) . further fragments were observed at m / z 133 ( m 29 ) and m / z 119 ( m 43 ) , both of which are reported as characteristic of nicotine , and correspond to losses of c2h5- and c3h7-radicals from the molecular ion by different processes of rearrangement ( duffield et al . , 1965 ) . as a reference for comparison of gc retention , we used a racemic mixture of nicotine ( nicotine has an asymmetric carbon atom in position 2 of the pyrrolidine - ring ) . the specific enantiomers , however , were not separable due to the chromatographic conditions used , and both enantiomers eluted as a single peak at rt = 10.54 min , exactly matching the retention time of component a. thus , we did not determine the chirality of nicotine from holoscotolemon spp . so far , only the ( s)-enantiomer is known to occur in nature.the ei - mass spectrum of the second major component ( e ) had a molecular ion at m / z 160 , again exhibiting loss of hydrogen ( base ion at m / z 159 ) , and losses of 15 ( leading to the ion at m / z 145 ) , 29 ( m / z 131 ) , and 56 ( m / z 104 ) mass units . the spectrum appeared to be similar , but not fully consistent , with reported spectra of anabaseine ( a nicotine - related tobacco nicotinoid ) from the literature ( e.g. , wheeler et al . , 1981 ) . however , a comparison of mass spectral fragmentation and retention times to an authentic sample of anabaseine [ 3,4,5,6-tetrahydro-2,3-bipyridine ] showed full correspondence . minor components on the basis of mass spectral fragmentation patterns of components b and c , two further nicotinic alkaloids were indicated , both showing mass spectra similar to that of nicotine : component b appeared to be a higher homolog to nicotine with m at m / z 176 and a base ion at m / z 98 , suggesting a piperidine - structure instead of the pyrrolidine - ring . the compound was tentatively identified as 3-(1-methyl-2-piperidinyl)-pyridine by mass spectral comparisons to literature , and to spectra from the nist - library . the mass spectral fragmentation of component c differed from that of nicotine mainly in the presence of prominent twin ions at m / z 105 and m / z 106 , being characteristic of anabasine [ = 3-(2-piperidinyl)-pyridine ] . the compound was identified by comparisons of spectra and retention times with an authentic sample of anabasine . only the mass spectrum of component d was clearly different from all other spectra , although again showing an intense molecular ion at m / z 156 ( base ion ) along with loss of hydrogen ( m / z 155 ) . the isotopic m + 1-ion ( about 12% relative intensity ) still indicated the presence of 10 carbon and 2 nitrogen atoms , suggesting a molecular formula of c10h8n2 , and thus corresponding to the fully aromatic homolog of anabasine and anabaseine , 2,3-bipyridyl [ 2-(3-pyridinyl ) pyridine ] . subsequent mass spectral comparisons confirmed a mass spectral fragmentation pattern indistinguishable from spectra of authentic 2,3-bipyridyl from literature and from the nist - library . extract profiles two different extract patterns were clearly distinguished : ( 1 ) nicotine - rich and ( 2 ) anabaseine - rich extracts . the extracts of holoscotolemon oreophilum and h. jaqueti were dominated by compound a ( nicotine : > 97% of the secretion , based on calculation of peak areas , see also table 2 and fig . 1 ) . components b ( 3-(1-methyl-2-piperidinyl)-pyridine ) and c ( anabasine ) were found constantly as accompanying minor or trace components ( each about 1% of the secretion ) . in h. lessiniense , anabaseine ( compound e ) was the major compound in the extracts ( > 95% , see table 2 and fig . 1 ) , accompanied by trace amounts of anabasine and 2,3-bipyridyl ( each about 13% of the secretion ) . in two out of five extracts , two trace components were also found at rt = 13.56 min and 14.55 min ( each about 1% of the secretion ) , both showing a pyridine - like pattern and a molecular weight of 174 , but both remain unidentified.it is noteworthy that some individuals did not show any components . in particular , extracts of three specimens ( all collected at one locality in serbia , see table 1 ) of h. jaqueti were investigated , and two of these ( one female , one juvenile ) consistently showed the nicotine - rich composition mentioned above . in the extract of the remaining specimen ( a juvenile ) , no components could be detected . for h. lessiniense , we analyzed extracts of five individuals ( all female ) , which were from two different collections ( see table 1 ) : three extracts ( two of collection no . 1 , one of collection no . 1 ) . for the largest species of the genus , h. oreophilum , only two individuals ( one male , one female ) were available to us , both of which discharged large amounts of nicotine . absolute amounts of secretions were determined for h. oreophilum , and were calculated to be about 15 g nicotine per individual ( 11.8 g measured for one specimen , 16 g for the other specimen).thus , only for h. oreophilum ( nicotine - rich ) , could male and female extracts be compared , showing no differences . for h. jaqueti ( nicotine - rich ) and h. lessiniense ( anabaseine - rich ) , only females and juveniles ( subadult females ) were available ; between subadults and adults , holoscotolemon unicolor contrasting the results obtained from h. jaqueti , h. oreophilum , and h. lessiniense , no components were detected in any of the nine extracts from adults of h. unicolor . this was surprising since these specimens were from several different collection sites in austria and were collected at different times of the year ( see table 1 ) . scent glands in h. unicolor , as well as openings ( ozopores ) appeared to be as well developed as in the other holoscotolemon species ( fig . 2 ) . however , in one extract of an early - instar juvenile , traces of three nitrogen - containing compounds were detected ( not shown ) , whereas extracts of 11 other juveniles did not show any compounds . the trace components in the single juvenile extract were tentatively identified as dimethyl - isoalkylpyrazines by mass spectral comparisons to literature , and to spectra from the nist - library ( compound 1 : a dimethyl - isobutylpyrazine , m at m / z 164 ; compounds 2 and 3 : isomeric dimethyl - isopentylpyrazines , m at m / z 178 ) . choloscotolemon oreophilum ; dholoscotolemon lessiniense ; e and f details of left ozopore in holoscotolemon jaqueti position and morphology of ozopores in species of genus holoscotolemon . choloscotolemon oreophilum ; dholoscotolemon lessiniense ; e and f details of left ozopore in holoscotolemon jaqueti origin of extract components we assume that the compounds found in the whole body extracts of holoscotolemon herein investigated are from the scent glands of these species . this view is based on several facts , and on corroborating evidence from previous studies . first , scent glands are the only glandular structures capable of producing such amounts of secretions ; they are large and well developed in all species of the genus holoscotolemon . corroborative evidence for the scent gland origin of extract components comes from extraction - attempts of individuals with obviously depleted gland reservoirs . we assume that we may have examined many empty specimens , as indicated by the lack of any compound in the gas chromatograms of extracts . from previous investigations , we know that depletion of gland reservoirs can be a rapid process , mainly depending on external irritation stimuli . refill of glandular reservoirs is slow ; according to holmberg ( 1970 ) , this process takes a few days in phalangiids . in our study , the relation of specimens with well - filled gland reservoirs to specimens with depleted reservoirs was about 1:1 for h. jaqueti and h. lessiniense . glandular depletion may be explained by irritating stimuli in the course of collection and transport . absolute amounts of secretion , as shown for nicotine in extracts of h. oreophilum , were calculated to be about 15 g per individual . such amounts correspond to a volume of 15 nl of nicotine ( which is a liquid with a density of 1.01 g / ml at room temperature ) , and hence to two hypothetical spherical droplets of about 130 m in diameter . moreover , droplets of that size would be consistent with the expected dimension and filling capacity of glandular reservoirs in holoscotolemon , at least if glandular dimensions in other opilionids of comparable size are considered ( e.g. , juberthie , 1976 ; schaider and raspotnig , 2009).in general , the whole body extraction - method is widely used and commonly accepted for the assessment of exocrine products in small arthropods that are too tiny to sample or dab their secretions directly from the gland orifices . the method has been standardized for investigations of exocrine glandular products in oribatid and astigmatid mites over many years ( sakata and norton , 2001 ; raspotnig et al . , 2001 , 2008 ) , and also has been used to assess scent gland exudates of opilionids of different groups ( raspotnig et al . , 2005 , 2010 2010a , b ) . for instance , in sironids ( cyphophthalmi ) direct sampling of secretions from ozopores leads to the same chemical results as working with hexane whole body extracts ( raspotnig et al . , nicotine and anabaseine in general , tobacco alkaloids are unusual components in arthropod exocrine secretions . nicotine is known only from sclerobunus robustus ( a travunioid laniatorean ) ( www.pherobase.com ) . anabaseine , on the other hand , is new for scent gland secretions of opiliones . nicotine is a defensive neurotoxin of some plants , and anabaseine is a neurotoxin from nemertine worms that use it for paralyzing prey and possibly for deterring predators ( kem , 1971 ; kem et al . , 1971 , 1976 ) . one example , however , is the poison glands of certain myrmicine ants ( wheeler et al . , 1981 ; leclercq et al . , 2001 ; co et al . , 2003 ) , where anabaseine also may act as a part of the trail pheromone ( attygalle et al . , 1998 ) . in certain hymenopterans , anabaseine is found as a by - product in anabasine - rich secretions ( co et al . , 2003 ; cruz - lopez et al . , 2006 ) . anabaseine is remarkable for exhibiting striking neurotoxic effects by interacting with nicotinic receptors , and a synthetic anabaseine - derivative has proved to be a promising test agent to study and cure cognitive function loss associated with several human diseases ( kem et al . , 1997 , 2006 ) . to our knowledge , h. lessiniense is the only arthropod species producing an exocrine secretion almost exclusively based on anabaseine . it is further interesting to note that h. lessiniense ( anabaseine - rich ) and h. oreophilum ( nicotine - rich ) , both occurring in the italian southern alps and separated by an air - line distance of just 300 km , use differently composed secretions ; this might indicate more specific communicative functions of scent gland secretions in cladonychiid harvestmen , in addition to assumingly defensive roles . pyridines and alkyl - pyrazines three minor components in the holoscotolemon - secretions were classified as pyridines , chemically close to nicotine and anabaseine . these compounds may represent by - products of the biosynthetic pathways to nicotine and anabaseine , but the possibility of their artificial formation from major compounds in the hot injector of the gas chromatograph can not be excluded . in fact , small amounts of 2,3-bipyridyl also could be detected when analyzing an authentic sample of anabasine , calling the scent gland origin of 2,3-bipyridyl into question . on the other hand , 2,3-bipyridyl is known to represent a second neurotoxic component in nemertine worms , in addition to anabaseine ( kem et al . , 1976 ) , and also occurs in exocrine glands of certain aphaenogaster and messor ants ( attygalle et al . , 1998 ; co et al . , 2003 ) . the alkyl - pyrazines detected in the extract of one single juvenile of h. unicolor also are widespread exocrine compounds in several species of ants and wasps ( cavill and houghton , 1974 ; borg - karlson and teng , 1980 ; wheeler et al . , 1982 ; tecle et al . , 1987 ; brophy , 1989 ; jones et al . in some cases , alkyl pyrazines seem to accompany alkaloids of the anabasine - type as minor components in exocrine secretions of some hymenopterans ( e.g. , cruz - lopez et al . , 2006 ) . however , our preliminary chemical data for h. unicolor , deviating from the chemistry of the other three holoscotolemon species , need further clarification , and we hope to unravel this question in the near future . chemosystematics in genus holoscotolemon our study provides an analysis of the scent - gland chemistry of all four alpine and carpathian holoscotolemon species . in this context as well as from a zoogeographical and evolutionary point of view , questions concerning the presently inhabited areas of these species are of interest , particularly with respect to the hypothetical regions where they outlasted the last ice - age ( wrm ) . holoscotolemon unicolor is endemic to the eastern alps and , following holdhaus ( 1954 ) , thaler ( 1966 , 1976 ) , and komposch ( 2009 ) , is a re - wanderer of long distance , which probably survived the last ice age in holoscotolemon oreophilum and h. lessiniense are local endemics of the southern alps , and should have survived the wrm ice - age in massifs de refuge in the areas of their present distribution in the sea alps ( h. oreophilum ) and monti lessini ( h. lessiniense ) , respectively . martens ( 1978 ) found no close relationship between h. lessiniense and h. unicolor , and believed the former species is closer to h. oreophilum . therefore , it is surprising that the available chemical information points toward a high similarity between the most western ( h. oreophilum ) and the most eastern species ( h. jaqueti ) . thus , tentatively , nicotine may represent a plesiomorphic compound in holoscotolemon ( and possibly also in travunioidea , see below ) while h. lessiniense , occurring in an area in between , shows a clearly distinct although related chemistry ( i.e. , anabaseine - rich ) , possibly representing the derivative status . scent gland chemistry in insidiatores the major part of laniatorean scent gland research has been performed on grassatorean laniatores , and this strong bias may have contributed to the picture of a typical , rather homogenous , phenolic and benzoquinone - rich phenol- and benzoquinone - rich secretions have been identified from representatives of all grassatorean families hitherto investigated , such as many gonyleptidae ( see gnaspini and hara , 2007 and references therein ; hara et al . , 2005 ; machado and pomini , 2008 ; fttinger et al . , 2010 ) , roach et al . , 1980 ) , stygnommatidae ( duffield et al . , 1981 ) , stygnopsidae ( pomini et al . , 2010 ; shear et al . , 2010b ) , and phalangodidae ( shear et al . , 2010a ) . even though components other than phenols and benzoquinones ( i.e. , some accompanying acyclic compounds and alkyl - dihydro - pyrans ) have been found in certain gonyleptidae ( hara et al . , 2005 ; rocha et al . , 2011 ) , nitrogen - containing compounds or even tobacco alkaloids do not seem to occur in the scent glands of the grassatores . on the other hand , there is growing evidence that insidiatorean secretions or at least those of superfamily travunioidea are based on a completely different chemistry that relies on nitrogen - containing compounds as major secretory constituents . ( 1984 ) who found two nitrogen - containing compounds ( - dimethyl - phenylethylamine and nicotine ) in the scent gland secretion of sclerobunus robustus , a travunioid species from both another continent ( america ) and a second travunioid family ( travuniidae ; kury 2000 onwards ) . for grouping of northern hemisphere triaenonychoidea such as sclerobunus with travunioidea see giribet and kury ( 2007 ) . preliminary chemical results from representatives of travuniids in europe ( unpublished ) also indicate consistently that tobacco alkaloids constitute major secretory components in species of travunia and abasola ( for possible synonymies see kury and mendes 2007 ) as well as in the so - far systematically unplaced but travunioid trojanella ( karaman , 2005 ) . in this respect , nitrogen - containing compounds : ( 1 ) may be widespread or even common in the travunioidea ; and , ( 2 ) according to the basal phylogenetic position of travunioidea within laniatores , these compounds also may represent the ancestral scent gland equipment of laniatoreans.opilionid scent gland secretions generally seem to be a promising pool of group- or even species - specific chemical characters , representing an independent set of data of phylogenetic value , in addition to characters from traditional morphology and molecular genetics . the importance of scent gland chemistry for studies of opilionid phylogeny has been emphasized previously ( roach et al . , 1980 ; duffield et al . , 1981 ) , and more recent papers have added to this topic ( raspotnig et al . , 2005 , 2010 ; jones et al . , so far , an evolutionary line may be traced from the naphthoquinone- and acyclic ketone - rich secretions of the cyphophthalmi to the similarly structured secretions in eupnoid and dyspnoid palpatores ( see introduction ) , but any evolutionary link to the chemically distinct secretions of laniatores is still missing . additionally , within the laniatores , the chemical gap between the alkaloid - rich secretions of the travunioidea on the one hand and the generally phenol- and benzoquinone - rich chemistry in the grassatores on the other hand is not yet well explained . in this regard , information on the unknown scent gland chemistry of the true triaenonychoidea ( sensu kury , 2000 onwards ) would be important , as this mainly southern hemisphere - distributed group of insidiatores ( see pinto - da - rocha and giribet , 2007 ) may represent the missing link in the chemosystematic puzzle of harvestmen .

the exocrine secretions from prominently developed prosomal scent glands in four species of the european laniatorean harvestman genus holoscotolemon ( laniatores , travunioidea , cladonychiidae ) were analyzed by gas chromatography mass spectrometry . two major alkaloidal compounds were detected : nicotine accounted for more than 97% of the secretion in holoscotolemon jaqueti and h. oreophilum , whereas the chemically related nicotinoid alkaloid anabaseine was the major compound in h. lessiniense . in addition , a series of minor nitrogen - containing components was found , namely 3-(1-methyl-2-piperidinyl)-pyridine and anabasine , in h. jaqueti and h. oreophilum , and anabasine together with 2,3-bipyridyl in h. lessiniense . by contrast , extracts of adult h. unicolor did not show any components . in one juvenile specimen of h. unicolor , however , low amounts of alkyl pyrazines ( dimethyl - isobutyl- and dimethyl - isopentylpyrazines ) were detected . nitrogen - containing components previously were found in sclerobunus robustus ( an american travunioid harvestman ) , so scent gland - derived alkaloids may be widespread or even common in the travunioidea . alkaloids have not been reported for other opilionid scent gland secretions outside the travunioidea , and we hypothesize that they may be the phylogenetically ancestral allomones in the laniatores , having been reduced and replaced by a phenol- and benzoquinone - rich chemistry in the more derived grassatorean taxa .

Introduction Methods and Materials Results Discussion

moreover , opilionid scent glands are the sources of a diversity of rare natural products , and the chemistry of their secretions is characteristic for specific opilionid groups . , 1985 ) , and naphthoquinones reported from one species of phalangiid eupnoi ( wiemer et al . , 1978 only one study is available on scent gland secretions of the dyspnoi , demonstrating the presence of naphthoquinones , methoxy - naphthoquinones , and anthraquinones in the scent glands of a nemastomatid species ( raspotnig et al . , 1984 ) , revealing a series of completely aberrant components from an american travunioid species , sclerobunus robustus . these compounds include terpenes , such as bornyl esters , camphene , and limonene , and two alkaloids , namely n , n - dimethyl - - phenylethylamine and the tobacco alkaloid nicotine . as a first contribution to scent gland research in european insidiatores , we here report on the scent gland chemistry of four species of holoscotolemon , covering a representative part of european cladonychiidae . collection of species specimens of holoscotolemon jaqueti ( corti , 1905 ) , h. lessiniense martens , 1978 , h. oreophilum martens , 1978 , and h. unicolor roewer , 1915 , were collected by hand under stones and litter , and from samples of leaf litter by means of a soil sifter . while h. jaqueti is a species with a disjunct south - east european and carpathian distribution ( hungary , slovakia , romania , ukraine , bosnia and hercegovina , serbia ) , h. unicolor is restricted to the eastern alps ( austria , slovenia , north eastern italy ) , and both h. lessiniense and h. oreophilum are local endemics of the southwestern alps ( italy ) . holoscotolemon oreophilum can be found in a small area in the sea alps / alpes maritimes and ligurian alps in italy , and h. lessiniense inhabits the monti lessini close to the lake garda in the southern alps . the investigated material was collected in serbia ( h. jaqueti ) , italy ( h. oreophilum , h. lessiniense ) , and austria ( h. unicolor ) ( table 1 ) . unicoloraustria : carinthia , villach , eichholzgraben ( 4638 n , 1350 e , 590 m)5 april 20101935 ( 1 ) , 1936 ( 1)austria : styria , graz , maria trost ( 47 06 n , 15 29 e , 445 m)20 april 20101973 ( 1 juv ) , 1983 ( 3 juv)25 april 20101995 ( 3 juv)10 may 20102000 ( 2 juv ) , 2001 ( 2 juv)austria : styria , aibl ( 46 41 n , 15 13 e , 400 m)27 june 20102127 ( 1 juv)austria : carinthia , soboth , road between krumbach and rothwein ( 4681 n , 1507 e , 1,000 m)23 june 20102243 ( 1 ) , 2244 ( 1 ) , 2245 ( 1)refers to intern labelling of extracts collection of holoscotolemon - species and preparation of extracts refers to intern labelling of extracts extraction and analysis of secretions scent gland secretion was obtained by whole body extraction of individuals in 150 l of hexane or ethyl acetate for about 30 min as previously described and standardized for other opilionids ( raspotnig et al . quantification of secretion components determination of absolute amounts of nicotine in secretions are based on the integration of peak areas in the chromatograms , and comparisons to a calibration curve established for authentic nicotine . chemical identification of extract components gc - ms analyses of extracts of adult holoscotolemon jaqueti , h. oreophilum , and h. lessiniense revealed two major ( components a and e ) , and three minor compounds ( components b , c , and d ) ( fig . * compounds at rt = 13.56 min and rt = 14.55 min were found inconsistently in extracts of holoscotolemon lessiniense , and possibly represent isomeric , not fully characterized pyridines with a molecular weight of m = 174table 2gas chromatographic and mass spectral data to components from extracts of holoscotolemon lessiniense , h. oreophilum , and h. jaquetipeakretention time ( min)ei - fragmentation ( m / z)identified asa10.54162 ( m , 49 ) , 161 ( 46 ) , 133 ( 79 ) , 119 ( 24 ) , 118 ( 16 ) , 92 ( 23 ) , 84 ( 100 ) , 65 ( 21 ) , 51 ( 22 ) , 42 ( 61)nicotine = ( s)-3-(1-methyl-2-pyrrolidinyl)-pyridineb11.85176 ( m , 12 ) , 175 ( 7 ) , 147 ( 7 ) , 133 ( 9 ) , 119 ( 24 ) , 98 ( 100 ) , 42 ( 28)3-(1-methyl-2-piperidinyl)-pyridinec12.29162 ( m , 26 ) , 161 ( 23 ) , 133 ( 45 ) , 119 ( 45 ) , 106 ( 49 ) , 105 ( 60 ) , 92 ( 19 ) , 84 ( 100 ) , 80 ( 22 ) , 78 ( 20 ) , 57 ( 29 ) , 51 ( 28 ) , 41 ( 38)anabasine = 3-(2-piperidinyl)-pyridined12.89157 ( 12 ) , 156 ( m , 100 ) , 155 ( 71 ) , 130 ( 20 ) , 128 ( 12 ) , 104 ( 7 ) , 79 ( 13 ) , 78 ( 22 ) , 51 ( 13)2,3-bipyridinyl = 2-(3-pyridinyl)-pyridinee13.35161 ( 12 ) , 160 ( m , 97 ) , 159 ( 100 ) , 145 ( 46 ) , 132 ( 14 ) , 131 ( 79 ) , 118 ( 8) , 105 ( 44 ) , 104 ( 76 ) , 78 ( 27 ) , 77 ( 29 ) , 51 ( 36 ) , 41 ( 29)anabaseine = 3,4,4,6-tetrahydro-2,3-bipyridinetentatively identified on the basis of mass spectral data chemical profiles of scent gland secretions in three species of holoscotolemon . * compounds at rt = 13.56 min and rt = 14.55 min were found inconsistently in extracts of holoscotolemon lessiniense , and possibly represent isomeric , not fully characterized pyridines with a molecular weight of m = 174 gas chromatographic and mass spectral data to components from extracts of holoscotolemon lessiniense , h. oreophilum , and h. jaqueti tentatively identified on the basis of mass spectral data major components component a showed an ei - mass spectrum consistent with that of nicotine [ 3-(1-methyl-2-pyrrolidinyl)-pyridine ] , exhibiting a molecular ion ( m ) at a mass charge / ratio ( m / z ) of 162 , loss of a hydrogen atom ( m / z 161 ) , and a base ion at m / z 84 . the specific enantiomers , however , were not separable due to the chromatographic conditions used , and both enantiomers eluted as a single peak at rt = 10.54 min , exactly matching the retention time of component a. thus , we did not determine the chirality of nicotine from holoscotolemon spp . so far , only the ( s)-enantiomer is known to occur in nature.the ei - mass spectrum of the second major component ( e ) had a molecular ion at m / z 160 , again exhibiting loss of hydrogen ( base ion at m / z 159 ) , and losses of 15 ( leading to the ion at m / z 145 ) , 29 ( m / z 131 ) , and 56 ( m / z 104 ) mass units . the extracts of holoscotolemon oreophilum and h. jaqueti were dominated by compound a ( nicotine : > 97% of the secretion , based on calculation of peak areas , see also table 2 and fig . components b ( 3-(1-methyl-2-piperidinyl)-pyridine ) and c ( anabasine ) were found constantly as accompanying minor or trace components ( each about 1% of the secretion ) . in h. lessiniense , anabaseine ( compound e ) was the major compound in the extracts ( > 95% , see table 2 and fig . 1 ) , accompanied by trace amounts of anabasine and 2,3-bipyridyl ( each about 13% of the secretion ) . in two out of five extracts , two trace components were also found at rt = 13.56 min and 14.55 min ( each about 1% of the secretion ) , both showing a pyridine - like pattern and a molecular weight of 174 , but both remain unidentified.it is noteworthy that some individuals did not show any components . in particular , extracts of three specimens ( all collected at one locality in serbia , see table 1 ) of h. jaqueti were investigated , and two of these ( one female , one juvenile ) consistently showed the nicotine - rich composition mentioned above . in the extract of the remaining specimen ( a juvenile ) , no components could be detected . for h. lessiniense , we analyzed extracts of five individuals ( all female ) , which were from two different collections ( see table 1 ) : three extracts ( two of collection no . for the largest species of the genus , h. oreophilum , only two individuals ( one male , one female ) were available to us , both of which discharged large amounts of nicotine . absolute amounts of secretions were determined for h. oreophilum , and were calculated to be about 15 g nicotine per individual ( 11.8 g measured for one specimen , 16 g for the other specimen).thus , only for h. oreophilum ( nicotine - rich ) , could male and female extracts be compared , showing no differences . for h. jaqueti ( nicotine - rich ) and h. lessiniense ( anabaseine - rich ) , only females and juveniles ( subadult females ) were available ; between subadults and adults , holoscotolemon unicolor contrasting the results obtained from h. jaqueti , h. oreophilum , and h. lessiniense , no components were detected in any of the nine extracts from adults of h. unicolor . scent glands in h. unicolor , as well as openings ( ozopores ) appeared to be as well developed as in the other holoscotolemon species ( fig . however , in one extract of an early - instar juvenile , traces of three nitrogen - containing compounds were detected ( not shown ) , whereas extracts of 11 other juveniles did not show any compounds . the trace components in the single juvenile extract were tentatively identified as dimethyl - isoalkylpyrazines by mass spectral comparisons to literature , and to spectra from the nist - library ( compound 1 : a dimethyl - isobutylpyrazine , m at m / z 164 ; compounds 2 and 3 : isomeric dimethyl - isopentylpyrazines , m at m / z 178 ) . choloscotolemon oreophilum ; dholoscotolemon lessiniense ; e and f details of left ozopore in holoscotolemon jaqueti position and morphology of ozopores in species of genus holoscotolemon . choloscotolemon oreophilum ; dholoscotolemon lessiniense ; e and f details of left ozopore in holoscotolemon jaqueti origin of extract components we assume that the compounds found in the whole body extracts of holoscotolemon herein investigated are from the scent glands of these species . first , scent glands are the only glandular structures capable of producing such amounts of secretions ; they are large and well developed in all species of the genus holoscotolemon . in our study , the relation of specimens with well - filled gland reservoirs to specimens with depleted reservoirs was about 1:1 for h. jaqueti and h. lessiniense . absolute amounts of secretion , as shown for nicotine in extracts of h. oreophilum , were calculated to be about 15 g per individual . , 2001 , 2008 ) , and also has been used to assess scent gland exudates of opilionids of different groups ( raspotnig et al . for instance , in sironids ( cyphophthalmi ) direct sampling of secretions from ozopores leads to the same chemical results as working with hexane whole body extracts ( raspotnig et al . it is further interesting to note that h. lessiniense ( anabaseine - rich ) and h. oreophilum ( nicotine - rich ) , both occurring in the italian southern alps and separated by an air - line distance of just 300 km , use differently composed secretions ; this might indicate more specific communicative functions of scent gland secretions in cladonychiid harvestmen , in addition to assumingly defensive roles . pyridines and alkyl - pyrazines three minor components in the holoscotolemon - secretions were classified as pyridines , chemically close to nicotine and anabaseine . in fact , small amounts of 2,3-bipyridyl also could be detected when analyzing an authentic sample of anabasine , calling the scent gland origin of 2,3-bipyridyl into question . the alkyl - pyrazines detected in the extract of one single juvenile of h. unicolor also are widespread exocrine compounds in several species of ants and wasps ( cavill and houghton , 1974 ; borg - karlson and teng , 1980 ; wheeler et al . in some cases , alkyl pyrazines seem to accompany alkaloids of the anabasine - type as minor components in exocrine secretions of some hymenopterans ( e.g. however , our preliminary chemical data for h. unicolor , deviating from the chemistry of the other three holoscotolemon species , need further clarification , and we hope to unravel this question in the near future . holoscotolemon unicolor is endemic to the eastern alps and , following holdhaus ( 1954 ) , thaler ( 1966 , 1976 ) , and komposch ( 2009 ) , is a re - wanderer of long distance , which probably survived the last ice age in holoscotolemon oreophilum and h. lessiniense are local endemics of the southern alps , and should have survived the wrm ice - age in massifs de refuge in the areas of their present distribution in the sea alps ( h. oreophilum ) and monti lessini ( h. lessiniense ) , respectively . martens ( 1978 ) found no close relationship between h. lessiniense and h. unicolor , and believed the former species is closer to h. oreophilum . therefore , it is surprising that the available chemical information points toward a high similarity between the most western ( h. oreophilum ) and the most eastern species ( h. jaqueti ) . thus , tentatively , nicotine may represent a plesiomorphic compound in holoscotolemon ( and possibly also in travunioidea , see below ) while h. lessiniense , occurring in an area in between , shows a clearly distinct although related chemistry ( i.e. scent gland chemistry in insidiatores the major part of laniatorean scent gland research has been performed on grassatorean laniatores , and this strong bias may have contributed to the picture of a typical , rather homogenous , phenolic and benzoquinone - rich phenol- and benzoquinone - rich secretions have been identified from representatives of all grassatorean families hitherto investigated , such as many gonyleptidae ( see gnaspini and hara , 2007 and references therein ; hara et al . , 2011 ) , nitrogen - containing compounds or even tobacco alkaloids do not seem to occur in the scent glands of the grassatores . ( 1984 ) who found two nitrogen - containing compounds ( - dimethyl - phenylethylamine and nicotine ) in the scent gland secretion of sclerobunus robustus , a travunioid species from both another continent ( america ) and a second travunioid family ( travuniidae ; kury 2000 onwards ) . preliminary chemical results from representatives of travuniids in europe ( unpublished ) also indicate consistently that tobacco alkaloids constitute major secretory components in species of travunia and abasola ( for possible synonymies see kury and mendes 2007 ) as well as in the so - far systematically unplaced but travunioid trojanella ( karaman , 2005 ) . in this respect , nitrogen - containing compounds : ( 1 ) may be widespread or even common in the travunioidea ; and , ( 2 ) according to the basal phylogenetic position of travunioidea within laniatores , these compounds also may represent the ancestral scent gland equipment of laniatoreans.opilionid scent gland secretions generally seem to be a promising pool of group- or even species - specific chemical characters , representing an independent set of data of phylogenetic value , in addition to characters from traditional morphology and molecular genetics . , so far , an evolutionary line may be traced from the naphthoquinone- and acyclic ketone - rich secretions of the cyphophthalmi to the similarly structured secretions in eupnoid and dyspnoid palpatores ( see introduction ) , but any evolutionary link to the chemically distinct secretions of laniatores is still missing . additionally , within the laniatores , the chemical gap between the alkaloid - rich secretions of the travunioidea on the one hand and the generally phenol- and benzoquinone - rich chemistry in the grassatores on the other hand is not yet well explained . in this regard , information on the unknown scent gland chemistry of the true triaenonychoidea ( sensu kury , 2000 onwards ) would be important , as this mainly southern hemisphere - distributed group of insidiatores ( see pinto - da - rocha and giribet , 2007 ) may represent the missing link in the chemosystematic puzzle of harvestmen .

one mechanism used by cells to prevent the overexpression of genes is to target their mrna for decay . although several destabilizing elements have been described to alter mrna stability , the most widely studied is the adenine / uridine - rich element ( are ) , which often includes a repeat of the auuua pentamer . ares are found in the 3 untranslated region of certain mrnas and have been shown to severely decrease the stability of the mrna in which they reside . the involvement of ares in the decay of mrna is described as are - mediated decay ( amd ; barreau et al . , 2005 ) . in recent years , many advances have been made in the field of amd relating to its effectors , regulators , and location . the physiological significance of ares and amd has also been revealed through several studies ( gingerich et al . , 2004 ) another form of mrna degradation that has received much attention lately involves micrornas ( mirnas ) . mirnas are derived from short hairpin rna fragments , which are processed through a specific pathway to yield oligomers complementary to specific messages . when these oligomers then interact with their targets , one of two outcomes is observed : either translation is repressed or the target mrna molecule is sentenced to degradation ( filipowicz et al . , 2008 ) . interestingly , recent studies have indicated that some players in the mirna pathway may interact with and affect the fate of are - containing messages ( jing et al . , 2005 ; vasudevan et al . , 2007 ) . in this mini - review , we will highlight what is known regarding amd , how the rapidly evolving mirna field may tie in , and where recent work may lead . given the fact that amd allows control over protein expression , it is not surprising that the various are binding proteins ( aubps ) play entirely different roles in regulating the stability of are mrnas ( bevilacqua et al . some direct are mrnas toward rapid decay by amd ( e.g. tristetraprolin [ ttp ] ; lai et al . , 1999 ; lykke - andersen and wagner , 2005 ) , others increase the stability of their mrna ligands ( e.g. hur ; brennan and steitz , 2001 ) , and still others may do both ( e.g. auf-1/hnrnp d ; barreau et al . , 2005 ) . a series of studies have also shown that certain aubps , such as tia-1/tiar and hur , are capable of influencing mrna translation ( barreau et al . , 2005 ) , and although it is not known if this activity of aubps is related to amd , it certainly merits further attention . interestingly , most aubps have not yet been shown to be the direct executors of amd but rather recruit and regulate effectors of this process ( table i , aubps ) . there exist only a limited number of known enzymes capable of degrading mrna , and so it is not surprising that many of them have been linked to amd ( table i , degradation machineries ; chen et al . , 2001 ; the most prominent of these are the ribonucleases ( rnases ) , of which there exist two types : exo- and endoribonucleases . the most common exoribonucleases , performing 3 to 5 degradation , exist in a large complex known as the exosome ( bousquet - antonelli et al . this complex , with various exonuclease subunits , also contains proteins that may be capable of binding directly to ares . it was found that the subunits pm - scl-75 , oip2 , and rrp41 can specifically bind to ares via their rnase ph domain ( mukherjee et al . this table lists the aubps ( for a more thorough listing of the stabilizing and destabilizing roles of aubps , see barreau et al . ) and degradation enzymes that have been implicated in amd . the links that these proteins have to amd , as well as to mirna - based processes , are highlighted , with select references ( those underlined are in relation to mirna and those not underlined are in relation to amd ) . recent studies have demonstrated that in some cases , 5 to 3 mrna decay is also significant ( stoecklin et al . , 2006 ) . the major player responsible for this nonexosomal ribonuclease activity is xrn1 ( larimer and stevens , 1990 ) . intriguingly , both xrn1 and pm - scl-75 have been shown to be essential for adequate amd ( yang et al . 2006 ) , suggesting that more than one pathway is being used by this process ( fig . 1 ) . regardless of the direction exonuclease cleavage occurs in , other factors , such as decapping enzymes and deadenylases , are also typically implicated , and these have also been shown to associate with aubps ( table i ) . gap - sh3 binding protein and the erythroid cell enriched endoribonuclease have actually been shown to target the 3 untranslated regions of are mrna ( wang and kiledjian , 2000 ; tourriere et al . , 2001 ; schoenberg , 2007 ) , making them possible suspects in amd . , aubps promoting degradation ( e.g. ttp ) may bind the are of the target mrna and help recruit decapping enzymes such as dcp1/2 . after decapping , the 5 to 3 exoribonuclease xrn1 may then carry out 5 to 3 decay . in the second some data implicate mirnas in this aubp interaction , such as the mir16ago2ttp complex ( jing et al . , 2005 ) . in the third , aubps may recruit deadenylases ( such as parn or ccr4 ) to remove the poly ( a ) tail from the 3 terminus of the mrna , and 3 to 5 degradation may then occur by way of the exosome . stabilizing aubps , such as hur , may be implicated in one or more of these pathways by competing with binding of destabilizing aubps or by preventing mirna mrna interactions . another class of endoribonucleases that has also received much attention lately is the argonaute ( ago ) proteins . these endonucleases are clearly linked to mirna - mediated gene silencing , and growing evidence supports that this newfound pathway of gene expression regulation is somehow related to amd . mirnas have been shown to influence gene expression both by modulating translation and by causing the degradation of target mrnas , although it is uncertain if the latter of these effects is a consequence of the former ( filipowicz et al . , 2008 ) . mirnas are typically found associated with various factors , which together form micrornps ( mirnps ) . a core component of mirnps is the ago protein , which exists in various isoforms , some of which are capable of interfering with translation and of degrading mrna by way of their endonuclease activity ( filipowicz et al . , 2008 ; it is intriguing to note that these two effects of mirna mirror those linked to aubps , suggesting that perhaps the amd and translational roles of aubps are mediated , or at least influenced , by mirna . amd and mirna - mediated decay involve some of the same players , such as the ccr4 deadenylase complex and the decapping enzymes dcp1/2 ( behm - ansmant et al . , 2006 ) . beyond this , a few important studies have actually shown interactions between the two processes . jing et al . ( 2005 ) found that dicer , a key player in the biogenesis of mirnas , is a required component for the degradation via amd of the are - containing message tnf. they reported that mir16 targets a sequence located outside the 34-nt are region ( vasudevan and steitz , 2007 ) that is needed for amd of tnf mrna and that this mirna indirectly associates with ttp through the ago complex . the authors speculated that are recognition by ttp aids mir16 in binding to a target sequence and that the mir16-associated complex components , such as ago2 , can then mediate the amd effect . could this be an example of a collaborative effort between a mirna and an aubp , permitting a more stable interaction with the target mrna , which then allows the recruitment of the degradation machinery ? although most mirna - mediated regulation requires perfect complementarity in the seed sequence of the mirna ( bases 28 ; filipowicz et al . , 2008 ) , the complementarity between mir16 and its target mrna does not exist in this region . perhaps the collaboration between mir16 and ttp is a means of regulating the effects of mir16 . if a known destabilizing aubp , such as ttp , can assist the mirna - mediated degradation of a target message , then it would be reasonable to speculate that a stabilizing aubp , such as hur , could interfere with mirna binding . ( 2006 ) found that hur was capable of rescuing translationally repressed mrna , most likely by interfering with the association of mir122 with are mrna . if ttp can assist a mirna in carrying out decay , then having aubps either interfere with or support the translational effect of mirna is just as likely . this supports the idea that rna binding proteins and mirnas may regulate each other 's effects by competing for binding or complementing the binding of one another ( george and tenenbaum , 2006 ) . they showed that a well - known aubp , fragile x mental retardation related protein 1 ( fxr1 ) , binds to the are of tnf mrna to promote translation during serum starvation in an ago2-dependent manner ( vasudevan and steitz , 2007 ) . for translation of the are mrna to increase , both fxr1 and ago2 had to be present , showing that it is both the cellular environment and an interplay between aubps and mirna factors that influence gene expression . a subsequent study demonstrated that this translational up - regulation depended on mir369 - 3 to bring fxr1 and ago2 to the are and that mirnas enable the transition between the repression and promotion of translation ( vasudevan et al . , 2007 ) . collectively , these studies show that translation may be influenced by players associated with both amd and mirnas and that only through cooperation can the desired outcome be obtained . their players have been shown to operate codependently , and the studies showing these relationships suggest that they work together in a variety of situations . another commonality between these two involves where in the cell they carry out their roles , and , not surprisingly , uncertainty surrounds these details of amd as well . as mentioned earlier , the decapping enzyme complex has been linked to both amd and mirna function . it was noticed that these enzymes , along with several other factors that promote decapping , localized to cytoplasmic foci , and these have since been named processing bodies ( pbs ; eulalio et al . in addition , several mrna degradation enzymes have been found to aggregate in another species of cytoplasmic granule , which specifically form under stressful conditions , named stress granules ( sgs ; anderson and kedersha , 2006 ) . both of these cytoplasmic bodies have raised interest because of their link to amd and to mirna - mediated effects , with the potential of being the specific loci where these processes are modulated . the interaction between certain aubps and pb - associated proteins raised the possibility of a direct link between amd and pbs . ttp has been shown to interact with dcp2 and other components of the decapping complex ( fenger - grn et al . , 2005 ) . ttp , as well as several aubps , has also been shown to associate with various other pb - associated factors , including xrn1 , ccr4 ( lykke - andersen and wagner , 2005 ; hau et al . , 2007 ) , and the exosome ( chen et al . , 2001 ) , particularly the pm - scl-75 subunit ( hau et al . , 2007 ) . these observations suggest that ttp and other aubps help recruit degradation factors to are mrnas . this , and the result that exosome components can directly bind ares ( mukherjee et al . , 2002 ; anderson and kedersha , 2006 ) , support the hypothesis of van hoof and parker ( 2002 ) that the exosome and , perhaps , other components of the degradation machinery are recruited both by aubps bound to the target mrna and by the rnas themselves . several knockdown studies have been done to assess the influence of different factors on the existence of pbs and on amd . for example , knockdown of xrn1 or dcp2 caused an accumulation of are mrnas in pbs and a decrease in degradation ( franks and lykke - andersen , 2007 ) , whereas the down - regulation of various components of 5 to 3 and 3 to 5 exonuclease systems also diminished the amount of amd ( lin et al . , 2007 ) . nevertheless , the results of these studies are not always complementary . gw182 is documented to be required for both pb formation ( yang et al . , 2004b ) and mirna activity ( rehwinkel et al . , when the expression of gw182 is reduced , however , there is no observable effect on amd , even when a significant decrease in the number of visibly detectable pbs is seen ( stoecklin and anderson , 2007 ) . this highlights the fact that despite the ability of some aubps to interact with pb - associated decay enzymes , there is not adequate information to confirm a functional link between amd and the recruitment of are mrnas by pbs . there is also a mounting discussion over the role pbs play in repressing translation ( coller and parker , 2005 ; parker and sheth , 2007 ) , which is significant for both mirna effects and the roles of aubps . equally intriguing are observations showing that pbs can interact with sgs and that this interaction may be responsible for deciding the amd fate of target mrna ( kedersha et al . , 2005 ) . sgs were originally observed as localization centers for specific mrnas in cells exposed to heat shock ( nover et al . , 1989 ) . in mammalian cells , various environmental stressors can induce the formation of sgs ( anderson and kedersha , 2006 ) . sgs are believed to stabilize and block the translation of certain mrnas ( kedersha et al . , 2005 ; mazroui et al . , 2007 ) and then , when the stress is relieved , sgs may direct these mrnas either back to the polysome or to machineries responsible for their degradation ( anderson and kedersha , 2006 ; kedersha and anderson , 2007 ) . several rna binding proteins that have been shown to be essential for sg formation localize to sgs such as tia-1/tiar ( kedersha and anderson , 2002 ) . hur , ttp , and gap - sh3 binding protein , among other aubps , also accumulate in these foci ( gallouzi et al . , 2000 ; tourriere et al . , 2003 ; stoecklin et al . , 2004 ) . although several pb components , such as dcp1/2 and gw182 , are not present in sgs , others , such as xrn1 , are ( anderson and kedersha , 2006 ) . additionally , overexpressing ttp and brf1 has been shown to cause stable interaction between pbs and sgs . is it possible that under stressful conditions , aubp - bound mrnas first localize to sgs and are then directed to pbs for decay ? the pb sg relationship has been further complicated after observations that the formation of pbs and sgs are independent ( kedersha et al . , moreover , some stresses that induce sg formation actually prevent both pb development and the decay of mrna ( mazroui et al . , 2007 ) . these results make it difficult to develop an unambiguous model regarding the cellular location of amd . similar localization issues are also a problem in developing a unified model for mirna - mediated repression ( filipowicz et al . , 2008 ) . when hur rescues mirna - repressed mrna , it does so by causing the mrna to leave pbs ( bhattacharyya et al . , it was also reported that in the presence of mirnas , ago proteins are capable of dynamically associating with sgs , where these enzymes play a role in translation silencing but not in message decay ( leung et al . , 2006 ) . although the compositions and proposed functions of pbs and sgs may differ , there is much evidence that they are both involved in both amd and in mirna - mediated repression . the interaction between certain aubps and pb - associated proteins raised the possibility of a direct link between amd and pbs . ttp has been shown to interact with dcp2 and other components of the decapping complex ( fenger - grn et al . , 2005 ) . ttp , as well as several aubps , has also been shown to associate with various other pb - associated factors , including xrn1 , ccr4 ( lykke - andersen and wagner , 2005 ; hau et al . , 2007 ) , and the exosome ( chen et al . , 2001 ) , particularly the pm - scl-75 subunit ( hau et al . , 2007 ) . these observations suggest that ttp and other aubps help recruit degradation factors to are mrnas . this , and the result that exosome components can directly bind ares ( mukherjee et al . , 2002 ; anderson and kedersha , 2006 ) , support the hypothesis of van hoof and parker ( 2002 ) that the exosome and , perhaps , other components of the degradation machinery are recruited both by aubps bound to the target mrna and by the rnas themselves . several knockdown studies have been done to assess the influence of different factors on the existence of pbs and on amd . for example , knockdown of xrn1 or dcp2 caused an accumulation of are mrnas in pbs and a decrease in degradation ( franks and lykke - andersen , 2007 ) , whereas the down - regulation of various components of 5 to 3 and 3 to 5 exonuclease systems also diminished the amount of amd ( lin et al . , 2007 ) . nevertheless , the results of these studies are not always complementary . gw182 is documented to be required for both pb formation ( yang et al . , 2004b ) and mirna activity ( rehwinkel et al . , 2005 ) . when the expression of gw182 is reduced , however , there is no observable effect on amd , even when a significant decrease in the number of visibly detectable pbs is seen ( stoecklin and anderson , 2007 ) . this highlights the fact that despite the ability of some aubps to interact with pb - associated decay enzymes , there is not adequate information to confirm a functional link between amd and the recruitment of are mrnas by pbs . there is also a mounting discussion over the role pbs play in repressing translation ( coller and parker , 2005 ; parker and sheth , 2007 ) , which is significant for both mirna effects and the roles of aubps . equally intriguing are observations showing that pbs can interact with sgs and that this interaction may be responsible for deciding the amd fate of target mrna ( kedersha et al . , 2005 ) . sgs were originally observed as localization centers for specific mrnas in cells exposed to heat shock ( nover et al . , 1989 ) . in mammalian cells , various environmental stressors can induce the formation of sgs ( anderson and kedersha , 2006 ) . sgs are believed to stabilize and block the translation of certain mrnas ( kedersha et al . , 2005 ; mazroui et al . , 2007 ) and then , when the stress is relieved , sgs may direct these mrnas either back to the polysome or to machineries responsible for their degradation ( anderson and kedersha , 2006 ; kedersha and anderson , 2007 ) . several rna binding proteins that have been shown to be essential for sg formation hur , ttp , and gap - sh3 binding protein , among other aubps , also accumulate in these foci ( gallouzi et al . , 2000 ; tourriere et al . , 2003 ; stoecklin et al . , 2004 although several pb components , such as dcp1/2 and gw182 , are not present in sgs , others , such as xrn1 , are ( anderson and kedersha , 2006 ) . additionally , overexpressing ttp and brf1 has been shown to cause stable interaction between pbs and sgs . is it possible that under stressful conditions , aubp - bound mrnas first localize to sgs and are then directed to pbs for decay ? the pb sg relationship has been further complicated after observations that the formation of pbs and sgs are independent ( kedersha et al . , moreover , some stresses that induce sg formation actually prevent both pb development and the decay of mrna ( mazroui et al . , 2007 ) . these results make it difficult to develop an unambiguous model regarding the cellular location of amd . similar localization issues are also a problem in developing a unified model for mirna - mediated repression ( filipowicz et al . , 2008 ) . when hur rescues mirna - repressed mrna , it does so by causing the mrna to leave pbs ( bhattacharyya et al . , 2006 ) . it was also reported that in the presence of mirnas , ago proteins are capable of dynamically associating with sgs , where these enzymes play a role in translation silencing but not in message decay ( leung et al . , 2006 ) . although the compositions and proposed functions of pbs and sgs may differ , there is much evidence that they are both involved in both amd and in mirna - mediated repression . as advances in the field of mrna decay are made , it is apparent that aubps are a crucial component of amd and that modification of aubps may regulate are mrna decay ( stoecklin et al . , 2004 ) . at the same time , the localization of amd - linked players is of great importance , and another potential complication in constructing a model for amd is the possibility that the granules discussed are more complex than they appear . with the observations that the formation of sgs can be initiated in an eif2 phosphorylation - dependent and -independent manner , it was proposed that different types of sgs may exist ( anderson and kedersha , 2006 ; mazroui et al . , 2006 ) . if true , then it is reasonable to hypothesize that different sgs can direct messages differently . this theory actually supports the various ways that sgs and pbs have been shown to interact . if there are various classes of sgs , then it would be reasonable to suspect that some support pb formation and amd , whereas others promote alternative functions . what are taken to be a type of sg at this time could ultimately be shown to exist primarily for the purpose of reinitiating translation of mrnas . ultimately , sgs may be implicated in the balance a cell mediates between survival and death after stress , and differing granule classes and interactions may transiently exist as the cell gauges its fate ( mazroui et al . , 2007 ) . similarly , although pbs and gw bodies were considered to be the same entity after their discovery , this conclusion may have been premature , as they may ultimately be distinct subsets of cytoplasmic bodies . it has been observed that gw bodies disappear with cell cycle arrest ( eulalio et al . , 2007 ) , whereas pbs remain ( vasudevan and steitz , 2007 ) . moreover , amd - linked players , such as the fxr1 and ago2 members of the mirna pathway , were originally thought to be components of pbs but have been shown to colocalize to gw bodies rather than dcp1-containing cytoplasmic foci ( vasudevan and steitz , 2007 ) . this may explain why pbs could be seen in yeast even though they do not have an analogue of gw182 ( ding and han , 2007 ) . even the recently proposed exosome granules , which may serve as a major site for amd , could be distinct from pbs ( lin et al . , 2007 ) . another important consideration is whether are mrnas are brought to these preexisting cytoplasmic granules or whether a concentration of amd - targeted mrna , bound to its various factors , is necessary for the formation of sgs or pbs . these discrepancies invite further investigation into the localization of amd . meanwhile , the relationship between amd and mirna - mediated decay warrants attention . not only are there similar aspects to the processes but the colocalization of certain players strongly supports an underlying coordination . ultimately , the details surrounding amd , that is , the players involved in mediating such decay , the mechanism , the timing , the localization , and its regulation , leave much mystery regarding this process . investigation into amd has so far demonstrated quite well the validity of the adage that the more one knows , the more one learns is left unknown . for a process linked to so many evolving fields , there is little doubt that the existing facts about amd may drastically evolve , all the while bringing us closer to appreciating exactly how such tight regulation of cell function can be dictated by a series of adenine / uridine repeats .

messenger ribonucleic acids ( mrnas ) containing adenine / uridine - rich elements ( ares ) in their 3 untranslated region are particularly labile , allowing for the regulation of expression for growth factors , oncoproteins , and cytokines . the regulators , effectors , and location of are - mediated decay ( amd ) have been investigated by many groups in recent years , and several links have been found between amd and microrna - mediated decay . we highlight these similarities , along with recent advances in the field of amd , and also mention how there is still much left unknown surrounding this specialized mode of mrna decay .

Introduction Regulators and effectors: a roster for AMD miRNA: the missing piece of the puzzle? Common grounds PBs: into the lion's den. SGs: live or let die. Unsolved mysteries

although several destabilizing elements have been described to alter mrna stability , the most widely studied is the adenine / uridine - rich element ( are ) , which often includes a repeat of the auuua pentamer . ares are found in the 3 untranslated region of certain mrnas and have been shown to severely decrease the stability of the mrna in which they reside . the involvement of ares in the decay of mrna is described as are - mediated decay ( amd ; barreau et al . in recent years , many advances have been made in the field of amd relating to its effectors , regulators , and location . the physiological significance of ares and amd has also been revealed through several studies ( gingerich et al . , 2004 ) another form of mrna degradation that has received much attention lately involves micrornas ( mirnas ) . mirnas are derived from short hairpin rna fragments , which are processed through a specific pathway to yield oligomers complementary to specific messages . interestingly , recent studies have indicated that some players in the mirna pathway may interact with and affect the fate of are - containing messages ( jing et al . , 2007 ) . in this mini - review , we will highlight what is known regarding amd , how the rapidly evolving mirna field may tie in , and where recent work may lead . given the fact that amd allows control over protein expression , it is not surprising that the various are binding proteins ( aubps ) play entirely different roles in regulating the stability of are mrnas ( bevilacqua et al . some direct are mrnas toward rapid decay by amd ( e.g. hur ; brennan and steitz , 2001 ) , and still others may do both ( e.g. a series of studies have also shown that certain aubps , such as tia-1/tiar and hur , are capable of influencing mrna translation ( barreau et al . , 2005 ) , and although it is not known if this activity of aubps is related to amd , it certainly merits further attention . interestingly , most aubps have not yet been shown to be the direct executors of amd but rather recruit and regulate effectors of this process ( table i , aubps ) . there exist only a limited number of known enzymes capable of degrading mrna , and so it is not surprising that many of them have been linked to amd ( table i , degradation machineries ; chen et al . , 2001 ; the most prominent of these are the ribonucleases ( rnases ) , of which there exist two types : exo- and endoribonucleases . the most common exoribonucleases , performing 3 to 5 degradation , exist in a large complex known as the exosome ( bousquet - antonelli et al . it was found that the subunits pm - scl-75 , oip2 , and rrp41 can specifically bind to ares via their rnase ph domain ( mukherjee et al . this table lists the aubps ( for a more thorough listing of the stabilizing and destabilizing roles of aubps , see barreau et al . ) and degradation enzymes that have been implicated in amd . the links that these proteins have to amd , as well as to mirna - based processes , are highlighted , with select references ( those underlined are in relation to mirna and those not underlined are in relation to amd ) . recent studies have demonstrated that in some cases , 5 to 3 mrna decay is also significant ( stoecklin et al . intriguingly , both xrn1 and pm - scl-75 have been shown to be essential for adequate amd ( yang et al . regardless of the direction exonuclease cleavage occurs in , other factors , such as decapping enzymes and deadenylases , are also typically implicated , and these have also been shown to associate with aubps ( table i ) . gap - sh3 binding protein and the erythroid cell enriched endoribonuclease have actually been shown to target the 3 untranslated regions of are mrna ( wang and kiledjian , 2000 ; tourriere et al . , aubps promoting degradation ( e.g. ttp ) may bind the are of the target mrna and help recruit decapping enzymes such as dcp1/2 . after decapping , the 5 to 3 exoribonuclease xrn1 may then carry out 5 to 3 decay . in the second some data implicate mirnas in this aubp interaction , such as the mir16ago2ttp complex ( jing et al . , 2005 ) . in the third , aubps may recruit deadenylases ( such as parn or ccr4 ) to remove the poly ( a ) tail from the 3 terminus of the mrna , and 3 to 5 degradation may then occur by way of the exosome . stabilizing aubps , such as hur , may be implicated in one or more of these pathways by competing with binding of destabilizing aubps or by preventing mirna mrna interactions . these endonucleases are clearly linked to mirna - mediated gene silencing , and growing evidence supports that this newfound pathway of gene expression regulation is somehow related to amd . mirnas have been shown to influence gene expression both by modulating translation and by causing the degradation of target mrnas , although it is uncertain if the latter of these effects is a consequence of the former ( filipowicz et al . , 2008 ) . mirnas are typically found associated with various factors , which together form micrornps ( mirnps ) . a core component of mirnps is the ago protein , which exists in various isoforms , some of which are capable of interfering with translation and of degrading mrna by way of their endonuclease activity ( filipowicz et al . , 2008 ; it is intriguing to note that these two effects of mirna mirror those linked to aubps , suggesting that perhaps the amd and translational roles of aubps are mediated , or at least influenced , by mirna . amd and mirna - mediated decay involve some of the same players , such as the ccr4 deadenylase complex and the decapping enzymes dcp1/2 ( behm - ansmant et al . , 2006 ) . jing et al . ( 2005 ) found that dicer , a key player in the biogenesis of mirnas , is a required component for the degradation via amd of the are - containing message tnf. although most mirna - mediated regulation requires perfect complementarity in the seed sequence of the mirna ( bases 28 ; filipowicz et al . if a known destabilizing aubp , such as ttp , can assist the mirna - mediated degradation of a target message , then it would be reasonable to speculate that a stabilizing aubp , such as hur , could interfere with mirna binding . this supports the idea that rna binding proteins and mirnas may regulate each other 's effects by competing for binding or complementing the binding of one another ( george and tenenbaum , 2006 ) . , 2007 ) . collectively , these studies show that translation may be influenced by players associated with both amd and mirnas and that only through cooperation can the desired outcome be obtained . their players have been shown to operate codependently , and the studies showing these relationships suggest that they work together in a variety of situations . another commonality between these two involves where in the cell they carry out their roles , and , not surprisingly , uncertainty surrounds these details of amd as well . as mentioned earlier , the decapping enzyme complex has been linked to both amd and mirna function . it was noticed that these enzymes , along with several other factors that promote decapping , localized to cytoplasmic foci , and these have since been named processing bodies ( pbs ; eulalio et al . in addition , several mrna degradation enzymes have been found to aggregate in another species of cytoplasmic granule , which specifically form under stressful conditions , named stress granules ( sgs ; anderson and kedersha , 2006 ) . both of these cytoplasmic bodies have raised interest because of their link to amd and to mirna - mediated effects , with the potential of being the specific loci where these processes are modulated . the interaction between certain aubps and pb - associated proteins raised the possibility of a direct link between amd and pbs . ttp , as well as several aubps , has also been shown to associate with various other pb - associated factors , including xrn1 , ccr4 ( lykke - andersen and wagner , 2005 ; hau et al . , 2007 ) , and the exosome ( chen et al . , 2001 ) , particularly the pm - scl-75 subunit ( hau et al . , 2007 ) . this , and the result that exosome components can directly bind ares ( mukherjee et al . several knockdown studies have been done to assess the influence of different factors on the existence of pbs and on amd . for example , knockdown of xrn1 or dcp2 caused an accumulation of are mrnas in pbs and a decrease in degradation ( franks and lykke - andersen , 2007 ) , whereas the down - regulation of various components of 5 to 3 and 3 to 5 exonuclease systems also diminished the amount of amd ( lin et al . , 2004b ) and mirna activity ( rehwinkel et al . , when the expression of gw182 is reduced , however , there is no observable effect on amd , even when a significant decrease in the number of visibly detectable pbs is seen ( stoecklin and anderson , 2007 ) . this highlights the fact that despite the ability of some aubps to interact with pb - associated decay enzymes , there is not adequate information to confirm a functional link between amd and the recruitment of are mrnas by pbs . there is also a mounting discussion over the role pbs play in repressing translation ( coller and parker , 2005 ; parker and sheth , 2007 ) , which is significant for both mirna effects and the roles of aubps . sgs were originally observed as localization centers for specific mrnas in cells exposed to heat shock ( nover et al . several rna binding proteins that have been shown to be essential for sg formation localize to sgs such as tia-1/tiar ( kedersha and anderson , 2002 ) . hur , ttp , and gap - sh3 binding protein , among other aubps , also accumulate in these foci ( gallouzi et al . although several pb components , such as dcp1/2 and gw182 , are not present in sgs , others , such as xrn1 , are ( anderson and kedersha , 2006 ) . the pb sg relationship has been further complicated after observations that the formation of pbs and sgs are independent ( kedersha et al . , moreover , some stresses that induce sg formation actually prevent both pb development and the decay of mrna ( mazroui et al . these results make it difficult to develop an unambiguous model regarding the cellular location of amd . similar localization issues are also a problem in developing a unified model for mirna - mediated repression ( filipowicz et al . , 2008 ) . when hur rescues mirna - repressed mrna , it does so by causing the mrna to leave pbs ( bhattacharyya et al . , it was also reported that in the presence of mirnas , ago proteins are capable of dynamically associating with sgs , where these enzymes play a role in translation silencing but not in message decay ( leung et al . although the compositions and proposed functions of pbs and sgs may differ , there is much evidence that they are both involved in both amd and in mirna - mediated repression . the interaction between certain aubps and pb - associated proteins raised the possibility of a direct link between amd and pbs . ttp has been shown to interact with dcp2 and other components of the decapping complex ( fenger - grn et al . ttp , as well as several aubps , has also been shown to associate with various other pb - associated factors , including xrn1 , ccr4 ( lykke - andersen and wagner , 2005 ; hau et al . , 2007 ) , and the exosome ( chen et al . , 2001 ) , particularly the pm - scl-75 subunit ( hau et al . these observations suggest that ttp and other aubps help recruit degradation factors to are mrnas . this , and the result that exosome components can directly bind ares ( mukherjee et al . , 2002 ; anderson and kedersha , 2006 ) , support the hypothesis of van hoof and parker ( 2002 ) that the exosome and , perhaps , other components of the degradation machinery are recruited both by aubps bound to the target mrna and by the rnas themselves . several knockdown studies have been done to assess the influence of different factors on the existence of pbs and on amd . for example , knockdown of xrn1 or dcp2 caused an accumulation of are mrnas in pbs and a decrease in degradation ( franks and lykke - andersen , 2007 ) , whereas the down - regulation of various components of 5 to 3 and 3 to 5 exonuclease systems also diminished the amount of amd ( lin et al . , 2007 ) . nevertheless , the results of these studies are not always complementary . gw182 is documented to be required for both pb formation ( yang et al . , 2004b ) and mirna activity ( rehwinkel et al . when the expression of gw182 is reduced , however , there is no observable effect on amd , even when a significant decrease in the number of visibly detectable pbs is seen ( stoecklin and anderson , 2007 ) . this highlights the fact that despite the ability of some aubps to interact with pb - associated decay enzymes , there is not adequate information to confirm a functional link between amd and the recruitment of are mrnas by pbs . there is also a mounting discussion over the role pbs play in repressing translation ( coller and parker , 2005 ; parker and sheth , 2007 ) , which is significant for both mirna effects and the roles of aubps . , 2005 ) . , 1989 ) . in mammalian cells , various environmental stressors can induce the formation of sgs ( anderson and kedersha , 2006 ) . , 2005 ; mazroui et al . , 2007 ) and then , when the stress is relieved , sgs may direct these mrnas either back to the polysome or to machineries responsible for their degradation ( anderson and kedersha , 2006 ; kedersha and anderson , 2007 ) . several rna binding proteins that have been shown to be essential for sg formation hur , ttp , and gap - sh3 binding protein , among other aubps , also accumulate in these foci ( gallouzi et al . , 2004 although several pb components , such as dcp1/2 and gw182 , are not present in sgs , others , such as xrn1 , are ( anderson and kedersha , 2006 ) . the pb sg relationship has been further complicated after observations that the formation of pbs and sgs are independent ( kedersha et al . , moreover , some stresses that induce sg formation actually prevent both pb development and the decay of mrna ( mazroui et al . these results make it difficult to develop an unambiguous model regarding the cellular location of amd . similar localization issues are also a problem in developing a unified model for mirna - mediated repression ( filipowicz et al . when hur rescues mirna - repressed mrna , it does so by causing the mrna to leave pbs ( bhattacharyya et al . it was also reported that in the presence of mirnas , ago proteins are capable of dynamically associating with sgs , where these enzymes play a role in translation silencing but not in message decay ( leung et al . although the compositions and proposed functions of pbs and sgs may differ , there is much evidence that they are both involved in both amd and in mirna - mediated repression . as advances in the field of mrna decay are made , it is apparent that aubps are a crucial component of amd and that modification of aubps may regulate are mrna decay ( stoecklin et al . , 2004 ) . at the same time , the localization of amd - linked players is of great importance , and another potential complication in constructing a model for amd is the possibility that the granules discussed are more complex than they appear . , 2006 ) . this theory actually supports the various ways that sgs and pbs have been shown to interact . if there are various classes of sgs , then it would be reasonable to suspect that some support pb formation and amd , whereas others promote alternative functions . what are taken to be a type of sg at this time could ultimately be shown to exist primarily for the purpose of reinitiating translation of mrnas . ultimately , sgs may be implicated in the balance a cell mediates between survival and death after stress , and differing granule classes and interactions may transiently exist as the cell gauges its fate ( mazroui et al . similarly , although pbs and gw bodies were considered to be the same entity after their discovery , this conclusion may have been premature , as they may ultimately be distinct subsets of cytoplasmic bodies . moreover , amd - linked players , such as the fxr1 and ago2 members of the mirna pathway , were originally thought to be components of pbs but have been shown to colocalize to gw bodies rather than dcp1-containing cytoplasmic foci ( vasudevan and steitz , 2007 ) . this may explain why pbs could be seen in yeast even though they do not have an analogue of gw182 ( ding and han , 2007 ) . even the recently proposed exosome granules , which may serve as a major site for amd , could be distinct from pbs ( lin et al . another important consideration is whether are mrnas are brought to these preexisting cytoplasmic granules or whether a concentration of amd - targeted mrna , bound to its various factors , is necessary for the formation of sgs or pbs . these discrepancies invite further investigation into the localization of amd . meanwhile , the relationship between amd and mirna - mediated decay warrants attention . ultimately , the details surrounding amd , that is , the players involved in mediating such decay , the mechanism , the timing , the localization , and its regulation , leave much mystery regarding this process . investigation into amd has so far demonstrated quite well the validity of the adage that the more one knows , the more one learns is left unknown . for a process linked to so many evolving fields , there is little doubt that the existing facts about amd may drastically evolve , all the while bringing us closer to appreciating exactly how such tight regulation of cell function can be dictated by a series of adenine / uridine repeats .

the term oral cavity refers to lips , buccal mucosa , alveolar ridges , retro molar trigone , hard palate , floor of the mouth and anterior two - thirds of the tongue . oral cancer or oral cavity cancer , a subtype of head and neck cancer , is any cancerous tissue growth located in the oral cavity . the most common oral cancer is squamous cell carcinoma ( scc ) that affects the tissue lining of the oral cavity . oral cancer is the eleventh most common cancer in the world with an estimated 267,000 cases and 128,000 deaths in around 2000 , two - third of which occur in developing countries . the incidence of oral cancer is increasing in several parts of the world , particularly in australia , japan and parts of europe . oro - pharyngeal cancer is a significant part of the global burden of cancer . incidence rates for oral cancer vary in men from 1 to 10 cases per 100,000 populations in many countries . the immune compromised patients after treatment of oral cancer may have a chance of infection by drug - resistant opportunistic microbes . such microbes that originate in the oral cavity , representative microorganisms include staphylococcus aureus , pseudomonas aeruginosa , and candida albicans . one of the most worrying characteristics of p. aeruginosa is its low antibiotic susceptibility . this low susceptibility is attributable to a concerted action of multidrug efflux pumps with chromosomally encoded antibiotic resistance genes . most of these organisms have become drug - resistant , which has resulted in difficulties in curing the related infectious diseases . the candida species educe the infectious disease candidiasis , which causes infections such as oral thrush and vaginitis as well as life - threatening diseases , known as candidemia . c. albicans are yeast that normally inhabits the human mouth and skin , where it generally uneventfully coexists with a variety of other microorganisms . an infection occurs when the balance of bacteria in the body is disrupted , especially in immunocompromised situations , allowing drug - resistant candida species to proliferate and overcome other healthy microorganisms . immunocompromised situations are frequently seen in older individuals , infants , people infected with hiv , and individuals with cancer ; oral cancer can reduce immunity in the maxillofacial region . the principal treatments for oral cancer are surgical excision , radiotherapy , and chemotherapy , employed alone or in combination . currently , the systemic applications of antibacterial drugs have shown better results on curing diseases than local application , which could induce drug - resistant bacteria in the particular area . in this study , we have investigated the reason behind the frequent cause of oral infection after the treatment of oral cancer in elderly chinese . out of several elderly patients ( 60 - 95 years old ) , 128 patients who had undergone treatment for oral - cavity related problems participated in the study at ninth people 's hospital , shanghai jiao tong university . we have excluded patients with other systemic diseases like autoimmune disease or diabetes to avoid misleading of our parameter . the participants were divided into two groups : group i - the patients undergone oral cancer treatment ( n=93 ; 41 men , 52 women ; average age 68.18.3 years ) ranging from 1 month to 7 years after the initial treatment , 43 members were involved in species identification , remaining 50 members involved in drug - resistant test . these patients have been treated with surgery , chemo and/or radiotherapy ; group ii - the control ( n=35 ; 15 men , 20 women ; average age 70.210.1 years ) , who had received treatment for oral cavities or with no history of any cancer treatment . the institution review board of affiliated ninth people 's hospital of shanghai jiao tong university , according to helsinki declaration ii , approved the study . written informed consent microbes were collected from the areas of surgery ( group i ) , tongue , gingiva , and palate by using sterilized dry cotton swabs . after wetting , the cotton swab was immediately put into an airtight sterilized test tube . to collect anaerobic microbes samples , about 1 ml of saliva samples was collected from each subject for the analysis . the microorganisms were cultured on nalidixic acid / cetrimide agar ( sigma - aldrich chemie gmbh , switzerland ) for the first screening of pseudomonas species , while mannital salt agar ( acumedia manufacturers , usa ) was used for the first screening of staphylococcus species and brilliance candida agar ( thermo fisher scientific inc . ) was used for the first screening of candida species . the nalidixic acid / cetrimide agar and mannital salt agar plates were incubated at 37c under aerobic conditions for 2 days , while candida - gs agar plates were incubated at 30c for 3 days . the morphological assessment was carried out from the colonies on the plates , and those with different shapes were collected , gram stained and observed under a light microscope for species detection . genomic dna from each colony was obtained using a wizard genomic extraction kit ( promega , madison , wi , usa ) . for staphylococcus species , cells were treated with 1 mg / ml of lysostaphin in tris - edta buffer ( te ; 10 mm tris - cl , 1 mm edta , ph 8.0 ) at 37c for 1 h before using the kit . final identification was done by polymerase chain reaction ( pcr ) amplification with bacterial universal primers for 16s rdna and 26s rdna for fungus , followed by dna sequencing . dna sequencing was performed using an abi prism 3100 genetic analyzer ( applied biosystems , tokyo , japan ) with a big dye cycle sequencing reaction kit ( ab applied biosystems ) . identification of experimentally determined nucleotide sequences using sequence databases was performed by basic local alignment search tool ( blast ) . the antimicrobial activity was tested with respective antibiotics used commonly in clinical practice ( figure 1 ) . antibiotics used to test the antimicrobial activity statistical analyses were conducted using spss 15.0 ( spss inc . , u.s.a . ) and any differences at p<0.05 level were considered as statistically significant . out of several elderly patients ( 60 - 95 years old ) , 128 patients who had undergone treatment for oral - cavity related problems participated in the study at ninth people 's hospital , shanghai jiao tong university . we have excluded patients with other systemic diseases like autoimmune disease or diabetes to avoid misleading of our parameter . the participants were divided into two groups : group i - the patients undergone oral cancer treatment ( n=93 ; 41 men , 52 women ; average age 68.18.3 years ) ranging from 1 month to 7 years after the initial treatment , 43 members were involved in species identification , remaining 50 members involved in drug - resistant test . these patients have been treated with surgery , chemo and/or radiotherapy ; group ii - the control ( n=35 ; 15 men , 20 women ; average age 70.210.1 years ) , who had received treatment for oral cavities or with no history of any cancer treatment . the institution review board of affiliated ninth people 's hospital of shanghai jiao tong university , according to helsinki declaration ii , approved the study . written informed consent microbes were collected from the areas of surgery ( group i ) , tongue , gingiva , and palate by using sterilized dry cotton swabs . after wetting , the cotton swab was immediately put into an airtight sterilized test tube . to collect anaerobic microbes samples , about 1 ml of saliva samples was collected from each subject for the analysis . the microorganisms were cultured on nalidixic acid / cetrimide agar ( sigma - aldrich chemie gmbh , switzerland ) for the first screening of pseudomonas species , while mannital salt agar ( acumedia manufacturers , usa ) was used for the first screening of staphylococcus species and brilliance candida agar ( thermo fisher scientific inc . ) was used for the first screening of candida species . the nalidixic acid / cetrimide agar and mannital salt agar plates were incubated at 37c under aerobic conditions for 2 days , while candida - gs agar plates were incubated at 30c for 3 days . the morphological assessment was carried out from the colonies on the plates , and those with different shapes were collected , gram stained and observed under a light microscope for species detection . genomic dna from each colony was obtained using a wizard genomic extraction kit ( promega , madison , wi , usa ) . for staphylococcus species , cells were treated with 1 mg / ml of lysostaphin in tris - edta buffer ( te ; 10 mm tris - cl , 1 mm edta , ph 8.0 ) at 37c for 1 h before using the kit . final identification was done by polymerase chain reaction ( pcr ) amplification with bacterial universal primers for 16s rdna and 26s rdna for fungus , followed by dna sequencing . dna sequencing was performed using an abi prism 3100 genetic analyzer ( applied biosystems , tokyo , japan ) with a big dye cycle sequencing reaction kit ( ab applied biosystems ) . identification of experimentally determined nucleotide sequences using sequence databases was performed by basic local alignment search tool ( blast ) . the antimicrobial activity was tested with respective antibiotics used commonly in clinical practice ( figure 1 ) . statistical analyses were conducted using spss 15.0 ( spss inc . , u.s.a . ) and any differences at p<0.05 level were considered as statistically significant . the samples were collected from surgical scar , saliva , gingiva , and palate of 78 elderly chinese ( 43 cases of group i and 35 cases of group ii ) . the graphical representation for the percentage of species identification from one species in one isolates or two species in same isolates were presented in two sets of group i & ii in each species ( figure 2 ) . statistical analysis revealed no significant difference between groups in the number of isolated microorganisms or the number of participants with these isolates . graphical representation of the percentage of species identification , identified as one species in on isolates or two species in same isoltates were presennted in two sets of group i & ii in each species the universal primers for fungi , 26s rdna sequencing was used to identify the candida species ( figure 3 ) . the candida species isolated from 78 participants ( both groups ) are identified as c. albicans ( 43.6% ) , c. glabrata ( 37.2% ) , c. krusei ( 23.1% ) , c. africana ( 9% ) and c. guilliermondii ( 6.4% ) . the number of candida species isolated from group i were greater than that of group ii . on comparison between the groups , the c. albicans were found to be the dominant species in both group i ( 55.8% ) and group ii ( 28.6% ) . however , there is a significant difference ( p<0.05 ) found between groups . similarly , c. glabrata were found to be the next dominant species with significant difference between group i ( 48.8% ) and group ii ( 22.8% ) participants ( table 1 ) . however , no significant difference was found for c. africana and c. guilliermondii species , rather very less percentage of the species were identified in both groups ( table 1 ) . graphical representation of the overall percentage of identified candida species identification of candida species and their percentage of occurrence there were significant differences between the two groups ( p<0.05 ) the universal primers for bacteria , 16s rdna sequencing was used for species identification ( figure 4 ) . the bacterial species isolated from the 78 participants ( both groups ) are identified as staphylococcus ( 59% ) , pseudomonas ( 53.8% ) , streptococcus ( 44% ) , neisseria ( 37.2% ) , actinomyces ( 29.5% ) and veillonella ( 25.6 ) , which are mostly found in the surgical scar and saliva . on comparison between two groups of isolates , the streptococcus , staphylococcus , pseudomonas and neisseria showed significant differences ( p<0.05 ) in percentage of these species between groups i and ii ( table 2 ) . in group i , the numbers of positive isolates of bacterial species were greater than that of the control group using traditional oral care methods . for veillonella and actinomyces , no significant differences were shown between the two groups ( table 2 ) . graphical representation of the overall percentage of identified bacterial species identification of bacterial species and their percentage of occurrence there were significant differences between the two groups ( p<0.05 ) the species identification reveals that there are higher percentages of dominant microorganisms found in group i than those of group ii . to investigate the reason behind this dominant character , we studied the occurrence of microorganisms against the antimicrobial agents by categorizing the remaining group i participants ( 50 cases ) with their history of treatment with commonly practiced antimicrobial agents ( table 1 ) after the treatment for oral cancer . these 50 participants were divided as 25 cases for the study against antifungal agents and remaining 25 against antibacterial agents . the participants after the treatment of oral cancer were taking commonly practiced antifungal agents such as itraconazole , miconazole , fluconazole , 5-fluorocytosine , amphotericin b , voriconazole and micafungin . the isolates were tested for the presence of candida species . except for the c. glabrata and c. krusei species , all other species were susceptible to the antifungal agents . interestingly , itraconazole had 100% resistance by c. glabrata and 60% resistance by c. krusei species . the c. glabrata showed resistance to miconazole ( 20% ) , fluconazole ( 20% ) , 5 fluorocytosine ( 8% ) . however , c. krusei showed 44% , 32% and 28% of resistance respectively . the other drugs were susceptible to all candida species ( table 3 ) . prevalence of candida species against respective antibiotics the antibacterial agents were tested for the drug - resistant activity of staphylococci subspecies , particularly on methicillin - resistant staphylococcus aureus ( mrsa ) , s. epidermidis and s. haemolyticus . the tested agents are commonly practiced antibiotics such as oxacillin , arbekacin , vancomycin , teicoplanin , linezolid , imipenem / cilastatin , amikacin and ciprofloxacin . surprisingly , mrsa showed drug - resistance to almost all antibiotics with variation in percentage of resistance . the mrsa showed the highest percentage of resistance to oxacillin ( 96% ) and arbekacin . however , the lowest percentage ( 4% ) of resistance was shown in relation to amikacin and ciproflaxacin ( table 4 ) . the other species , s. epidermidis and s. haemolyticus , showed resistance to oxacillin with 60 and 40% , respectively . however , s. haemolyticus showed the lowest percentage ( 4% ) of resistance in relation to arbekacin . the samples were collected from surgical scar , saliva , gingiva , and palate of 78 elderly chinese ( 43 cases of group i and 35 cases of group ii ) . the graphical representation for the percentage of species identification from one species in one isolates or two species in same isolates were presented in two sets of group i & ii in each species ( figure 2 ) . statistical analysis revealed no significant difference between groups in the number of isolated microorganisms or the number of participants with these isolates . graphical representation of the percentage of species identification , identified as one species in on isolates or two species in same isoltates were presennted in two sets of group i & ii in each species the universal primers for fungi , 26s rdna sequencing was used to identify the candida species ( figure 3 ) . the candida species isolated from 78 participants ( both groups ) are identified as c. albicans ( 43.6% ) , c. glabrata ( 37.2% ) , c. krusei ( 23.1% ) , c. africana ( 9% ) and c. guilliermondii ( 6.4% ) . the number of candida species isolated from group i were greater than that of group ii . on comparison between the groups , the c. albicans were found to be the dominant species in both group i ( 55.8% ) and group ii ( 28.6% ) . however , there is a significant difference ( p<0.05 ) found between groups . similarly , c. glabrata were found to be the next dominant species with significant difference between group i ( 48.8% ) and group ii ( 22.8% ) participants ( table 1 ) . however , no significant difference was found for c. africana and c. guilliermondii species , rather very less percentage of the species were identified in both groups ( table 1 ) . graphical representation of the overall percentage of identified candida species identification of candida species and their percentage of occurrence there were significant differences between the two groups ( p<0.05 ) the universal primers for bacteria , 16s rdna sequencing was used for species identification ( figure 4 ) . the bacterial species isolated from the 78 participants ( both groups ) are identified as staphylococcus ( 59% ) , pseudomonas ( 53.8% ) , streptococcus ( 44% ) , neisseria ( 37.2% ) , actinomyces ( 29.5% ) and veillonella ( 25.6 ) , which are mostly found in the surgical scar and saliva . on comparison between two groups of isolates , the streptococcus , staphylococcus , pseudomonas and neisseria showed significant differences ( p<0.05 ) in percentage of these species between groups i and ii ( table 2 ) . in group i , the numbers of positive isolates of bacterial species were greater than that of the control group using traditional oral care methods . for veillonella and actinomyces , no significant differences were shown between the two groups ( table 2 ) . graphical representation of the overall percentage of identified bacterial species identification of bacterial species and their percentage of occurrence there were significant differences between the two groups ( p<0.05 ) the species identification reveals that there are higher percentages of dominant microorganisms found in group i than those of group ii . to investigate the reason behind this dominant character , we studied the occurrence of microorganisms against the antimicrobial agents by categorizing the remaining group i participants ( 50 cases ) with their history of treatment with commonly practiced antimicrobial agents ( table 1 ) after the treatment for oral cancer . these 50 participants were divided as 25 cases for the study against antifungal agents and remaining 25 against antibacterial agents . the participants after the treatment of oral cancer were taking commonly practiced antifungal agents such as itraconazole , miconazole , fluconazole , 5-fluorocytosine , amphotericin b , voriconazole and micafungin . the isolates were tested for the presence of candida species . except for the c. glabrata and c. krusei species , all other species were susceptible to the antifungal agents . interestingly , itraconazole had 100% resistance by c. glabrata and 60% resistance by c. krusei species . the c. glabrata showed resistance to miconazole ( 20% ) , fluconazole ( 20% ) , 5 fluorocytosine ( 8% ) . the other drugs were susceptible to all candida species ( table 3 ) . prevalence of candida species against respective antibiotics the antibacterial agents were tested for the drug - resistant activity of staphylococci subspecies , particularly on methicillin - resistant staphylococcus aureus ( mrsa ) , s. epidermidis and s. haemolyticus . the tested agents are commonly practiced antibiotics such as oxacillin , arbekacin , vancomycin , teicoplanin , linezolid , imipenem / cilastatin , amikacin and ciprofloxacin . surprisingly , mrsa showed drug - resistance to almost all antibiotics with variation in percentage of resistance . the mrsa showed the highest percentage of resistance to oxacillin ( 96% ) and arbekacin . however , the lowest percentage ( 4% ) of resistance was shown in relation to amikacin and ciproflaxacin ( table 4 ) . the other species , s. epidermidis and s. haemolyticus , showed resistance to oxacillin with 60 and 40% , respectively . however , s. haemolyticus showed the lowest percentage ( 4% ) of resistance in relation to arbekacin . in this study , a prevalence of drug - resistant microorganisms in the oral cavity after treatment of oral cancer was performed in aged chinese . the study was carried out in ninth people 's hospital , school of medicine , shanghai jiao tong university , shanghai . the participants were the patients from this hospital who had undergone oral cancer treatment and others without any history of cancer but that could be patients who had undergone treatment for other oral diseases . this study was aimed to investigate the reason behind the frequent cause of oral infection after the treatment of oral cancer in elderly individuals . several lines of evidence support our views that there is a possibility of drug - resistant microbes present in immunocompromised patients , particularly after the treatment of oral cancer . in general , infections are commonly found in oral cancer patients after surgical excision of the tumor . this might be due to wound exposure during and after the operation , even if sutured , when microorganisms may infect oral regions , oropharynx , nasal cavity , and paranasal sinuses areas . patients after oral cavity surgery often appear to have complications after bacterial infections , as well . colonization of pathogenic bacteria in oral cavity is thought to increase the risk of infections such as pneumonia and bacteraemia . it is therefore of high importance the prevention from or cure for the infections . during the development of tumor , the tumor cells or soluble products produced by tumor cells the high percentages of microbial species identified from the isolates of participants were candida , staphylococcus , streptococcus and pseudomonas species ( figure 2 ) . the highest numbers of these species were found in patients after the treatment of oral cancer in comparison with the non - cancer patients ( tables 1 and 2 ) . similar results were obtained in a phase 1 clinical trial with the application of an antibacterial dressing spray in the prevention of post - operative infection in oral cancer patients . the abuse of antimicrobial drugs brings severe adverse effects to people , for example , allergy , toxic reaction , and opportunistic infections . as a result , a new and ideal preventive method is needed for people who have surgery to reduce the chances of bacterial infections . in this study , after revealing the high percentage of microorganisms found particularly in post - treatment of oral cancer , we started to focus on screening or identification of drug - resistant microbes within the species of already identified microbes . the antimicrobial agents ( figure 1 ) , which are common in clinical practice , were taken for this study . the isolates were tested for the presence of candida species and subspecies of staphylococci , particularly on mrsa , s. epidermidis and s. haemolyticus . except for the c. glabrata and c. krusei species , all other candida species were susceptible to the antifungal agents ( table 3 ) . the mrsa showed the highest percentage of resistance to oxacillin ( 96% ) and arbekacin . the other species , s. epidermidis and s. haemolyticus , showed resistance to oxacillin with 60 and 40% , respectively . all other antibiotics were found susceptible to these species ( table 4 ) . according to a study performed in the united kingdom , 13 ( 41.9% ) of 31 s. aureus isolates from the oral mucosa and pockets of patients with another study , in the united states , reported that the prevalence of mrsa organisms in the nasal and oral cavities of nursing home residents was 20 - 35% . furthermore , a japanese group investigated mrsa colonization in neonatal intensive care units and found that 207 ( 49.9% ) of 415 newborns had mrsa organisms . when compared with these results , the drug - resistance ( 96% ) of mrsa against oxacillin was extremely high , possibly due in part to the tendency to prescribe long - term , high - dose antibiotic treatment in japan . our study revealed a group of opportunistic - microorganisms such as c. glabrata , c. krusei and subspecies of staphylococci , particularly methicillin - resistant s. aureus ( mrsa ) , s. epidermidis and s. haemolyticus . nevertheless , anti - tumor drugs used in tumor treatment will also lead to immune and bone marrow suppression . continuous monitoring and a basic infection control strategy , including standard precautions , are important for older individuals , especially those receiving follow - up care for oral cancer . there is always a risk that they may become immunocompromised hosts easily susceptible to oral infections . clinicians must also pay careful attention to opportunistic - microorganisms during the treatment of oral cancer , and to realize the fact of immune compromise in elderly patients .

objectivethe immune compromised patients after treatment of oral cancer may have a chance of infection by drug - resistant opportunistic microbes . we investigated the occurrence of opportunistic microorganisms in aged individuals receiving follow - up examinations after treatment of oral cancer in china . material and methodsthese patients were used as test group and the respective age grouped healthy individuals as control group . in this study , the oral cavity microorganisms such as bacteria and yeast were taken for the analysis . after the screening of representative microorganisms , their aptitude of pervasiveness against drugs was studied . here , we used antimicrobial agents which are common in clinical practice . we also performed studies to investigate the presence of toxin genes in methicillin - resistant s. aureus ( mrsa ) . resultsthe results indicate that the prevalence of drug - resistant microbes was more pronounced in oral cancer patients after initial treatment above 70 years old . the oxacillin resistance of s. aureus isolate confirms that the prevalence of mrsa is increasing in accordance to age - factor and immune compromise in elderly patients . conclusionsthis study reveals the occurrence of drug - resistant opportunistic microorganisms in oral cavity after treatment for oral cancer in aged individuals . special attention should be directed to mrsa during the treatment of oral cancer , and to realize the fact of immune compromise in elderly patients .

INTRODUCTION MATERIAL AND METHODS Patient study Sample collection Microbial cultivation Microbial species detection and antimicrobial testing Statistical analysis RESULTS Identification of microorganisms Candida species Bacterial species Occurrence of microorganism against respective antimicrobial agents DISCUSSION CONCLUSION

the term oral cavity refers to lips , buccal mucosa , alveolar ridges , retro molar trigone , hard palate , floor of the mouth and anterior two - thirds of the tongue . oral cancer or oral cavity cancer , a subtype of head and neck cancer , is any cancerous tissue growth located in the oral cavity . the most common oral cancer is squamous cell carcinoma ( scc ) that affects the tissue lining of the oral cavity . oral cancer is the eleventh most common cancer in the world with an estimated 267,000 cases and 128,000 deaths in around 2000 , two - third of which occur in developing countries . the incidence of oral cancer is increasing in several parts of the world , particularly in australia , japan and parts of europe . incidence rates for oral cancer vary in men from 1 to 10 cases per 100,000 populations in many countries . the immune compromised patients after treatment of oral cancer may have a chance of infection by drug - resistant opportunistic microbes . such microbes that originate in the oral cavity , representative microorganisms include staphylococcus aureus , pseudomonas aeruginosa , and candida albicans . most of these organisms have become drug - resistant , which has resulted in difficulties in curing the related infectious diseases . the candida species educe the infectious disease candidiasis , which causes infections such as oral thrush and vaginitis as well as life - threatening diseases , known as candidemia . an infection occurs when the balance of bacteria in the body is disrupted , especially in immunocompromised situations , allowing drug - resistant candida species to proliferate and overcome other healthy microorganisms . immunocompromised situations are frequently seen in older individuals , infants , people infected with hiv , and individuals with cancer ; oral cancer can reduce immunity in the maxillofacial region . the principal treatments for oral cancer are surgical excision , radiotherapy , and chemotherapy , employed alone or in combination . currently , the systemic applications of antibacterial drugs have shown better results on curing diseases than local application , which could induce drug - resistant bacteria in the particular area . in this study , we have investigated the reason behind the frequent cause of oral infection after the treatment of oral cancer in elderly chinese . out of several elderly patients ( 60 - 95 years old ) , 128 patients who had undergone treatment for oral - cavity related problems participated in the study at ninth people 's hospital , shanghai jiao tong university . the participants were divided into two groups : group i - the patients undergone oral cancer treatment ( n=93 ; 41 men , 52 women ; average age 68.18.3 years ) ranging from 1 month to 7 years after the initial treatment , 43 members were involved in species identification , remaining 50 members involved in drug - resistant test . these patients have been treated with surgery , chemo and/or radiotherapy ; group ii - the control ( n=35 ; 15 men , 20 women ; average age 70.210.1 years ) , who had received treatment for oral cavities or with no history of any cancer treatment . written informed consent microbes were collected from the areas of surgery ( group i ) , tongue , gingiva , and palate by using sterilized dry cotton swabs . after wetting , the cotton swab was immediately put into an airtight sterilized test tube . to collect anaerobic microbes samples , about 1 ml of saliva samples was collected from each subject for the analysis . the microorganisms were cultured on nalidixic acid / cetrimide agar ( sigma - aldrich chemie gmbh , switzerland ) for the first screening of pseudomonas species , while mannital salt agar ( acumedia manufacturers , usa ) was used for the first screening of staphylococcus species and brilliance candida agar ( thermo fisher scientific inc . ) was used for the first screening of candida species . the morphological assessment was carried out from the colonies on the plates , and those with different shapes were collected , gram stained and observed under a light microscope for species detection . the antimicrobial activity was tested with respective antibiotics used commonly in clinical practice ( figure 1 ) . out of several elderly patients ( 60 - 95 years old ) , 128 patients who had undergone treatment for oral - cavity related problems participated in the study at ninth people 's hospital , shanghai jiao tong university . the participants were divided into two groups : group i - the patients undergone oral cancer treatment ( n=93 ; 41 men , 52 women ; average age 68.18.3 years ) ranging from 1 month to 7 years after the initial treatment , 43 members were involved in species identification , remaining 50 members involved in drug - resistant test . these patients have been treated with surgery , chemo and/or radiotherapy ; group ii - the control ( n=35 ; 15 men , 20 women ; average age 70.210.1 years ) , who had received treatment for oral cavities or with no history of any cancer treatment . the institution review board of affiliated ninth people 's hospital of shanghai jiao tong university , according to helsinki declaration ii , approved the study . written informed consent microbes were collected from the areas of surgery ( group i ) , tongue , gingiva , and palate by using sterilized dry cotton swabs . after wetting , the cotton swab was immediately put into an airtight sterilized test tube . to collect anaerobic microbes samples , about 1 ml of saliva samples was collected from each subject for the analysis . the microorganisms were cultured on nalidixic acid / cetrimide agar ( sigma - aldrich chemie gmbh , switzerland ) for the first screening of pseudomonas species , while mannital salt agar ( acumedia manufacturers , usa ) was used for the first screening of staphylococcus species and brilliance candida agar ( thermo fisher scientific inc . ) was used for the first screening of candida species . the morphological assessment was carried out from the colonies on the plates , and those with different shapes were collected , gram stained and observed under a light microscope for species detection . the antimicrobial activity was tested with respective antibiotics used commonly in clinical practice ( figure 1 ) . the samples were collected from surgical scar , saliva , gingiva , and palate of 78 elderly chinese ( 43 cases of group i and 35 cases of group ii ) . the graphical representation for the percentage of species identification from one species in one isolates or two species in same isolates were presented in two sets of group i & ii in each species ( figure 2 ) . graphical representation of the percentage of species identification , identified as one species in on isolates or two species in same isoltates were presennted in two sets of group i & ii in each species the universal primers for fungi , 26s rdna sequencing was used to identify the candida species ( figure 3 ) . on comparison between the groups , the c. albicans were found to be the dominant species in both group i ( 55.8% ) and group ii ( 28.6% ) . the bacterial species isolated from the 78 participants ( both groups ) are identified as staphylococcus ( 59% ) , pseudomonas ( 53.8% ) , streptococcus ( 44% ) , neisseria ( 37.2% ) , actinomyces ( 29.5% ) and veillonella ( 25.6 ) , which are mostly found in the surgical scar and saliva . on comparison between two groups of isolates , the streptococcus , staphylococcus , pseudomonas and neisseria showed significant differences ( p<0.05 ) in percentage of these species between groups i and ii ( table 2 ) . in group i , the numbers of positive isolates of bacterial species were greater than that of the control group using traditional oral care methods . to investigate the reason behind this dominant character , we studied the occurrence of microorganisms against the antimicrobial agents by categorizing the remaining group i participants ( 50 cases ) with their history of treatment with commonly practiced antimicrobial agents ( table 1 ) after the treatment for oral cancer . these 50 participants were divided as 25 cases for the study against antifungal agents and remaining 25 against antibacterial agents . the participants after the treatment of oral cancer were taking commonly practiced antifungal agents such as itraconazole , miconazole , fluconazole , 5-fluorocytosine , amphotericin b , voriconazole and micafungin . the isolates were tested for the presence of candida species . except for the c. glabrata and c. krusei species , all other species were susceptible to the antifungal agents . prevalence of candida species against respective antibiotics the antibacterial agents were tested for the drug - resistant activity of staphylococci subspecies , particularly on methicillin - resistant staphylococcus aureus ( mrsa ) , s. epidermidis and s. haemolyticus . the tested agents are commonly practiced antibiotics such as oxacillin , arbekacin , vancomycin , teicoplanin , linezolid , imipenem / cilastatin , amikacin and ciprofloxacin . surprisingly , mrsa showed drug - resistance to almost all antibiotics with variation in percentage of resistance . the mrsa showed the highest percentage of resistance to oxacillin ( 96% ) and arbekacin . however , the lowest percentage ( 4% ) of resistance was shown in relation to amikacin and ciproflaxacin ( table 4 ) . however , s. haemolyticus showed the lowest percentage ( 4% ) of resistance in relation to arbekacin . the samples were collected from surgical scar , saliva , gingiva , and palate of 78 elderly chinese ( 43 cases of group i and 35 cases of group ii ) . the graphical representation for the percentage of species identification from one species in one isolates or two species in same isolates were presented in two sets of group i & ii in each species ( figure 2 ) . statistical analysis revealed no significant difference between groups in the number of isolated microorganisms or the number of participants with these isolates . the candida species isolated from 78 participants ( both groups ) are identified as c. albicans ( 43.6% ) , c. glabrata ( 37.2% ) , c. krusei ( 23.1% ) , c. africana ( 9% ) and c. guilliermondii ( 6.4% ) . on comparison between the groups , the c. albicans were found to be the dominant species in both group i ( 55.8% ) and group ii ( 28.6% ) . the bacterial species isolated from the 78 participants ( both groups ) are identified as staphylococcus ( 59% ) , pseudomonas ( 53.8% ) , streptococcus ( 44% ) , neisseria ( 37.2% ) , actinomyces ( 29.5% ) and veillonella ( 25.6 ) , which are mostly found in the surgical scar and saliva . on comparison between two groups of isolates , the streptococcus , staphylococcus , pseudomonas and neisseria showed significant differences ( p<0.05 ) in percentage of these species between groups i and ii ( table 2 ) . in group i , the numbers of positive isolates of bacterial species were greater than that of the control group using traditional oral care methods . graphical representation of the overall percentage of identified bacterial species identification of bacterial species and their percentage of occurrence there were significant differences between the two groups ( p<0.05 ) the species identification reveals that there are higher percentages of dominant microorganisms found in group i than those of group ii . to investigate the reason behind this dominant character , we studied the occurrence of microorganisms against the antimicrobial agents by categorizing the remaining group i participants ( 50 cases ) with their history of treatment with commonly practiced antimicrobial agents ( table 1 ) after the treatment for oral cancer . these 50 participants were divided as 25 cases for the study against antifungal agents and remaining 25 against antibacterial agents . the participants after the treatment of oral cancer were taking commonly practiced antifungal agents such as itraconazole , miconazole , fluconazole , 5-fluorocytosine , amphotericin b , voriconazole and micafungin . the isolates were tested for the presence of candida species . except for the c. glabrata and c. krusei species , all other species were susceptible to the antifungal agents . prevalence of candida species against respective antibiotics the antibacterial agents were tested for the drug - resistant activity of staphylococci subspecies , particularly on methicillin - resistant staphylococcus aureus ( mrsa ) , s. epidermidis and s. haemolyticus . the tested agents are commonly practiced antibiotics such as oxacillin , arbekacin , vancomycin , teicoplanin , linezolid , imipenem / cilastatin , amikacin and ciprofloxacin . surprisingly , mrsa showed drug - resistance to almost all antibiotics with variation in percentage of resistance . however , the lowest percentage ( 4% ) of resistance was shown in relation to amikacin and ciproflaxacin ( table 4 ) . however , s. haemolyticus showed the lowest percentage ( 4% ) of resistance in relation to arbekacin . in this study , a prevalence of drug - resistant microorganisms in the oral cavity after treatment of oral cancer was performed in aged chinese . the participants were the patients from this hospital who had undergone oral cancer treatment and others without any history of cancer but that could be patients who had undergone treatment for other oral diseases . this study was aimed to investigate the reason behind the frequent cause of oral infection after the treatment of oral cancer in elderly individuals . several lines of evidence support our views that there is a possibility of drug - resistant microbes present in immunocompromised patients , particularly after the treatment of oral cancer . in general , infections are commonly found in oral cancer patients after surgical excision of the tumor . this might be due to wound exposure during and after the operation , even if sutured , when microorganisms may infect oral regions , oropharynx , nasal cavity , and paranasal sinuses areas . patients after oral cavity surgery often appear to have complications after bacterial infections , as well . colonization of pathogenic bacteria in oral cavity is thought to increase the risk of infections such as pneumonia and bacteraemia . it is therefore of high importance the prevention from or cure for the infections . during the development of tumor , the tumor cells or soluble products produced by tumor cells the high percentages of microbial species identified from the isolates of participants were candida , staphylococcus , streptococcus and pseudomonas species ( figure 2 ) . the highest numbers of these species were found in patients after the treatment of oral cancer in comparison with the non - cancer patients ( tables 1 and 2 ) . similar results were obtained in a phase 1 clinical trial with the application of an antibacterial dressing spray in the prevention of post - operative infection in oral cancer patients . the abuse of antimicrobial drugs brings severe adverse effects to people , for example , allergy , toxic reaction , and opportunistic infections . in this study , after revealing the high percentage of microorganisms found particularly in post - treatment of oral cancer , we started to focus on screening or identification of drug - resistant microbes within the species of already identified microbes . the antimicrobial agents ( figure 1 ) , which are common in clinical practice , were taken for this study . the isolates were tested for the presence of candida species and subspecies of staphylococci , particularly on mrsa , s. epidermidis and s. haemolyticus . except for the c. glabrata and c. krusei species , all other candida species were susceptible to the antifungal agents ( table 3 ) . the other species , s. epidermidis and s. haemolyticus , showed resistance to oxacillin with 60 and 40% , respectively . all other antibiotics were found susceptible to these species ( table 4 ) . according to a study performed in the united kingdom , 13 ( 41.9% ) of 31 s. aureus isolates from the oral mucosa and pockets of patients with another study , in the united states , reported that the prevalence of mrsa organisms in the nasal and oral cavities of nursing home residents was 20 - 35% . when compared with these results , the drug - resistance ( 96% ) of mrsa against oxacillin was extremely high , possibly due in part to the tendency to prescribe long - term , high - dose antibiotic treatment in japan . our study revealed a group of opportunistic - microorganisms such as c. glabrata , c. krusei and subspecies of staphylococci , particularly methicillin - resistant s. aureus ( mrsa ) , s. epidermidis and s. haemolyticus . continuous monitoring and a basic infection control strategy , including standard precautions , are important for older individuals , especially those receiving follow - up care for oral cancer . clinicians must also pay careful attention to opportunistic - microorganisms during the treatment of oral cancer , and to realize the fact of immune compromise in elderly patients .

the term pneumoconiosis was coined by zenker to define a group of diseases caused by inhalation of dust . silicosis is a fibrosing disease of the lung caused by the inhalation , retention , and pulmonary reaction to crystalline silica . silicosis is known to cause random nodules , progressive massive fibrosis ( pmf ) , and obstructive airway disease . anthracosis or coal workers ' pneumoconiosis is seen in coal workers and is caused by inhalation and deposition of black pigments of which carbon is a major constituent . air pollution , biomass smoke , and cigarette smoke are also known to cause deposition of black pigment leading to environmental anthracosis . environmental anthracosis usually leads to anthracofibrosis ( anthracosis with bronchial narrowing ) and obstructive airway disease . but anthracosilicosis , a form of mixed dust pneumoconiosis , has not been reported due to nonoccupational cause . we present six cases of nonoccupational anthracofibrosis / anthcosilicosis from ladakh in jammu and kashmir , india . six cases from the ladakh region in jammu and kashmir ( india ) that presented to a tertiary care institute in delhi from january 2012 to july 2015 with similar clinico - radiological - pathological features were analyzed . of these six cases , four were analyzed from the available records and two were analyzed prospectively . a 58-year - old nonsmoker lady had presented with breathlessness on exertion and cough with melanoptysis for 5 years . there was no significant past history except having taken antitubercular therapy 3 years ago for the same complaints with no improvement in the symptoms . on examination , the available computed tomography ( ct ) of the chest showed partially calcified bilateral uppers lobe masses , air trapping , and random nodules all over the lung fields [ figure 1b d ] . on comparison of two ct scans , the masses had increased by 7 mm on the right side and 6 mm on the left side . the spirometry showed forced vital capacity ( fvc ) of 1.43 lit ( 67% predicted ) , forced expiratory volume in the 1 second ( fev1 ) of 1.10 lit ( 62% predicted ) with fev1/fvc of 0.77 . bronchoscopy had shown narrowing of both the upper lobe bronchi and distortion of the bronchial tree with deposition of anthracotic pigments throughout the bronchial tree [ figure 2a c ] . transbronchial lung biopsy ( tblb ) showed nodular aggregates of polygonal cells with abundant black pigment in the cytoplasm . there was a suspicion of melanoma ; hence , immunohistochemistry ( ihc ) was performed . ihc was positive for cd68 while negative for cd56 , hmb-45 , and ki , and hence , melanoma was ruled out . a final diagnosis of anthracofibrosis was made , as polarized light microscopy , which is pathognomonic of silicosis had not been performed in this case . ( case 1 ) : a ) chest radiograph showing bilateral reticulonodular opacities , upper lobe fibroparenchymal lesion and heterogenous opacity in left mid zone . b ) ct chest showing partially calcified soft tissue mass in the right upper lobe 3458 mm ( shown by arrow ) and left upper lobe ( 3551 mm ) . c ) fibrotic changes seen in both upper lobes d ) randomly distributed nodules in bilateral lung ( case 1 ) : ( a and c ) bronchoscopy showing deposition of anthracotic pigments thought out bronchial tree on both sides and narrowing upper lobe bronchi due to b ) external compression with distortion of bronchial lumen . stroma was made up of fibrocollagenous tissue with neutrophils and mononuclear cells consistent diagnosis of anthracosis a 57-year - old nonsmoker lady from ladakh in jammu and kashmir , india had presented with breathlessness on exertion and minimal cough for 23 years with occasional history of wheezing . there was also history of antitubercular treatment 910 years ago for 9 months . on examination , the chest radiograph showed reticulonodular opacities with right upper lobe collapse [ figure 3a ] . the ct showed an ill - defined mass - like lesion in the right hilum with air trapping and multiple random nodules scattered in both the lung fields [ figure 3b d ] . the spirometry showed fvc of 2.36 lit ( 105% predicted ) , fev1 of 0.99 lit ( 53% predicted ) , fev1/fvc - 0.42 . both the right and left bronchial anatomies were distorted , with mucosal edema and anthracotic pigment . the bronchoscope could not be negotiated into the right intermedius bronchus due to distorted anatomy . whole body positron emission tomography ( pet)-ct scan showed confluent lymph nodes with increased fluorodeoxyglucose ( fdg ) uptake in the right hilum [ table 1 ] . as per the records , she was being followed up for 2 years and treated with inhaled bronchodilator . the spirometry had shown improvement in fvc by 760 ml and fev1 by 720 ml . after 2 years a repeat bronchoscopy , ct scan chest , and pet scan had also been performed . there was no significant change in ct scan , bronchoscopy , and pet scan compared to previous reports . a diagnosis of anthracofibrosis was made on the basis of nonprogressive lesions , bronchoscopy , and histopathology . ( case 2 ) : a ) chest radiograph showing reticulo - nodular opacities in bilateral lung with segmental collapse of right upper lobe . b ) ct chest showing ill - defined mass lesion of ( 3.22.7 cm ) in right hilum encasing the right upper lobe bronchus causing an abrupt cut off . d ) multiple randomly distributed nodules scatted in both lungs a 62-year - old old nonsmoker lady had presented with breathlessness on exertion of 5 years and cough with minimum sputum for 23 years . on examination , the ct had shown speculated mass lesion in the apical segments of the right upper lobe with air trapping and random nodules [ figure 4b d ] . the spirometry had shown fvc of 1.99 lit ( 84% predicted ) , fev1 of 1.35 lit ( 69% predicted ) with fev1/fvc of 0.68 . bronchoscopy had shown constriction and deposition of anthracotic pigments in the right upper lobe bronchus . whole body pet - ct scan showed an increased fdg uptake in the lung lesions and lymph nodes . transthoracic lung biopsy of the lung mass was performed , which showed necrosis and anthrocotic pigment deposition consistent with anthracofibrosis . the sample was not subjected to polarized light and hence , a final diagnosis of anthracofibrosis was made . ( case 3 ) : a ) chest radiograph showing bilateral small nodular opacities in all zones with one heteogenous opacity in right upper lobe . ( c and d ) multiple randomly distributed nodules scatted in bilateral lung field a 55-year - old nonsmoker lady had presented with dyspnea on exertion and cough with melanoptysis for the last 56 years . there was history of bronchodilator therapy for 3 years with some improvement in the symptoms . on examination , the ct of the chest showed an irregular soft tissue mass in both the upper lobes with random nodules in both the lungs [ figure 5b d ] . the spirometry showed fvc of 2.75 lit ( 131% predicted ) , fev1 of 1.49 lit ( 85% predicted ) , and fev1/fvc of 0.54 . the diffusion capacity for carbon monoxide ( dlco ) was -1.73 ml / kg / min ( 102% predicted ) . bronchoscopy showed extensive anthracotic pigments scattered throughout the bronchial tree with complete occlusion of the left upper division with mass - like lesion [ figure 6a and b ] . bronchial biopsy was inconclusive ; hence , trucut transthoracic lung biopsy was performed , which showed a tumor of polygonal and spindle cells . a suspicion of malignant melanoma hence , a repeat biopsy was performed , which showed features of anthracosis [ figure 6c ] and deposition of silicotic pigment ( polarizing birefringent - positive ) [ figure 6d ] . ( case 4 ) : a ) chest radiograph showing reticulo - nodular opacities in bilateral lung with fibrotic lesion on right parahilar region with mediastinal widening . b ) ct chest showing irregular soft tissue ( 3 2.8 2.6 cm ) mass with speculated margin , calcification and collapse of left upper lobe . d ) multiple tiny random nodules were scattered in both lungs ( case 4 ) : a ) bronchoscopy showing extensive anthracotic pigments deposition throughout the bronchial tree of both sides ( a ) with complete occlusion of left upper division with mass like lesion and b ) distortion of bronchial lumen . c ) biopsy showing alveolar septae thicken due to infiltration of numerous polygonal and spindle cell with large amount of black ( anthracotic ) pigment . d ) the fibrous tissue was heavily infiltrated with silicotic pigment deposition ( polarizing birefregent positive ) a 42-year - old nonsmoker lady was investigated by the local physician with chest radiograph , ct scan of the thorax , and other routine investigation , and diagnosed to have left lower lobe hydatid cyst . the ct showed diffuse fine nodules in both the lung fields , with a left lower lobe hydatid cyst [ figure 7b d ] . the spirometry showed fvc of 2.94 lit ( 105% predicted ) , fev1 of 2.33 lit ( 93% predicted ) with fev1/fvc 0.76 . she was treated with oral albendazole 10 mg / kg per day and planned for surgery of hydatid cyst . video - assisted thoracoscopy with excision of the hydatid cyst was performed and simultaneous biopsy was taken from the lung parenchyma . apart from hydatid cyst , the lung biopsy showed anthracotic and polarizing birefringent pigment deposition [ figure 8a and b ] , suggestive of anthracosilicosis . ( case 5 ) : a ) chest radiograph showing bilateral small nodular opacities in all zones with well , defined circular heterogeneous opacity in left lower lobe para and retrocardiac region . cect chest showing b ) well defined cavity containing soft tissue density in posterior basal segment of left lower lobe and ( c and d ) randomly distributed fine nodules involving bilateral lung parenchyma ( case 5 ) : a ) lung biopsy showing fibrous exudate , macular patches around the bronchovascular bundle , circumscribed necrotic area with anthracotic and b ) polarizing birefregent pigment deposition a 62-year - old nonsmoker lady presented with breathlessness on exertion , chest pain , and cough with on - and - off melanoptysis for 1 year . on examination , the ct showed multiple centrilobular nodules scattered in both the lungs with fibroparenchymal changes and air trapping [ figure 9b ] . the spirometry showed fvc of 1.54 lit ( 63% predicted ) , fev1 of 1.22 lit ( 60% predicted ) with fev1/fvc of 0.79 . bronchoscopy showed anthrocotic pigments deposition in both sides with distortion of the bronchial mucosa [ figure 9c and d ] . bronchial biopsy showed nonspecific changes in the bronchial mucosa with anthracotic and silicotic pigment depositions . ( case 6 ) : a ) chest radiograph showing bilateral small nodular opacities in all zones with fibroparenchymal changes with pleural thickening . b ) cect chest showing randomly distributed nodules scatted in bilateral lung fields with fibroparenchymal changes and pleural reaction . ( c and d ) bronchoscopy showing anthracotic pigments deposition throughout bronchial tree of both sides with luminal distortion a 58-year - old nonsmoker lady had presented with breathlessness on exertion and cough with melanoptysis for 5 years . there was no significant past history except having taken antitubercular therapy 3 years ago for the same complaints with no improvement in the symptoms . on examination , the available computed tomography ( ct ) of the chest showed partially calcified bilateral uppers lobe masses , air trapping , and random nodules all over the lung fields [ figure 1b d ] . on comparison of two ct scans , the masses had increased by 7 mm on the right side and 6 mm on the left side . the spirometry showed forced vital capacity ( fvc ) of 1.43 lit ( 67% predicted ) , forced expiratory volume in the 1 second ( fev1 ) of 1.10 lit ( 62% predicted ) with fev1/fvc of 0.77 . bronchoscopy had shown narrowing of both the upper lobe bronchi and distortion of the bronchial tree with deposition of anthracotic pigments throughout the bronchial tree [ figure 2a c ] . transbronchial lung biopsy ( tblb ) showed nodular aggregates of polygonal cells with abundant black pigment in the cytoplasm . there was a suspicion of melanoma ; hence , immunohistochemistry ( ihc ) was performed . ihc was positive for cd68 while negative for cd56 , hmb-45 , and ki , and hence , melanoma was ruled out . transthoracic trucut biopsy was consistent with anthracosis [ figure 2d ] . a final diagnosis of anthracofibrosis was made , as polarized light microscopy , which is pathognomonic of silicosis had not been performed in this case . ( case 1 ) : a ) chest radiograph showing bilateral reticulonodular opacities , upper lobe fibroparenchymal lesion and heterogenous opacity in left mid zone . b ) ct chest showing partially calcified soft tissue mass in the right upper lobe 3458 mm ( shown by arrow ) and left upper lobe ( 3551 mm ) . c ) fibrotic changes seen in both upper lobes d ) randomly distributed nodules in bilateral lung ( case 1 ) : ( a and c ) bronchoscopy showing deposition of anthracotic pigments thought out bronchial tree on both sides and narrowing upper lobe bronchi due to b ) external compression with distortion of bronchial lumen . stroma was made up of fibrocollagenous tissue with neutrophils and mononuclear cells consistent diagnosis of anthracosis a 57-year - old nonsmoker lady from ladakh in jammu and kashmir , india had presented with breathlessness on exertion and minimal cough for 23 years with occasional history of wheezing . there was also history of antitubercular treatment 910 years ago for 9 months . on examination , the chest radiograph showed reticulonodular opacities with right upper lobe collapse [ figure 3a ] . the ct showed an ill - defined mass - like lesion in the right hilum with air trapping and multiple random nodules scattered in both the lung fields [ figure 3b d ] . the spirometry showed fvc of 2.36 lit ( 105% predicted ) , fev1 of 0.99 lit ( 53% predicted ) , fev1/fvc - 0.42 . both the right and left bronchial anatomies were distorted , with mucosal edema and anthracotic pigment . the bronchoscope could not be negotiated into the right intermedius bronchus due to distorted anatomy . whole body positron emission tomography ( pet)-ct scan showed confluent lymph nodes with increased fluorodeoxyglucose ( fdg ) uptake in the right hilum [ table 1 ] . as per the records , she was being followed up for 2 years and treated with inhaled bronchodilator . the spirometry had shown improvement in fvc by 760 ml and fev1 by 720 ml . after 2 years a repeat bronchoscopy , ct scan chest , and pet scan had also been performed . there was no significant change in ct scan , bronchoscopy , and pet scan compared to previous reports . a diagnosis of anthracofibrosis was made on the basis of nonprogressive lesions , bronchoscopy , and histopathology . ( case 2 ) : a ) chest radiograph showing reticulo - nodular opacities in bilateral lung with segmental collapse of right upper lobe . b ) ct chest showing ill - defined mass lesion of ( 3.22.7 cm ) in right hilum encasing the right upper lobe bronchus causing an abrupt cut off . a 62-year - old old nonsmoker lady had presented with breathlessness on exertion of 5 years and cough with minimum sputum for 23 years . on examination , the ct had shown speculated mass lesion in the apical segments of the right upper lobe with air trapping and random nodules [ figure 4b d ] . the spirometry had shown fvc of 1.99 lit ( 84% predicted ) , fev1 of 1.35 lit ( 69% predicted ) with fev1/fvc of 0.68 . bronchoscopy had shown constriction and deposition of anthracotic pigments in the right upper lobe bronchus . whole body pet - ct scan showed an increased fdg uptake in the lung lesions and lymph nodes . transthoracic lung biopsy of the lung mass was performed , which showed necrosis and anthrocotic pigment deposition consistent with anthracofibrosis . the sample was not subjected to polarized light and hence , a final diagnosis of anthracofibrosis was made . ( case 3 ) : a ) chest radiograph showing bilateral small nodular opacities in all zones with one heteogenous opacity in right upper lobe . a 55-year - old nonsmoker lady had presented with dyspnea on exertion and cough with melanoptysis for the last 56 years . there was history of bronchodilator therapy for 3 years with some improvement in the symptoms . on examination , the ct of the chest showed an irregular soft tissue mass in both the upper lobes with random nodules in both the lungs [ figure 5b d ] . the spirometry showed fvc of 2.75 lit ( 131% predicted ) , fev1 of 1.49 lit ( 85% predicted ) , and fev1/fvc of 0.54 . the diffusion capacity for carbon monoxide ( dlco ) was -1.73 ml / kg / min ( 102% predicted ) . bronchoscopy showed extensive anthracotic pigments scattered throughout the bronchial tree with complete occlusion of the left upper division with mass - like lesion [ figure 6a and b ] . bronchial biopsy was inconclusive ; hence , trucut transthoracic lung biopsy was performed , which showed a tumor of polygonal and spindle cells . hence , a repeat biopsy was performed , which showed features of anthracosis [ figure 6c ] and deposition of silicotic pigment ( polarizing birefringent - positive ) [ figure 6d ] . ( case 4 ) : a ) chest radiograph showing reticulo - nodular opacities in bilateral lung with fibrotic lesion on right parahilar region with mediastinal widening . b ) ct chest showing irregular soft tissue ( 3 2.8 2.6 cm ) mass with speculated margin , calcification and collapse of left upper lobe . d ) multiple tiny random nodules were scattered in both lungs ( case 4 ) : a ) bronchoscopy showing extensive anthracotic pigments deposition throughout the bronchial tree of both sides ( a ) with complete occlusion of left upper division with mass like lesion and b ) distortion of bronchial lumen . c ) biopsy showing alveolar septae thicken due to infiltration of numerous polygonal and spindle cell with large amount of black ( anthracotic ) pigment . d ) the fibrous tissue was heavily infiltrated with silicotic pigment deposition ( polarizing birefregent positive ) a 42-year - old nonsmoker lady was investigated by the local physician with chest radiograph , ct scan of the thorax , and other routine investigation , and diagnosed to have left lower lobe hydatid cyst . the ct showed diffuse fine nodules in both the lung fields , with a left lower lobe hydatid cyst [ figure 7b d ] . the spirometry showed fvc of 2.94 lit ( 105% predicted ) , fev1 of 2.33 lit ( 93% predicted ) with fev1/fvc 0.76 . she was treated with oral albendazole 10 mg / kg per day and planned for surgery of hydatid cyst . video - assisted thoracoscopy with excision of the hydatid cyst was performed and simultaneous biopsy was taken from the lung parenchyma . apart from hydatid cyst , the lung biopsy showed anthracotic and polarizing birefringent pigment deposition [ figure 8a and b ] , suggestive of anthracosilicosis . ( case 5 ) : a ) chest radiograph showing bilateral small nodular opacities in all zones with well , defined circular heterogeneous opacity in left lower lobe para and retrocardiac region . cect chest showing b ) well defined cavity containing soft tissue density in posterior basal segment of left lower lobe and ( c and d ) randomly distributed fine nodules involving bilateral lung parenchyma ( case 5 ) : a ) lung biopsy showing fibrous exudate , macular patches around the bronchovascular bundle , circumscribed necrotic area with anthracotic and b ) polarizing birefregent pigment deposition a 62-year - old nonsmoker lady presented with breathlessness on exertion , chest pain , and cough with on - and - off melanoptysis for 1 year . on examination , the vitals were within normal limits . the ct showed multiple centrilobular nodules scattered in both the lungs with fibroparenchymal changes and air trapping [ figure 9b ] . the spirometry showed fvc of 1.54 lit ( 63% predicted ) , fev1 of 1.22 lit ( 60% predicted ) with fev1/fvc of 0.79 . bronchoscopy showed anthrocotic pigments deposition in both sides with distortion of the bronchial mucosa [ figure 9c and d ] . bronchial biopsy showed nonspecific changes in the bronchial mucosa with anthracotic and silicotic pigment depositions . ( case 6 ) : a ) chest radiograph showing bilateral small nodular opacities in all zones with fibroparenchymal changes with pleural thickening . b ) cect chest showing randomly distributed nodules scatted in bilateral lung fields with fibroparenchymal changes and pleural reaction . ( c and d ) bronchoscopy showing anthracotic pigments deposition throughout bronchial tree of both sides with luminal distortion we have described six cases of nonoccupational anthracofibrosis / anthracosilicosis from the ladakh region of jammu and kashmir in north india . nonoccupational silicosis has been reported from various parts of the world including ladakh . in the study from ladakh , the presence of soot and silica was demonstrated in the environment of ladakh but the radiographic abnormality in the study was attributed to only silica . in fact , anthracosis and anthracofibrosis have been described from many parts of the country but not from ladakh . also , this is the first time that anthracosilicosis has been described in the nonindustrial population . in our study , we could demonstrate soot in 6/6 cases and both soot and silica in the pathological samples of only 3/6 cases . however , we believe that all the patients had anthracosilicosis because there is high predilection of silica exposure in the ladakh region in jammu and kashmir , india ( discussed later ) . also , these three cases where silica could not be demonstrated had pmf with random nodules , a finding common with silica dust but not with anthracofibrosis . ladakh in jammu and kashmir , india is among the highest of the world 's inhabited plateaus situated at > 10,000 feet above the sea level . this high altitude and geographic location of ladakh gives rise to the long duration of winter of nearly 45 months in years with sub - zero temperature . one of the reasons for anthracosilicosis may be exposure to soot from the burning of wood and dung in winter to keep the rooms warm . houses in ladakh have fireplaces in the center of room , which use partially burnt wood / dung with smoke during the noncooking period . people usually remain indoors during this prolonged harsh winter ; cooking is also usually done in the same room [ figure 10a ] . the windows are also very small and the houses lack chimneys [ figure 10b ] . women are exposed more to this smoke and soot compared to men because of cooking and indoor confinement . dust storm is common in ladakh mostly during spring season . during these storms a thick blanket of fine dust covers the affected villages , and inhabitants are exposed to considerable amounts of the dust for several days . the frequency , duration , and severity of these dust storms vary considerably from one village to another . in a study by norboo et al . , dust taken from the living rooms from one of the village 's houses had particles ranging from 0.55 m diameters and about half of it was silica . women are again more prone to exposure because they do most of the farming work , sweep the dusty houses , and carry baskets of earth for the traditional toilets . these unique environmental features make the population in ladakh , especially women , prone to nonoccupational anthracosilicosis . a ) roof and wall of the living room showing deposition of black soots and smoke particles . c ) new type of chula with chimmey using presently and old one nobroo et al . had mentioned about taking preventive measures in their study . in the last 30 years , a few preventive measures have been taken , i.e. some of the villages have switched over to electrical heating measures and chimneys have been installed in certain houses in the towns and cities [ figure 10c ] . however , preventive measures are still required . these are : awareness about environmental hygiene , education about installation of chimneys and exhaust fans in all the villages , and using noncombustive substance for cooking and heating in remote areas as well . a systemic study also needs to be undertaken in order to find out the exact role of silica and soot each and prevent this irreversible disease . on ct 4/6 cases showed mass lesions of > 3 cm size , i.e. , pmf with calcified mediastinal lymph nodes , which is one of the commonest lesions seen in complicated pneumoconiosis . in two of the cases ( cases 1 and 4 ) , an increase in the size of the mass lesion was demonstrated . pet scan of two of the patients ( cases 2 and 3 ) showed an increased fdg uptake as well . in two of the cases , even histopathology mimicked malignancy ( cases 1 and 4 ) . although increase in the size of the lesion , increased metabolic uptake and anthracotic pigments are known to occur in pmf , these findings can be mistaken for malignancy if the patient is not expected to have pmf . there were random nodules and centrilobular nodules in all the six cases , which might have been misdiagnosed as miliary or pulmonary tuberculosis . in this series , three out of six patients ( cases 1 , 2 , and 4 ) had received antituberculosis treatment possibly erroneously . increased awareness about environmental anthracosilicosis clinical finding , ct scan , and/or spirometry showed obstructive airway disease in all cases except for case 5 possibly because she was diagnosed incidentally early in the course of illness while undergoing investigation for hydatid . she would have had a similar fate after a few years , had she not been diagnosed and advocated the preventive measures . the obstructive abnormalities in other cases were possibly due to both soot and silica since both are known to cause obstructive airway disease . silica leads to obstructive airway disease because of inflammation of large and small airways with emphysema , whereas anthracosis leads to chronic bronchitis . the treatment of airway disease in these patients would go a long way in treating this on bronchoscopy , five cases showed multiple anthracotic pigments with narrowing and distortion of the bronchus , which is pathognomonic of anthracofibrosis . on histopathology , abundant black anthracotic pigment was present in all the cases that was suggestive of anthracosis . the presence of birefringent particles on polarized light microscopy , which is diagnostic of silicosis is essential when the role of environmental dust is not known . the earlier study reported from ladakh in jammu and kashmir , india did not correlate the environmental dust with pathological findings . so , though we have not accurately completed the unfinished work of sayeed et al . , it is an important step for inciting a prospective research in order to improve the environmental anomaly of this region . anthracosilicosis is a common finding among older female ladakh residents due to exposure to free silica and biomass fuel smoke in the environment . finally , environmental measures and a proper study need to be undertaken for knowing a relative role of silica versus soot in causing the lung disease and preventing this irreversible condition .

background : nonoccupational anthracosis and silicosis has been reported from various parts of the world including ladakh in jammu and kashmir , india ; however , anthracosilicosis has only been reported in industrial workers till date.materials and methods : six cases from the ladakh region in jammu and kashmir , india with similar clinico - radiological - pathological features , i.e. , anthracosilicosis / anthracofibrosis have been analyzed . of these , four were analyzed retrospectively and two prospectively.result:all the patients were homemakers and resided in ladakh in jammu and kashmir , india since birth with an age range of 4262 years and an average age of 56 years . their average duration of symptoms was 4 years . spirometry showed small and/or large airway disease in 5/6 cases . on computed tomography ( ct ) , 4/6 cases showed progressive massive fibrosis ( pmf ) with calcified mediastinal lymph nodes . there were random or centrilobular nodules in all the six cases . bronchoscopy in 5/6 cases showed multiple anthracotic pigments with narrowing and distortion of the bronchus ( anthracofibrosis ) . malignancy was suspected clinico - radiologically in four cases and pathologically in two cases . on histopathology , anthracosis was demonstrated in all and silicosis in three cases.conclusion:anthracosilicosis can occur due to environmental exposure . ladakh in jammu and kashmir , india is the only place across the globe with unique environmental features having the presence of both free silica and biomass fuel . the disease was observed predominantly in older women . awareness would prevent unnecessary investigation for malignancy . treatment with the bronchodilator is useful as it has evidence of airway disease . finally , environmental measures and a proper study need to be undertaken for knowing the relative role of silica versus soot in causing the lung disease and preventing this irreversible condition .

INTRODUCTION CASE REPORTS Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

silicosis is a fibrosing disease of the lung caused by the inhalation , retention , and pulmonary reaction to crystalline silica . silicosis is known to cause random nodules , progressive massive fibrosis ( pmf ) , and obstructive airway disease . environmental anthracosis usually leads to anthracofibrosis ( anthracosis with bronchial narrowing ) and obstructive airway disease . but anthracosilicosis , a form of mixed dust pneumoconiosis , has not been reported due to nonoccupational cause . we present six cases of nonoccupational anthracofibrosis / anthcosilicosis from ladakh in jammu and kashmir , india . six cases from the ladakh region in jammu and kashmir ( india ) that presented to a tertiary care institute in delhi from january 2012 to july 2015 with similar clinico - radiological - pathological features were analyzed . of these six cases , four were analyzed from the available records and two were analyzed prospectively . on examination , the available computed tomography ( ct ) of the chest showed partially calcified bilateral uppers lobe masses , air trapping , and random nodules all over the lung fields [ figure 1b d ] . the spirometry showed forced vital capacity ( fvc ) of 1.43 lit ( 67% predicted ) , forced expiratory volume in the 1 second ( fev1 ) of 1.10 lit ( 62% predicted ) with fev1/fvc of 0.77 . bronchoscopy had shown narrowing of both the upper lobe bronchi and distortion of the bronchial tree with deposition of anthracotic pigments throughout the bronchial tree [ figure 2a c ] . c ) fibrotic changes seen in both upper lobes d ) randomly distributed nodules in bilateral lung ( case 1 ) : ( a and c ) bronchoscopy showing deposition of anthracotic pigments thought out bronchial tree on both sides and narrowing upper lobe bronchi due to b ) external compression with distortion of bronchial lumen . stroma was made up of fibrocollagenous tissue with neutrophils and mononuclear cells consistent diagnosis of anthracosis a 57-year - old nonsmoker lady from ladakh in jammu and kashmir , india had presented with breathlessness on exertion and minimal cough for 23 years with occasional history of wheezing . the ct showed an ill - defined mass - like lesion in the right hilum with air trapping and multiple random nodules scattered in both the lung fields [ figure 3b d ] . the spirometry showed fvc of 2.36 lit ( 105% predicted ) , fev1 of 0.99 lit ( 53% predicted ) , fev1/fvc - 0.42 . the bronchoscope could not be negotiated into the right intermedius bronchus due to distorted anatomy . whole body positron emission tomography ( pet)-ct scan showed confluent lymph nodes with increased fluorodeoxyglucose ( fdg ) uptake in the right hilum [ table 1 ] . as per the records , she was being followed up for 2 years and treated with inhaled bronchodilator . d ) multiple randomly distributed nodules scatted in both lungs a 62-year - old old nonsmoker lady had presented with breathlessness on exertion of 5 years and cough with minimum sputum for 23 years . on examination , the ct had shown speculated mass lesion in the apical segments of the right upper lobe with air trapping and random nodules [ figure 4b d ] . the spirometry had shown fvc of 1.99 lit ( 84% predicted ) , fev1 of 1.35 lit ( 69% predicted ) with fev1/fvc of 0.68 . bronchoscopy had shown constriction and deposition of anthracotic pigments in the right upper lobe bronchus . whole body pet - ct scan showed an increased fdg uptake in the lung lesions and lymph nodes . transthoracic lung biopsy of the lung mass was performed , which showed necrosis and anthrocotic pigment deposition consistent with anthracofibrosis . on examination , the ct of the chest showed an irregular soft tissue mass in both the upper lobes with random nodules in both the lungs [ figure 5b d ] . the spirometry showed fvc of 2.75 lit ( 131% predicted ) , fev1 of 1.49 lit ( 85% predicted ) , and fev1/fvc of 0.54 . bronchoscopy showed extensive anthracotic pigments scattered throughout the bronchial tree with complete occlusion of the left upper division with mass - like lesion [ figure 6a and b ] . d ) multiple tiny random nodules were scattered in both lungs ( case 4 ) : a ) bronchoscopy showing extensive anthracotic pigments deposition throughout the bronchial tree of both sides ( a ) with complete occlusion of left upper division with mass like lesion and b ) distortion of bronchial lumen . d ) the fibrous tissue was heavily infiltrated with silicotic pigment deposition ( polarizing birefregent positive ) a 42-year - old nonsmoker lady was investigated by the local physician with chest radiograph , ct scan of the thorax , and other routine investigation , and diagnosed to have left lower lobe hydatid cyst . the ct showed diffuse fine nodules in both the lung fields , with a left lower lobe hydatid cyst [ figure 7b d ] . the spirometry showed fvc of 2.94 lit ( 105% predicted ) , fev1 of 2.33 lit ( 93% predicted ) with fev1/fvc 0.76 . video - assisted thoracoscopy with excision of the hydatid cyst was performed and simultaneous biopsy was taken from the lung parenchyma . apart from hydatid cyst , the lung biopsy showed anthracotic and polarizing birefringent pigment deposition [ figure 8a and b ] , suggestive of anthracosilicosis . ( case 5 ) : a ) chest radiograph showing bilateral small nodular opacities in all zones with well , defined circular heterogeneous opacity in left lower lobe para and retrocardiac region . on examination , the ct showed multiple centrilobular nodules scattered in both the lungs with fibroparenchymal changes and air trapping [ figure 9b ] . the spirometry showed fvc of 1.54 lit ( 63% predicted ) , fev1 of 1.22 lit ( 60% predicted ) with fev1/fvc of 0.79 . bronchoscopy showed anthrocotic pigments deposition in both sides with distortion of the bronchial mucosa [ figure 9c and d ] . ( case 6 ) : a ) chest radiograph showing bilateral small nodular opacities in all zones with fibroparenchymal changes with pleural thickening . ( c and d ) bronchoscopy showing anthracotic pigments deposition throughout bronchial tree of both sides with luminal distortion a 58-year - old nonsmoker lady had presented with breathlessness on exertion and cough with melanoptysis for 5 years . on examination , the available computed tomography ( ct ) of the chest showed partially calcified bilateral uppers lobe masses , air trapping , and random nodules all over the lung fields [ figure 1b d ] . the spirometry showed forced vital capacity ( fvc ) of 1.43 lit ( 67% predicted ) , forced expiratory volume in the 1 second ( fev1 ) of 1.10 lit ( 62% predicted ) with fev1/fvc of 0.77 . bronchoscopy had shown narrowing of both the upper lobe bronchi and distortion of the bronchial tree with deposition of anthracotic pigments throughout the bronchial tree [ figure 2a c ] . c ) fibrotic changes seen in both upper lobes d ) randomly distributed nodules in bilateral lung ( case 1 ) : ( a and c ) bronchoscopy showing deposition of anthracotic pigments thought out bronchial tree on both sides and narrowing upper lobe bronchi due to b ) external compression with distortion of bronchial lumen . stroma was made up of fibrocollagenous tissue with neutrophils and mononuclear cells consistent diagnosis of anthracosis a 57-year - old nonsmoker lady from ladakh in jammu and kashmir , india had presented with breathlessness on exertion and minimal cough for 23 years with occasional history of wheezing . the ct showed an ill - defined mass - like lesion in the right hilum with air trapping and multiple random nodules scattered in both the lung fields [ figure 3b d ] . the spirometry showed fvc of 2.36 lit ( 105% predicted ) , fev1 of 0.99 lit ( 53% predicted ) , fev1/fvc - 0.42 . whole body positron emission tomography ( pet)-ct scan showed confluent lymph nodes with increased fluorodeoxyglucose ( fdg ) uptake in the right hilum [ table 1 ] . as per the records , she was being followed up for 2 years and treated with inhaled bronchodilator . a 62-year - old old nonsmoker lady had presented with breathlessness on exertion of 5 years and cough with minimum sputum for 23 years . on examination , the ct had shown speculated mass lesion in the apical segments of the right upper lobe with air trapping and random nodules [ figure 4b d ] . the spirometry had shown fvc of 1.99 lit ( 84% predicted ) , fev1 of 1.35 lit ( 69% predicted ) with fev1/fvc of 0.68 . bronchoscopy had shown constriction and deposition of anthracotic pigments in the right upper lobe bronchus . whole body pet - ct scan showed an increased fdg uptake in the lung lesions and lymph nodes . transthoracic lung biopsy of the lung mass was performed , which showed necrosis and anthrocotic pigment deposition consistent with anthracofibrosis . ( case 3 ) : a ) chest radiograph showing bilateral small nodular opacities in all zones with one heteogenous opacity in right upper lobe . a 55-year - old nonsmoker lady had presented with dyspnea on exertion and cough with melanoptysis for the last 56 years . on examination , the ct of the chest showed an irregular soft tissue mass in both the upper lobes with random nodules in both the lungs [ figure 5b d ] . the spirometry showed fvc of 2.75 lit ( 131% predicted ) , fev1 of 1.49 lit ( 85% predicted ) , and fev1/fvc of 0.54 . bronchoscopy showed extensive anthracotic pigments scattered throughout the bronchial tree with complete occlusion of the left upper division with mass - like lesion [ figure 6a and b ] . d ) multiple tiny random nodules were scattered in both lungs ( case 4 ) : a ) bronchoscopy showing extensive anthracotic pigments deposition throughout the bronchial tree of both sides ( a ) with complete occlusion of left upper division with mass like lesion and b ) distortion of bronchial lumen . c ) biopsy showing alveolar septae thicken due to infiltration of numerous polygonal and spindle cell with large amount of black ( anthracotic ) pigment . d ) the fibrous tissue was heavily infiltrated with silicotic pigment deposition ( polarizing birefregent positive ) a 42-year - old nonsmoker lady was investigated by the local physician with chest radiograph , ct scan of the thorax , and other routine investigation , and diagnosed to have left lower lobe hydatid cyst . the ct showed diffuse fine nodules in both the lung fields , with a left lower lobe hydatid cyst [ figure 7b d ] . the spirometry showed fvc of 2.94 lit ( 105% predicted ) , fev1 of 2.33 lit ( 93% predicted ) with fev1/fvc 0.76 . video - assisted thoracoscopy with excision of the hydatid cyst was performed and simultaneous biopsy was taken from the lung parenchyma . apart from hydatid cyst , the lung biopsy showed anthracotic and polarizing birefringent pigment deposition [ figure 8a and b ] , suggestive of anthracosilicosis . ( case 5 ) : a ) chest radiograph showing bilateral small nodular opacities in all zones with well , defined circular heterogeneous opacity in left lower lobe para and retrocardiac region . the ct showed multiple centrilobular nodules scattered in both the lungs with fibroparenchymal changes and air trapping [ figure 9b ] . the spirometry showed fvc of 1.54 lit ( 63% predicted ) , fev1 of 1.22 lit ( 60% predicted ) with fev1/fvc of 0.79 . bronchoscopy showed anthrocotic pigments deposition in both sides with distortion of the bronchial mucosa [ figure 9c and d ] . ( case 6 ) : a ) chest radiograph showing bilateral small nodular opacities in all zones with fibroparenchymal changes with pleural thickening . ( c and d ) bronchoscopy showing anthracotic pigments deposition throughout bronchial tree of both sides with luminal distortion we have described six cases of nonoccupational anthracofibrosis / anthracosilicosis from the ladakh region of jammu and kashmir in north india . nonoccupational silicosis has been reported from various parts of the world including ladakh . in the study from ladakh , the presence of soot and silica was demonstrated in the environment of ladakh but the radiographic abnormality in the study was attributed to only silica . in fact , anthracosis and anthracofibrosis have been described from many parts of the country but not from ladakh . also , this is the first time that anthracosilicosis has been described in the nonindustrial population . in our study , we could demonstrate soot in 6/6 cases and both soot and silica in the pathological samples of only 3/6 cases . however , we believe that all the patients had anthracosilicosis because there is high predilection of silica exposure in the ladakh region in jammu and kashmir , india ( discussed later ) . also , these three cases where silica could not be demonstrated had pmf with random nodules , a finding common with silica dust but not with anthracofibrosis . ladakh in jammu and kashmir , india is among the highest of the world 's inhabited plateaus situated at > 10,000 feet above the sea level . this high altitude and geographic location of ladakh gives rise to the long duration of winter of nearly 45 months in years with sub - zero temperature . one of the reasons for anthracosilicosis may be exposure to soot from the burning of wood and dung in winter to keep the rooms warm . houses in ladakh have fireplaces in the center of room , which use partially burnt wood / dung with smoke during the noncooking period . dust storm is common in ladakh mostly during spring season . during these storms a thick blanket of fine dust covers the affected villages , and inhabitants are exposed to considerable amounts of the dust for several days . the frequency , duration , and severity of these dust storms vary considerably from one village to another . , dust taken from the living rooms from one of the village 's houses had particles ranging from 0.55 m diameters and about half of it was silica . women are again more prone to exposure because they do most of the farming work , sweep the dusty houses , and carry baskets of earth for the traditional toilets . these unique environmental features make the population in ladakh , especially women , prone to nonoccupational anthracosilicosis . in the last 30 years , a few preventive measures have been taken , i.e. some of the villages have switched over to electrical heating measures and chimneys have been installed in certain houses in the towns and cities [ figure 10c ] . however , preventive measures are still required . these are : awareness about environmental hygiene , education about installation of chimneys and exhaust fans in all the villages , and using noncombustive substance for cooking and heating in remote areas as well . a systemic study also needs to be undertaken in order to find out the exact role of silica and soot each and prevent this irreversible disease . on ct 4/6 cases showed mass lesions of > 3 cm size , i.e. , pmf with calcified mediastinal lymph nodes , which is one of the commonest lesions seen in complicated pneumoconiosis . in two of the cases ( cases 1 and 4 ) , an increase in the size of the mass lesion was demonstrated . pet scan of two of the patients ( cases 2 and 3 ) showed an increased fdg uptake as well . in two of the cases , even histopathology mimicked malignancy ( cases 1 and 4 ) . although increase in the size of the lesion , increased metabolic uptake and anthracotic pigments are known to occur in pmf , these findings can be mistaken for malignancy if the patient is not expected to have pmf . there were random nodules and centrilobular nodules in all the six cases , which might have been misdiagnosed as miliary or pulmonary tuberculosis . increased awareness about environmental anthracosilicosis clinical finding , ct scan , and/or spirometry showed obstructive airway disease in all cases except for case 5 possibly because she was diagnosed incidentally early in the course of illness while undergoing investigation for hydatid . the obstructive abnormalities in other cases were possibly due to both soot and silica since both are known to cause obstructive airway disease . silica leads to obstructive airway disease because of inflammation of large and small airways with emphysema , whereas anthracosis leads to chronic bronchitis . the treatment of airway disease in these patients would go a long way in treating this on bronchoscopy , five cases showed multiple anthracotic pigments with narrowing and distortion of the bronchus , which is pathognomonic of anthracofibrosis . on histopathology , abundant black anthracotic pigment was present in all the cases that was suggestive of anthracosis . the presence of birefringent particles on polarized light microscopy , which is diagnostic of silicosis is essential when the role of environmental dust is not known . the earlier study reported from ladakh in jammu and kashmir , india did not correlate the environmental dust with pathological findings . anthracosilicosis is a common finding among older female ladakh residents due to exposure to free silica and biomass fuel smoke in the environment . finally , environmental measures and a proper study need to be undertaken for knowing a relative role of silica versus soot in causing the lung disease and preventing this irreversible condition .

absence seizures , typical for many different patients with absence epilepsy , principally differ from convulsive seizures . to illustrate , a number of widely used and efficient anticonvulsant drugs enhance absence seizures in patients and in the genetic absence models and ethosuximide is only effective in suppressing absence seizures and completely ineffective in other types of seizures [ 13 ] . absence epilepsy is characterized by the occurrence of spontaneous occurring spike - wave discharges ( swds ) and periods of reduced alertness or responsiveness . the spike of the swd represents an excitatory postsynaptic potential and bursts of action potentials ( figure 1(b ) ) of thalamocortical and corticothalamic cells , the wave a slow wave in the eeg , is the subsequent inhibitory phase [ 4 , 5 ] . the generalized and widespread bilaterally synchronous swds are the result of highly synchronized oscillations in corticothalamocortical networks . swds , as can be found in wag / rij rats , have a local cortical origin in the perioral region of the somatosensory cortex [ 612 ] , which is now confirmed in gaers [ 13 , 14 ] , another well validated and often used strain of rats with absence epilepsy [ 15 , 16 ] . local injections in the perioral region of the somatosensory cortex of the t - type ca channel blocker ethosuximide was rather effective in suppressing swds , but also in long - evans rats with swds , and in wistar rats treated with a low dose of ptz . the cortex is part of a network in which the reticular thalamic nucleus ( rtn ) is an essential part . the rtn is responsible for the initiation of sleep spindles not only in rats , but also in other species [ 19 , 20 ] . it needs to be acknowledged that previously the rtn was considered as the pacemaker for swds as well [ 21 , 22 ] . nowadays the origin of the initiation site of swds is the subject of quite some research efforts , in rats as well as in humans and it is widely assumed that the cortex contains a focal hyperexcitable region in which swds have their origin , although the initiation site and the number of foci might be different among patients and between humans and rats [ 10 , 14 , 2329 ] . they allow the investigation of the neurobiological underpinning of this type of epilepsy and the accompanying swds [ 9 , 16 , 3136 ] . the mice have often spontaneous mutations such as in lethargic and tottering mice and transgenic and gene replacement models are also often used ( for review see [ 35 , 36 ] ) . these mice have spontaneous epileptic seizures of which the behavioral and electrographic features and anticonvulsant sensitivity might sometimes be similar to those of human absence seizures . acutely induced absence seizures can also be studied in acute pharmacological models , -hydroxybutyrate ( ghb ) , penicillin but most often a low dose of pentylenetetrazole ( ptz ) is used . these are the ay-9944 [ 38 , 39 ] and the methylazoxymethanol acetate ( mam)-ay-9944 models in rats . administration of the 7-dehydrocholesterol reductase inhibitor , ay9944 , early in life to either rats or mice results in the spontaneous recurrent occurrence of atypical absence seizures which last throughout the animal 's lifetime . the mam - ay9944 model is a double hit model of medically refractory atypical absence epilepsy since cortical dysplasias are first induced in rat by prenatal administration of mam and then ay9944 is administered during the first 3 weeks postnatally . the wag / rij model is one of the well documented models [ 9 , 10 , 16 , 34 , 42 ] . the swds of wag / rij rats start to appear in the eeg when the rats are about 2 - 3 months . at the age of six months , the model has some features , which further increase the interest.wag/rij have a changed expression of genes coding for the low threshold t - type ca channel ( ica , t ) compared to aci control rats and absence epileptic patients have a mutation of genes coding for ica , t [ 4446].reduction of expression of the 3 subunit protein in gabaa receptors in the rtn of wag / rij rats and mutation of genes coding for subunits of gabaa receptors in people with absence epilepsy were found [ 48 , 49].wag / rij rats have complex phenotype : they are comorbid for depression [ 5052 ] . wag / rij have a changed expression of genes coding for the low threshold t - type ca channel ( ica , t ) compared to aci control rats and absence epileptic patients have a mutation of genes coding for ica , t [ 4446 ] . reduction of expression of the 3 subunit protein in gabaa receptors in the rtn of wag / rij rats and mutation of genes coding for subunits of gabaa receptors in people with absence epilepsy were found [ 48 , 49 ] . wag / rij rats have complex phenotype : they are comorbid for depression [ 5052 ] . we assume that the ih pacemaker channels localized in the somatosensory cortex contribute to the initiation of the occurrence of swds and that a second set of pacemaker channels can be found in the thalamus . the latter is responsible for the occurrence of thalamic delta activity and sleep spindles , as can be assumed from the classical studies of the firing pattern in thalamic relay cells , tonic and burst firing modes [ 53 , 54 ] . a small portion of thalamic relay cells show bursting during swds , in which ih might also play a role . the thalamus , a recipient of many cortical efferents , is also part of a resonant circuitry for swds that can be activated by the somatosensory cortex . in this way , the rtn and its widespread connections to other thalamic nuclei form a pathway in the propagation of cortically triggered swds and sustain oscillations . the pacemaker channels in the mammalian brain are named ih channels , the pacemaker channels in other tissue are named if [ 30 , 55 ] . both are cationic pacemaker channels . hyperpolarization - activated cyclic nucleotide - gated cationic if or ih pacemaker channels maintain spontaneous periodic activation , and they were discovered in heart ( if ) and brain ( ih ) . four isoforms of this channel are known ( hcn1hcn4 , hyperpolarization - activated and cyclic nucleotide - gated ) [ 5661 ] . hcn channels resemble voltage - dependent k channels ; they are tetrameric and consist of monomeric subunits , each of which contains six transmembrane domains ( s1s6 ) with a p - loop ( figure 2 ) between the s5 and s6 domains [ 60 , 62 ] . four hcn1 subunits join and form a hcn1 homotetrameric channel , four hcn2 subunits form a hcn2 homotetrameric channel , and so forth . different hcn subunits may also join to form a heteromultimeric complex , this substantially increases the diversity of if , or ih channels [ 6264 ] . the primary analysis of the hcn channel sequence [ 5658 , 60 , 62 ] indicated the presence of five endogenous cysteine residues in the transmembrane domains . three of these are localized closer to the extracellular side , the cys303 residue in the s5 transmembrane domain , the cys318 residue in the aminoacid chain connecting s5 and s6 , and cys347 , which is in direct proximity to the glycine - tyrosine - glycine ( gyg ) sequence that forms the channel pore . two other cysteine residues are localized closer to the cytoplasmic , or inner , side ( cys298 in s5 and cys374 in s6 ) ( figure 2 ) . it is known that cysteine has a hydrosulphate group and its reduced form may easily participate in chemical reactions , like alkylation , acylation , and arylation , or even cross - inking with another nearby cysteine to form a disulphide bridge [ 65 , 66 ] . to illustrate , hcn1 and hcn2 subunit isoforms , coexpressed in the cerebral cortex and hippocampus , are substantially different in their biophysical features . also , hcn1 channels become activated at a membrane potential that is 20 mv more positive than the potential required for hcn2 activation [ 68 , 69 ] . the hcn2 channel demonstrates a clear response ( + 17 mv ) to camp binding at the cyclic nucleotide binding domain ( cnbd ) on the c - terminus , the hcn1 channel demonstrates a minimal response to camp ( + 4 mv ) . therefore it can be safely assumed that hcn1 and hcn2 perform quite different functions in the brain regions where they predominate . heteromultimeric channels with new properties can be found throughout the central nervous system [ 68 , 69 ] . coexpression of hcn1 and hcn2 with heteromultimeric channel formation leads to ih currents , which are kinetically activated via a voltage - dependent mechanism and tend to possess functional properties that are intermediate between hcn1 and hcn2 homomeric channels . coexpressed heteromultimeric channels demonstrate a relatively larger shift in response to camp ( + 14 mv ) than the monomeric hcn1 channel . neither the kinetic curve nor dose - effect curve in response to camp of coexpressed heteromultimeric ih channels are a linear summation of the independent populations of hcn1 and hcn2 homomeric channels . several chimers of hcn1 and hcn2 homomeric channels have been created with a modified nh2 terminus , transmembrane domain , and different areas of the cooh terminus . cnbd exchange between hcn1 and hcn2 results in only a small influence on the main gating mechanism , and exerts a relatively poor effect on camp modulation . the difference in the camp modulatory effect depends instead on the interaction between cnbd and the aminoacid sequence ( 80 amino acids ) that connects the last transmembrane domain ( s6 ) and cnbd . the glycine - tyrosine - glycine ( gyg ) sequence found between the s5 and s6 domains forms the channel pore ( figure 2 ) . the pore is formed by this aminoacid sequence originating from all four subunits when they are united in the tetrameric channel . the pore of the hcn channel is permeable not only for k but also for na ions . the main diagnostic sign of absence epilepsy is the presence of swds in human or animal electroencephalogram ( eeg ) . the similarity in morphology of swds [ 32 , 71 ] and discharges generated by ih channel ( figure 1(b ) ) suggests that the ih channel is a contributing factor to the morphology of the spike and wave complex . several other authors have already hypothesized that this nonselective ih channel plays also a crucial role in the age - dependent development in the occurrence in swds [ 70 , 7276 ] . considering that different subtypes are differently present in cortex and thalamus , hcn1 is expressed selectively in specific brain regions , among others the neocortex and certainly on the large pyramidal neurons [ 58 , 77 , 78 ] , while hcn2 is more widely expressed throughout the brain , including the thalamus ; these different parts will be discussed separately . studies aimed at elucidating the role of ih channel activity in absence epilepsy compared ih channels in cortical neurons of epileptic and control strains of rats : wag / rij , wistar ( of the same genetic background as wag / rij but less prone to absence epilepsy ) and aci rats ( an inbred strain practically devoid of epileptic activity ) [ 73 , 79 , 80 ] . patch - clamp recording from the whole cell of the second - third cortical layer of pyramidal neurons was supplemented by immunohistochemical , western blot , and pcr studies of hcn1hcn4 subunits of the ih channel . the fast component of ih activation in neurons of wag / rij rats was significantly reduced ( a 50% decrease in the current density ) and was four time slower than in the neurons of nonepileptic wistar or aci rats . a decrease by 34% was found in the level of the hcn1 subunit protein in the cerebral cortex of wag / rij rats as compared to wistar rats , but hcn1 mrna expression was not different . the protein and mrna levels of the other three ih channel subunits ( hcn2hcn4 ) were not altered . this loss of neocortical hcn1 function may contribute to an increased cortical excitability since there are substantially fewer hcn1 subunits in the combined complex of the ih channel in the cortical zone containing the focal region in wag / rij strain compared to the two control strains . a loss of hcn1 was also found during ontogeny : it occurs primarily in the apical dendrites of layer 5 pyramidal neurons in the cortex , leading to a spatially uniform 2-fold reduction in dendritic hcn current throughout the entire somatodendritic axis . dual whole - cell recordings from the soma and apical dendrites demonstrate that loss of hcn1 increases somatodendritic coupling and significantly reduces the frequency threshold for generation of dendritic ca spikes by back propagating action potentials . as a result of increased dendritic ca electrogenesis , a large population of wag / rij layer 5 neurons showed intrinsic high - frequency burst firing . using morphologically realistic models of layer 5 pyramidal neurons from control wistar and wag / rij animals , it was shown that the experimentally observed loss of dendritic ih recruits dendritic ca channels to amplify action potential - triggered dendritic ca spikes and increase burst firing . thus , loss of function of dendritic hcn1 channels in layer 5 pyramidal neurons provides a somatodendritic mechanism for increasing the synchronization of cortical output , and is therefore likely to play an important role in the generation of swds accompanying absence seizures . it has been shown that neonatal handling and maternal deprivation postnatally ( 121 days ) resulted in reduced number of absences , decreased interspike interval , and changes in power of the frequency spectrum of swds in adult wag / rij rats . whole cell patch - clamp recordings from the cells of the fifth pyramidal layer , in situ hybridization , and western blot analysis of the cortex of adult wag / rij rats showed an increase in the hcn1 protein level in the somatosensory cortex of handled and mother - deprived rats as compared to control rats . this increase was selective for the hcn1 subunit and did not affect the expression of hcn2hcn4 subunit proteins ; neither did expression of the mrna of any subunit ( hcn2 , hcn3 , and hcn4 ) . these results indicate that relatively mild changes in the environment of neonatal rats have long - lasting consequences for the occurrence of paroxysmal activity later in life and suggest that increased concentration or better , an increased proportion of hcn1 subunits in ih channel composition in a small area of the somatosensory cortex is related to a reduction in the number of swds . an increased number of hcn1 subunits in the channel due to their extensive expression ( increased hcn1 protein level without changes in hcn2 , hcn3 , and hcn4 mrna levels ) , and consequently , a decreased number of hcn2 , hcn3 , and hcn4 subunits in the channel complex , as mentioned above [ 68 , 69 ] , result in impaired activity of the ih channel , reduced cortical excitability , and less swds , as it has been demonstrated experimentally [ 73 , 74 , 81 ] . besides the age - dependent decrease in the number of hcn1 subunits and in protein expression in the deep layers of the focal region , a chronic drug treatment aimed at antiepileptogenesis in the same model confirmed that the reduction of swds was accompanied by an increase in hcn1 , producing levels that resemble nonepileptic controls . in addition , neonatal manipulations increased the number of hcn1 subunits and decreased swds , providing evidence for the negative correlation between swds on the one side and this ih channel otherwise . blocking the ih channels of layer v neurons by zd7288 ( 4-ethylphenylamino-1,2-dimethyl-6-methyl - aminopyrimidinium chloride ) , as well as a loss of hcn1 subunits , increases somatodendritic coupling , increases ca conductance by reducing the frequency threshold for induction of dendritic ca electrogenesis , increasing synchronization of cortical output and burst firing . it is , however , possible that this property of ih channels is only present in neurons of the the subgranular layers , since ih channels of thalamic neurons have different biophysical properties . it can be concluded that an age - dependent loss of neocortical hcn1 function contributes to an increased cortical excitability in the form of more and longer spontaneous occurring swds and that various early manipulations , such as maternal deprivation but also treatments aiming at antiepileptogenesis confirm the negative relation between hcn1 and swds . the in vitro studies done in the late eighties and in the nineties of the last century were influential for the ideas than have been developed on the role of the interaction between ih and ica , t . basically , the interaction of ih and ica , t channels has been assumed to be the basis for burst firing in thalamocortical ( tc ) cells . briefly , upon hyperpolarization of the tc cell membrane by a reduction of depolarizing inputs beneath the resting membrane potential , ih is activated and produces a slow pacemaker depolarization until the threshold voltage of activation of it is reached . the pacemaker depolarization subsequently activates it that leads to the generation of a low - threshold ca potential , bringing the membrane potential positive to firing threshold . less ih ( less inward current ) than at the beginning of the ca spike will be activated because ih partly deactivated during the depolarization associated with the ca potential . as a consequence , the membrane potential will hyperpolarize to a potential more negative than the it activation threshold . the cell will thus reach the peak of the hyperpolarization and reactivate ih , restarting the cycle . thus , while it is responsible for the large - amplitude depolarizations , ih mediates both the slow pacemaker depolarization necessary to activate it and the hyperpolarization that follows low - threshold ca potentials . the burst firing mode is activated during drowsiness , with tc and ct cells undergoing a progressive hyperpolarization from drowsiness and the early stages of sleep to the late sleep stage of deep slow wave sleep . the frequency of the burst firing mode is determined by the amount of hyperpolarization , such that sleep spindle oscillations ( 712 hz ) occur at more depolarized membrane potentials than delta oscillations . it has been assumed based on in vitro studies that this thalamic burst firing mode can also be the physiological correlate of swd . however , studies in gaers have demonstrated that tc relay cells were not associated with rhythmic ipsps , ca currents , or burst discharges , but only with the occasional firing of a single action potential [ 87 , 88 ] , and that during swds a minor portion of tcr cells ( 7% in gaers , ) show ca currents and burst discharges . the bursting of such a small portion of tcr cells appears to be sufficient for the sustainment of swds . in agreement with this finding is that local injections of the low - threshold ca blocker ethosuximide are not very active when injected in the thalamus . moreover , there are no in vivo studies in genetic models which investigated the role of thalamic ih . however , burst firing in tc cells is crucial for the presence of delta waves . in in vitro rat , cat , and ferret studies , isolation of tc relay cells from synaptic input from ct and rtn leads to a hyperpolarization of the cells and onset of self - sustained oscillations in the delta ( 0.54 hz ) frequency band [ 85 , 89 , 90 ] . likewise hyperpolarization of tc neurons by means of hyperpolarizing current injection leads to burst firing of the neurons also in the delta frequencies , and this goes together with the activation of ih [ 85 , 89 ] . studies on the role of ih in spindle oscillations have been performed in thalamic slices with neurons that either show spontaneous spindling , such as in the ferret lgnd , or where thalamic spindle activity is evoked by applying hyperpolarizing current pulses mimicking ipsps arriving during spindle waves in the intact rtn / pgn - tc network . induced spindle activity in tc neurons has been shown to go together with ih activation , lending evidence for an involvement of ih in spindle generation [ 93 , 94 ] . further evidence for a role of ih in spindle oscillations comes from ih manipulation studies in thalamic slices . intracellular injection of depolarizing current into spindling lgnd neurons resulted in inactivation of it , abolition of rebound ca spikes and cessation of the spindle oscillations . the type of burst firing oscillation ( delta and spindle oscillations ) is determined by the membrane potential , that is on its turn set by ih [ 70 , 93 ] . ih thus plays an important role in setting the membrane potential that determines the type of burst firing oscillation present in the tc cells . but different from delta oscillations , ih and the pacemaker potential created by low - threshold ca , does not seem to be of critical importance for spindles since blockade of ih does not lead to the cessation of the oscillation , as seen in delta oscillations [ 85 , 89 , 96 , 97 ] , but to a failure in spindle termination [ 89 , 92 ] . this questions the exact role of ih in spindle generation ; moreover in vivo studies towards the role of ih in sleep spindle generation are lacking as well . it can be concluded that the interaction of ih and it seems critical for the generation of delta oscillations , ih seems involved in sleep spindle generation , and however its exact mechanisms are beyond the scope of this paper . a dominant role for ih in thalamic bursting underlying swd however , the possibility exists that ih and it interact in the portion of tc cells that do show bursting in which the ih and it interact as a tandem . the role of thalamic ih was investigated in wag / rij rats : high hcn1 mrna and protein expression without alterations in hcn2hcn4 expression was found in thalamic neurons . these authors found also that that burst discharges in thalamocortical neurons locked to swds were prolonged during blockade of ih by cs and zd7288 , similar as has been found for sleep spindles . this was associated with hyperpolarizing shift in ih activation , a decrease in camp responsiveness of ih in tc neurons and resulting impairment to shift from burst to tonic firing , which in turn produced long burst activity after recruitment of ih during swds . on the basis of these results and shifts of ih channel reactivity induced by high concentrations of camp , budde et al . hypothesized that increased hcn1 expression in the epileptic thalamus is associated with decreased ih channel reactivity to camp in thalamic neurons and leads to a loss of control over the channel , which , in turn , prolongs high ih channel activity during absence seizures . interesting results were also obtained during the study of the regulation of the pacemaker ih channel in the thalamus of gaers rats ; the function of the ih channel and subunit composition in tc - neurons were studied before ( presymptomatic ) and when ( symptomatic ) swds were present in the eeg . the voltage - dependent ih current remained unaltered in mature somatosensory neurons , both in vivo and in vitro . however , an increase in ih current by the phasic , practically physiologic , impulse of camp was diminished by approximately 40% and the half - maximal camp concentration was approximately fivefold increased . this diminished sensitivity of ih current to its main cellular modulator preceded the beginning of epilepsy , lasted during its chronic phase , and accompanied the elevated expression of mrna of the camp - insensitive hcn1 subunit of the channel ( > 50% ) without alterations in hcn2hcn4 mrna expression . changes in excitability of tc neurons were analyzed by measuring the delayed upregulation of the ih current . this current was caused by ca - triggered camp synthesis and is essential for finishing synchronized oscillations in vitro . repeated recovery of the ca spike induced the normal deceleration of upregulation of the ih current in mature gaers neurons , sufficient to reduce the spontaneous rhythmic burst discharges . these adaptive mechanisms take place despite camp turnover and involve increased intracellular ca concentration in response to repeated low - threshold ca discharges . the weakening of the binding of camp to hcn channels in the thalamus proceeds , and likely promotes , epileptogenesis in gaers rats , whereas compensatory mechanisms stabilizing the function of ih channels contribute to the completion of the swds in chronic absence epilepsy . a quantitative model ( figure 3 ) describes how ih and it cooperate and how the subcompositions of the subunits determine the net effect ( depolarization ) of hyperpolarizing inputs . the tandem consisting of ih and ica , t works as follows : the hyperpolarization of the cell membrane opens a ih channel and the membrane is depolarized by an amount n. next the hyperpolarization opens the ica , t channel and ca enters the cell , increases activity of adenylate cyclase , and increases camp concentration as the result of selective activation of pka ( camp - dependent protein kinase - a ) isoforms . the camp binds to the cnbd domain and enhances the ion flow and amount of membrane depolarization ( gating mechanism ) to a value m. the total amount of depolarization ih dep = n + m. when the ih channel consists of four hcn1 subunits , as the case in the cortex for the large pyramidal cells , then the interaction of camp with the cnbd hcn1 subunit strengthens the gating mechanism and the depolarization is increased by 4 mv . in this case , n + ( 4 4 ) mv = n + 16 mv . when camp interacts with cnbd of an hcn2 subunit , as is the case in the thalamus , then the gating mechanism is amplified and the depolarization is increased by 17 mv . the subunit composition can be either : 3(hcn1 ) + 1(hcn2 ) ; ih dep = n + ( 3 4 ) mv + ( 1 17 ) mv = n + 29 mv 2(hcn1 ) + 2(hcn2 ) ; ih dep = n + ( 2 4 ) mv + ( 2 17 ) mv = n + 42 mv 1(hcn1 ) + 3(hcn2 ) ; ih dep = n + ( 1 4 ) mv + ( 3 17 ) mv = n + 55 mv 4(hcn2 ) ; ih dep = n + ( 4 17 ) mv = n + 68 mv . 3(hcn1 ) + 1(hcn2 ) ; ih dep = n + ( 3 4 ) mv + ( 1 17 ) mv = n + 29 mv 2(hcn1 ) + 2(hcn2 ) ; ih dep = n + ( 2 4 ) mv + ( 2 17 ) mv = n + 42 mv 1(hcn1 ) + 3(hcn2 ) ; ih dep = n + ( 1 4 ) mv + ( 3 17 ) mv = n + 55 mv 4(hcn2 ) ; ih dep = n + ( 4 17 ) mv = n + 68 mv . this implies that the composition of the heteromeric ih channel , as can be found for example , in the thalamus , determines the amount of depolarization and the subsequent activation of voltage - activated ca channels . with age , the contribution of hcn1 subunits decreases ( through a process called trafficking ) and is replaced by hcn2 subunits . it does also explain that during aging the reaction to hyperpolarizing inputs might increase the amount of depolarization ; in other words , during aging the thalamus will react with larger depolarizations upon the same hyperpolarizing inputs . our hypothesis that burst firing in tc neurons is caused by ( tandem ) hyperpolarization - activated ih channel and hyperpolarization - activated ( ica , t ) channel and that burst firing of at least some tc cells plays a role in swd occurrence , is supported by pharmacological studies indicating that increased gabaergic inhibition in the ventral basal ( vb ) complex of the thalamus , as induced by local injections of gaba - agonists , enhances swds . remember that opposite effects ( a decrease in swds ) were obtained by injections of gaba agonists in the focal zone of the cortex [ 15 , 99 , 100 ] , suggesting that here depolarization rather than hyperpolarization promotes burst firing [ 76 , 101 ] . the literature reviewed in this paragraph suggests that ih and low - threshold t - type ca channels in tc cells work in tandem in order to generate burst - like activity , the assumed neurophysiological firing pattern that forms the basis of delta waves , sleep spindles and perhaps swds . the latter remains uncertain since the vast majority of tc cells do not show burst firing activity during swds . hyperpolarization opens the ih channel , and cationic current depolarizes the membrane to the threshold and induces a ca spike . the entrance of ca ions into the cell induces ca - dependent camp synthesis , and camp dramatically increases channel activity through binding to the cnbd locus of hcn subunits . the hcn1 subunit responds weakly to camp binding ; therefore , a decrease in the proportion of hcn1 subunits in the channel increases pacemaker activity and an increase in the proportion of hcn1 subunits in the channel decreases pacemaker activity . the analysis of experimental data shows that one of the basic mechanisms of the long - term regulation of ih pacemaker activity is modification and consolidation of a number of hcn1 subunits in the pacemaker channel by the help of the transduction signal . the first type of receptors mediating fast postsynaptic gaba reactions ( ipsp 's ) are ionotropic gabaa receptors , including in its structure a chlorine channel . , these receptors can decrease ca conductance and inhibit camp production via intracellular mechanisms mediated by g proteins . presynaptic inhibition may occur as a result of gabab receptor binding on nerve terminals causing a decrease in the influx of ca , thereby reducing the release of neurotransmitters . postsynapticaly , the g protein - gated inwardly rectifying k ( girk ) channels are activated by stimulation of gi / o - coupled gabab receptors and provide an important mechanism for the slow inhibitory modulation of cellular excitability in the brain [ 102104 ] . the gabaa receptors can be allosterically facilitated ( increase of chlorine current ) by benzodiazepines [ 105107 ] . pharmacological studies with specific agonists and antagonists of the benzodiazepine ( bdz ) receptor focused on the gabaa receptor complex . a series of drugs belonging to the class of the -carbolines were evaluated , mainly partial agonists such as zk 91296 and zk 112119 ( abecarnil ) and compared with the full agonist diazepam [ 108110 ] . diazepam showed all behavioral and electrophysiological changes characteristic for the bdz , zk 91296 reduced seizure activity without inducing signs of sedation , sleepiness , myorelaxation , and changes in behavior or eeg spectral content . another member of the -carboline family , the partial inverse agonist fg 7142 , aggravated epileptic activity , with slightly enhanced immobile behavior , suggesting anxiogenic properties . the results not only demonstrate that the multiple effects of the bdz could be separated by these compounds , but also that the antiepileptic activity is not related to changes in spectral content of the eeg . because of its selective activity , zk 91296 appears to be more suitable than diazepam in reducing seizure activity . finally , fg 7142 seems a genuine partial inverse agonist which has some , but not all , of the inverse effects of a full agonist . next the effects of the -carboline ( abecarnil , zk 112119 ) were determined in two experiments : in the first the antiabsence profile of abecarnil was evaluated in wag / rij rats . in the second experiment it was found that , similarly to classical bdz , abecarnil possessed a strong antiepileptic character and also changed the background eeg to more high - frequency waves and less spindle activity . abecarnil induced only small , marginally significant increases in slow - wave sleep while reducing rem sleep as a proportion of total sleep . these observations are consistent with the proposed partial agonist activity of abecarnil , a drug with interesting therapeutic implications . rather similar results on the antiabsence action of the full agonists of bdz receptors have been found in gaers , emphasizing the similarities in the pharmacological profile of the two genetic absence models . therefore , the effects of the bdz hypnotic midazolam and the imidazopyridine hypnotic zolpidem ( both 10 mg / kg ) on sleep - wake states and on the number of swds were evaluated in wag / rij rats . the initial decrease of swds after midazolam , however , was followed by a rebound . the antiabsence activity of zolpidem mimics that of midazolam and is well correlated with the equipotent hypnotic action and anticonvulsant effect in the isoniazid test . the assumed tolerance to bdz was investigated by injecting diazepam ( 5 mg / kg ) twice daily for 23 days in wag / rij rats . after this , the rats received the agonist , diazepam , or the antagonist , flumazenil ( ro 15 - 1788 ) . the effects were rather different after chronic administration : both compounds increased the number of swds , suggesting that repeated administration of diazepam abolished its antiepileptic action and even reversed its action . the antiepileptic effect of diazepam , acutely administrated , has been found also by others in gaers . the next allosteric facilitator of gaba inhibition at the gabaa receptor is the group of neurosteroids . they have clinical relevance partly because the ovarian hormones acting as neurosteroids are held responsible for the variation of seizure frequency during the menstrual cycle , around puberty , and menopause . the variations in seizure frequency have been attributed partly to the antiepileptic action of progesterone : it reduces seizures during the luteal phase . however , studies on catamenial epilepsy ( derived from the greek word katomenios , meaning monthly ) , clusters of seizures around specific points in the menstrual cycle , have not always distinguished between patients with focal and generalized seizures . grnewald et al . described a patient whose absence seizures exacerbated when she was given progesterone . this indicates that , in contrast to the well - known anticonvulsant and antiepileptic properties of progesterone in most animal models and in almost all seizure types , absence epilepsy might be an exception in the sense that progesterone increases absence seizures . the role of the sex hormones , but also of ganaxolone and other neurosteroids , has been thoroughly investigated in the genetic and pharmacological absence models . ganaxolone ( 3-hydroxy-3-methyl-5-pregnan-20-one ) is a synthetic neurosteroid with anticonvulsant properties in a number of seizure models as well as the ability to enhance function of the gabaa receptor complex via the neurosteroid binding site . the effect of ganaxolone on absence seizures was assessed in the low - dose ptz and the ghb model . ganaxolone resulted in a significant prolongation of absence seizure in both models and in doses above 20 mg / kg it produced bilaterally synchronous swds associated with behavioral arrest . these data suggest that augmentation of gabaa receptor complex function by neurosteroids has the potential to result in or exacerbate absence seizures . systemic administration of the neurosteroids allopregnanolone and pregnenolone sulfate was studied in wag / rij rats . allopregnanolone is also a positive modulator of the gabaa receptor , it increased dose dependently the number and total duration of swds . pregnenolone sulfate , also a positive modulator of nmda receptors , also increased those parameters , though only at the highest dose used ( 100 mg / kg ) and only in the first hour post - injection . these proepileptic activities of these neurosteroids contrasts with the anticonvulsant effects of many of these neurosteroids and they point to a different pharmacological profile of epilepsy with convulsive or nonconvulsive seizures . in the next experiment the influence of endogenous neurosteroids on swds was investigated in female wag / rij rats in a chronic eeg study during a complete ovarian cycle . a circadian pattern emerged for the number of swds : a nadir during the first hours of the light period , and an acrophase during the first hours of the dark period . this daily maximum was increased at proestrus day compared with the other days of the cycle , coinciding with an increased progesterone plasma level specifically at these hours of this day . this clearly suggests that even shifts in physiological concentrations of progesterone are already large enough to enhance swds . there was no difference in the first few hours of the light period in the number of swds between proestrus and the three other days , suggesting that estradiol has no effect on swds . in the second study , the effects of systemic administration of progesterone and 17-estradiol on swds and spontaneous behavior were investigated . it was confirmed what was found in the long term eeg study : progesterone increased the number and total duration of swds ; 17-estradiol was completely ineffective . the administration of ru 38486 , an antagonist of intracellular progesterone receptors , had no effect on swds and did not block the stimulatory effect of progesterone . the intracellular estrogen receptor antagonist tamoxifen did not evoke alterations in the number or duration of swds either . the results indicate that estradiol is not affecting swds and that progesterone aggravates swds , but the progesterone effect is not mediated through intracellular receptors . since progesterone is rapidly metabolized in the brain to the positive modulator of gabaa receptor allopregnanolone , which is also known to increase swds in wag / rij rats , it is rather likely that the epileptoformic effects of progesterone are mediated through this metabolite . in order to elucidate whether the regulatory effect of progesterone depends indeed on its conversion to allopregnanolone , the effect of finasteride , a 5-reductase inhibitor , on progesterone - induced increase in swds was studied . finasteride alone had no effect on the number of swd , up to 24 h following its administration . pretreatment of rats with finasteride , however , blocked the progesterone - induced enhancement of swd . this indicates that the action of progesterone is mediated by its neuroactive metabolite allopregnanolone , through strengthening of gabaa receptor activity . the administration of both progesterone and allopregnanolone exacerbated seizures in the ay model ( an atypical absence epilepsy model ) in agreement with the outcomes in wag / rij rats , whereas 17-estradiol attenuated swds ( this ovarian hormone does not modulate absences in the typical absence model ) . neither mifepristone nor tamoxifen blocked the effects of progesterone and 17-estradiol , respectively , on swds duration in the atypical absence model , suggesting that these two sex hormones are working in a manner independent of their intracellular neurosteroid receptor . anyway , the differential role of estradiol in typical and atypical absence seizures emphasizes the differences between these types of epilepsy . the effects of neurosteroids on swds in wag / rij rats were also studied by local microinjections . allopregnanolone and ganaxolone ( the two positive allosteric modulators of the gabaa receptor complex acting on the steroid recognition site ) and pregnenolone sulphate , ( the negative allosteric modulator of gabaa receptors and a positive modulator of nmda receptors ) were investigated . focal bilateral microinjection of the two gabaa positive modulators into some thalamic nuclei ( nucleus ventralis posteromedialis , nrt , nucleus ventralis posterolateralis ) increased swds . both compounds reduced number and duration of swds when microinjected into the perioral region of the primary somatosensory cortex , the focal region . this demonstrates that there is not a general increase in inhibition , instead there seems to be an increased inhibition on the level of the vb , not a decrease in inhibition in the rtn and the focal zone in the cortex . the effects of pregnenolone sulphate were more complex and depended on the dose and the site of administration : generally , at low doses in rtn and focal cortical region an increase in number and mean duration was found , and a reduction at high doses . the effects in the ventralis posteromedialis and posterolateralis were a decrease and no effect , respectively , on swds . it was concluded that neurosteroids might play a role in absence epilepsies and that their action depends on the activity in specific neuronal areas . more specifically , the results show that a lack of inhibition at the focal cortical site may be a cause of the increased excitability . the effects of specific gabaa receptor agonist and antagonist , muscimol and bicuculline , respectively , were studied by intracerebroventricular injections . eeg registrations and behavioral observations showed that muscimol dose dependently increased the nonconvulsive absence seizures . besides this , it induced eeg spikes and body twitches . all effects of muscimol were blocked by bicuculline and vice versa , which suggests that the observed effects are genuine gabaa effects . the other agonists of gabaa receptors such as 4,5,6,7-tetrahydroisooxazolo(5,4-c)pyridin-3-ol ( thip ) , l - baclofen [ 115 , 125 ] enhanced the duration of swds in a dose - dependent fashion . all these described results implicate that nonconvulsive epilepsy can be caused by a gabaa receptor hyperfunction , although this is an oversimplification . in general , the opposite action of gabamimetic drugs points to a pharmacological difference between convulsive and absence types of epilepsy . the local injection studies demonstrated that there is gabaa hyperfunction in the vb , but a gabaa hypofunction in the focal cortical region . the gabab receptor antagonist cgp 35348 was intraperitoneally given in doses of 100 , 300 , and 900 mg / kg to old wistar rats , endowed with similar swds as wag / rij rats and gaers . the effects on sleep and behavior were investigated as well . the low and middle dose of the drug produced an increase in the duration of rem sleep , the rem sleep latency was correspondingly reduced . non - rem sleep and total sleep duration increased and an s - shaped dose - response relationship was found . number and duration of swds were reduced after all doses of cgp 35348 , confirming the strong suppressive action of this gabab antagonist on absences . others have described similar effects in gaers and in the lethargic ( lh / lh ) mutant mice model . antagonists of the gabab receptors suppressed swds whereas agonists of gabab receptors exacerbated them . furthermore , gabab receptor binding and synaptically evoked gabab receptor - mediated inhibitions of nmda responses were selectively increased in lh / lh mice . therefore , enhanced gabab receptor - mediated synaptic responses may underlie absence seizures in the lh / lh mice , and gabab receptor antagonists hold promise as antiabsence drugs . the microinjections of the gabab agonist [ ( )-baclofen ] and the antagonist cgp 35348 in anterior ventral lateral thalamic nucleus , rtn and nucleus reunions decrease and increase , respectively , absence seizures in ( lh / lh ) mice . next the antiabsence effect of the gabab receptor antagonist sch 50911 was investigated in the lethargic ( lh / lh ) mutant mouse and in two rat models in which absence seizures were induced by administration of either ghb or a low - dose ( 20 mg / kg ) ptz . sch 50911 abolished seizures in all three models in a dose - dependent fashion . in each model sch 50911 was more potent than the gabab receptor antagonist cgp 35348 , ethosuximide , trimethadione , and valproic acid . sch 50911 was equipotent as the gabab antagonist cgp 46381 in the lh / lh mouse model . further experiments examined the effectiveness of a range of specific gabab - receptor agonists and antagonists of varying specificity , as well as the specific ghb - receptor antagonist ncs 382 , in the ghb and low - dose ptz models . all specific gabab - receptor antagonists as well as the specific ghb - receptor antagonist produced blockade of absence seizures in both models ; pretreatment with gabab receptor agonists resulted in generalized absence status epilepticus lasting for hours . these data confirm that specific gabab - receptor antagonists have antiabsence activity , that the same holds for specific ghb - receptor antagonists , and raise the possibility that both ghb- and gabab - antagonist drugs have the potential to be useful therapeutic agents in generalized absence seizures . systemic injections of the gabab receptor antagonists suppress completely swds in gaers . in the same strain , bilateral injections of the selective gabab receptor agonist r - baclofen into the specific relay nuclei and the rtn increased swds dose dependently , whereas injections of a gabab antagonist cgp 35348 into the same sites decreased these seizures dose dependently . the effect of r - baclofen on swds could be blocked by a subsequent injection of cgp 35348 at the same site . injections of r - baclofen or cgp 35348 in the midline thalamus had no effect . bilateral injections of r - baclofen into the specific relay nuclei of nonepileptic rats induced synchronized rhythmic oscillations on the cortical eeg . these results suggest that gabab receptors in the ventrolateral thalamus and in the rtn are involved in an oscillatory activity which underlies swds . administration of the gabab receptor antagonists cgp 36742 and cgp 56999 , and by bilateral microinjections of the same compounds into the lateral nuclei of the thalamus . in rats susceptible to audiogenic seizures , intraperitoneal administration of both gabab receptor antagonists , at doses which suppressed absence seizures , facilitated the elicitation of sound - induced tonic seizures . in nonepileptic control rats , intraperitoneal injections of higher doses of cgp 36742 and cgp 56999 induced delayed clonic convulsions , which were suppressed by pretreatment with baclofen . c - fos protein was expressed after gabab receptor antagonist - induced clonic seizures in the cortex , hippocampus , amygdala , perirhinal , and piriform cortex . the authors concluded that gabab receptor antagonists induce convulsions in cortical and limbic structures but suppress nonconvulsive absence seizures by blocking thalamic gabab receptors . somewhat different conclusions were drawn by staak and pape in wag / rij rats : their in vivo studies in anesthetized wag / rij rats in combination with single - unit recordings and microiontophoretic techniques in the ventrobasal thalamic complex showed that the application of the gabab receptor antagonist cgp 55845a exerted a statistically insignificant modulatory effect on neuronal activity during spontaneous swds but significantly attenuated the bicuculline - evoked aggravation of swd - related firing . this indicates that gabaa receptor - mediated events are recruited with each swd and that swd - related activity can be evoked with no significant contribution of gabab receptors . it is obvious that their results voted against a predominant or even exclusive contribution of gabab receptors to spontaneous swds in thalamic relay nuclei in the wag / rij strain , but rather point to a critical role of gabaa receptor activation . whether this difference is due to the neurolept anaesthetic is not immediately clear . the gabab receptor antagonists cgp55845a and cgp62349 suppressed the development of the absence syndrome to a larger extent in mice than in rats with ghbl - induced absence epilepsy in a chronic treatment protocol in which both ghbl and the gabab antagonists were given for 33 days . the absence syndrome was observed after 3 weeks of treatment in the saline group . a third antagonists cgp71982 suppressed it later than the other two antagonists ( fifth week ) , suggesting that gabab receptors are also involved in epileptogenesis . the effects of combined and single administration of the gabab receptor antagonist cgp 36742 and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa ) receptor antagonist , 7,8-methylenedioxy-1-(4-aminophenyl)-4-methyl-3-acetyl-4,5-dihydro-2,3-benzodiazepine ( ly 300164 ) on swds were also investigated in wag / rij rats in order whether the combined treatment might yield additive effects . cgp 36742 was effective as it reduced the number and mean duration of swds dose dependently . ly 300164 had minor effects : only the highest dose ( 16 mg / kg ) reduced the number of swds in a short time window . the ed(50 ) values for the inhibition of swds by ly 300164 and cgp 36742 in a time window 3060 min after injection were 15.5 and 16.6 mg / kg , respectively . the ed(50 ) of cgp 36742 was reduced to 8.0 mg / kg when this antagonist was administered in combination with ly 300164 ( 6 importantly , cgp 36742 and ly 300164 administered either alone or in combination had no apparent effects on behavior . their isobolographic method used may provide information for a rational approach to polytherapy in general and specifically for the treatment of generalized absence epilepsy . a similar effect for specific antagonist of gabab receptor has been received in the ay-9944 model of atypical absence epilepsy . the gabab receptor antagonist cgp 35348 , administered i.p . , decreased dose dependently the mean burst times . there are two mechanisms of the physiological inactivation of gaba : high affinity reuptake of gaba by specific gaba transporters and enzymatic inactivation of gaba by gaba - transaminase . the inhibition of both mechanisms of gaba inactivation by specific agents leads to a substantial increase of gaba concentration in the synaptic cleft . in a series of clinical reports the development of nonconvulsive status epilepticus ( ncse ) with electroclinical features consistent with those of atypical absence seizures after adjunctive antiepileptic therapy of tiagabine ( an inhibitor of gaba reuptake ) was described . the patient , a boy of 7 years , suffered from frequent myoclonic seizures and rare atypical absences , tonic , atonic , and simple partial seizures of unknown etiology . in two other reports , it was described that tiagabine induced ncse in patients with focal epilepsies and in one patient with juvenile myoclonic epilepsy . a 32-year - old patient with absence epilepsy and primary generalized tonic - clonic seizures since 11 years of age was described , who developed her first ncse while treated with 45 mg of tiagabine daily . three other cases with focal lesional epilepsy had nonconvulsive status epilepticus induced by treatment with tiagabine . these clinical cases were predicted based on results in the genetic absence models : effects of tiagabine in various doses were investigated on eeg , two types of swds , and behavior of wag / rij rats . wag / rij rats have two types of swds , the most common ones , bilateral symmetrical and generalized ( type i ) , and more occipital , shorter lasting swds ( type ii ) . according to expectations from other gabamimetics , tiagabine enhanced in a dose - related way both the number and mean duration of both types of swds . the low dose of 1 mg / kg had almost no effects , but doses of 3 and 10 mg / kg were effective . furthermore , tiagabine in the latter two doses increased the power in the higher band of the background eeg , whereas no significant changes in behavior of the rats were found . in general , this experiment supports that nonconvulsive epilepsy is associated with a gaba hyperfunction . it also underlines the biochemical differences of convulsive and nonconvulsive animal models of epilepsy since tiagabine is rather effective in blocking convulsive seizures ; it belongs to the category of drugs effective in convulsive animal models and not in nonconvulsive models of epilepsy . the limbic system is generally not included in any theory ( for review see ) about the pathogenesis of absence seizures . however , some data demonstrated that the alterations in the limbic system attribute to the expression of absence epileptic phenotype in genetic models of absence epilepsy . tolmacheva and van luijtelaar investigated whether local intrahippocampal administration of the neurosteroid progesterone and the gaba reuptake inhibitor tiagabine might affect the occurrence of swds . wag / rij rats received intracerebral injections of progesterone , 45% -cyclodextrin ( cd ) , saline , or tiagabine . eeg recordings made before and after injection showed that progesterone , cd , and tiagabine administration to the hippocampus reduced swds for 60 min following administration without behavioral or electroencephalographic side - effects . both progesterone administration into the cortex and saline injection into the hippocampus yielded no changes in the occurrence of swds . these data suggest that activation of gabaergic transmission in the hippocampus has an inhibitory effect on corticothalamocortical circuits underlying the generation of swds and might be critically involved in the regulation of absence seizures in the corticothalamocortical network . tiagabine increases absence seizures also in the other genetic absence models such as after systemic administration in gaers and in the lethargic mouse models [ 2 , 142 ] . the intensification of the efficiency of the gabaergic system by vigabatrin , a gaba - transaminase inhibitor , causes also an increase of absences . yang et al . presented three patients with de novo absence epilepsy after administration of carbamazepine and vigabatrin with different types of epilepsy . in a second clinical study 14 patients ( seven male ) , aged 1546 years , and with a mean duration of epilepsy of 16.4 years video - eeg demonstrated typical absence status epilepticus in five , atypical status epilepticus in five , atypical myoclonic status epilepticus in three and typical myoclonic status epilepticus in one . epilepsy had been misclassified as cryptogenic partial in eight cases and cryptogenic generalized in four . the correct diagnosis proved to be juvenile absence epilepsy in six patients , juvenile myoclonic epilepsy in four , epilepsy with generalized tonic - clonic seizures on awakening in two , and childhood absence epilepsy in two . all patients had been treated with carbamazepine ( cbz ) and had experienced seizure aggravation or new seizure types before referral . potential precipitating factors included dose increase of carbamazepine or of carbamazepine and phenytoin ; initiation of carbamazepine , vigabatrin , or gabapentin ; decrease of phenobarbital . this series shows that severe pharmacodynamic aggravation of seizures in idiopathic generalized epilepsy may result in absence status epilepticus or myoclonic status epilepticus , often with atypical features . the effects of vigabatrin on type ii swds in the eeg of aci rats were studied in order to learn more about the effects of altering gaba concentration on swds . the incidence and mean duration of type ii swds increased after vigabatrin as compared to saline . the peak frequency of the type ii swds decreased after vigabatrin ( 5.6 hz ) as compared to saline treatment ( 7.5 hz ) . these results are in agreement with predictions of destexhe 's theoretical model , modulating both gabaa and gabab conductance 's . the effect of vigabatrin on the starting and the stopping mechanisms ( the latter was determined by the hazard function ) was also investigated in wag / rij rats . this experiment showed that a high gaba level changed the stopping mechanism of the absence epileptic seizures , creating much better conditions for very long seizures to occur . with respect to the starting mechanism , it was found that both with a high and a low gaba level , there was evidence for a recovery mechanism that decreases the probability that a new seizure starts . another possibility is that the brain protects itself from a subsequent absence seizure , an endogenous protection mechanism ; this has been described for the hippocampus by kelly and mcintyre and for acute ptz seizures . initially this probability is lower with high gaba concentrations , but gradually it converges to the same constant baseline probability as in the condition with a low gaba concentration . other inhibitors of gaba catabolism such as -vinyl gaba ( gvg ) also increase swds , as demonstrated in gaers [ 115 , 125 , 150 ] . thus , pharmacological research of gabaa and gabab receptors and of gaba inactivation mechanisms specifies the basic strengthened role of the gabaergic system on absence seizures . such conclusion can be made on the basis of that in the overwhelming majority agonists of gaba receptors after systemic injections increase swd activity ( an exception are the benzodiazepines ) , and antagonists reduce them . besides strengthening of gabaergic neurotransmission , a more exact prolongation of gaba effects by inhibition of gaba inactivation mechanisms also increases absences . local injections of gaba mimetic drugs may show opposite effects , so that one can conclude that absence seizure is not due to a global increase in inhibition , but to localized increased and decreased sites of inhibition . the increase of swds by gaba agonists confirms our assumption that the hyperpolarization - induced ih pacemaker in the thalamus contributes to the occurrence of swds . the composition of the ih channel isoforms in the focal region of the cortex , the putative presence of ih on gaba interneurons in different part of the brain , together with the found opposite actions of gaba agonists in cortex and thalamus , might give room for another collaboration between ih and ( ica , t ) . one of the methods to estimate the efficiency of neurotransmitter systems is to determine the binding constants of a labeled ligand with a receptor ; it characterizes the activity of a receptor . by the help of specific concentration - dependent kinetic reactions and equations describing them , the dissociation constant ( kd ) and density of a receptor ( bmax ) rate of direct and reverse reactions of interaction ( i.e. , rate of association and dissociation ) depends on the concentration of the ligand . the dissociation constant ( kd ) is the concentration ligand at which the rate of direct and reverse reactions is equal , then the rate of association is equal to the rate of dissociation . the general pool of receptors in a membrane can be in two states : closed state ( receptors immerse in the cell membrane , with such type of receptors binding is not carried out ) and in open state , when receptor binding takes place . an increase of receptor density ( bmax ) implies an increase in the quantity of the active centers of binding . a reduction of kd ( without changes in bmax ) means increase in affinity of receptors , strengthening of efficiency of the reaction of the receptors to the most sensitive and intensive part of a receptor . an increase of bmax ( without change in kd ) means strengthening of receptors reaction efficiency to all concentrations tested . in vitro , the binding parameters of h - ro 5 - 4864 , a ligand labeling the peripheral benzodiazepine receptor , were determined for brain homogenates of wag / rij and aci rats . no difference in kd , but a significant decrease ( 25% ) in wag / rij rats receptors bmax , was found . autoradiography was used to study the binding of [ h]-gaba with gabaa and gabab receptors in brains from gaers : both the density of gabaa or gabab receptors and affinity of these receptors to gaba were determined . no difference in the density of gabaa and gabab receptors and affinity of these receptors to gaba between control and epileptic animals were found . the regional distribution of radioactive ligand binding for different receptors of the gabaa - benzodiazepine - picrotoxin chloride channel complex was measured on tissue section by autoradiography in brains taken from gaers and controls . the ligands employed included [ h]muscimol for high affinity gaba agonists sites ; [ h]sr 95531 for the low - affinity gaba sites ; [ h]flunitrazepam for the benzodiazepine sites ; and [ s]t - butyl bicyclophosphorothionate ( tbps ) for the picrotoxin site . there were no significant differences between gaers and control animals in [ h]flunitrazepam and [ s]tbps binding . however , there was a decreased [ h]muscimol and [ h]sr 95531 bmax in the ca2 region of the hippocampus in gaers . it is necessary to note once more that the hippocampus is traditionally not considered as a structure in which swds can be measured [ 4 , 21 , 153 ] , although recent data in gaers and wag / rij rats point towards alternations in the limbic system as a consequence of the hundreds daily swds [ 41 , 140 ] . another study explored a possible role of gabab receptors in absence seizures in lethargic ( lh / lh ) mice . [ h]baclofen binding to neocortical plasma membranes prepared from lh / lh and wild ( + /+ ) age - matched congenic mice was measured . the bmax of gabab receptors was 20% larger in lh / lh than in + /+ mice in an age - independent manner . interestingly , the subset of lh / lh mice with larger seizure frequency ( 4070 seizures/15 min ) had a significantly greater bmax than the subset with lower seizure frequency ( 110 seizures/15 min ) . the increase of gabab receptor density was selective , because binding to glutamate receptors ( [ h]glutamate binding ) and to gabaa receptors ( [ h]muscimol binding ) was not significantly different in the two strains . the authors suggest that higher density of gabab receptors in lh / lh mice underlies the expression of absence seizures in this model . gaba and the enzyme of its synthesis , glutamic acid decarboxylase ( gad ) , was investigated in cortex and thalamus of gaers by immunocytochemistry ; the gabaa receptors were evaluated by autoradiography of h - flunitrazepam binding and by immunocytochemistry using specific antibodies against the 2-3 subunits of gabaa receptor protein . gaba and gad immunocytochemistry did not show any difference in density or distribution of immunoreactive elements ( fibers , terminals and neurons ) between epileptic and control animals , but autoradiographic and immunocytochemical studies showed a decreased enhancement of h - flunitrazepam binding and of 2-3 subunits of gabaa receptor in the sensory - motor cortex and anterior thalamic areas of the epileptic strain . gabab receptors were measured as h - baclofen binding in a crude synaptic membrane preparation and there was no difference between epileptic and control animals . the data suggest an impairment of the gabaa system in studied brain regions of epileptic rats , due to a reduction of receptor 2-3 subunits and coupling to benzodiazepine receptors despite the normal synthesis and location of the neurotransmitter . the effect of ghba - induced swds on the function of various components of the gabaa receptor complex in the cortex of the rat was determined in a series of in vitro experiments . ghba itself without swds had no effect on the binding of [ h]muscimol , [ h]flunitrazepam , and [ s]tbps or on the uptake of cl into synaptoneurosomes in the in vitro studies . however , at the onset of ghba - induced swds , there was a significant decrease in the binding of [ s]tbps , associated with a significant decrease in muscimol - stimulated uptake of cl with no other biochemical change . one minute after onset of ghba - induced absence seizures , a significant increase in the binding of [ h]muscimol was noted . ten minutes later the decrease in muscimol - stimulated uptake of cl had normalized , while the changes in binding of [ h]muscimol and [ s]tbps persisted . the analysis of extra- and intracellularly recorded synaptic responses revealed an intracortical hyperexcitability which was accompanied by a significant reduction in the efficiency of gabaergic inhibition . these data indicate that the imbalance between intracortical excitatory and inhibitory mechanisms may at least contribute to the expression and augmentation of swds in wag / rij rats , and this was also found in an in vitro neurophysiologic study . leaning on the known fact , that postsynaptic gabab receptor - mediated events are reduced after prior treatment with pertussis toxin ( ptx ) , the influence of ptx ( 0.4 microg ) , denatured - ptx or vehicle saline bilaterally injected into the relay nuclei of the thalamus in anesthetized gaers were studied for up to 6 days , following which the brains were removed and gabab or gabaa receptor autoradiography was performed . by 6 days the swd of the rats treated with ptx was suppressed by 96% compared with vehicle - injected rats with a significant ( 62% ) reduction even after 1 day . after 6 days gabab , but not gabaa , receptor binding was significantly reduced by 7080% in the ventrolateral and ventral posterolateral thalamic nuclei . in the same model , the high affinity binding of gabaa / bzd receptors with ( h)ro 15 - 1788 in the brain of naive rats and after administration of fg 7142 did not differ in gaers and nonepileptic control rats . the authors suggested that the hypersensitivity of gaers to various inverse agonists of the gabaa / bdz receptor involves cortical gabaa receptors and is not related to differential activity of a subunit - selective receptor . miniature gabaa ipscs recorded in the vb complex of the thalamus , and in cortical layer ii / iii neurons were similar in gaers and nonepileptic controls , whereas in gaers rtn neurons had a 25% larger amplitude and 40% faster decay . in addition , baclofen was significantly less effective in decreasing the frequency of rtn mipscs in gaers than in control , whereas no difference was observed for cortical and vb mipscs between the two strains . paired - pulse depression was 45% smaller in gaers rtn , but not in vb , and was insensitive to gabab antagonists . these results point to subtle , nucleus - specific , gabaa receptor abnormalities underlying swds rather than a full block of these receptors across the whole corticothalamocortical network . their occurrence prior to seizure onset suggests that they might be critically involved in epileptogenesis . we have studied muscimol - induced cl conductivity in synaptoneurosomes prepared from the frontal and somatosensory cortex of rats with three types of epileptic activity : tonic - clonic pentylenetetrazole kindling in wistar rats , nonconvulsive absence ptz kindling in wistar rats ( an absence seizure model ) , wag / rij and wistar control rats . the occurrence of kindling prior to tonic - clonic seizures in the wistar rats decreased the muscimol - induced cl conductivity compared to control ( figure 6(a ) ) . wag / rij rats showed an increase in cl conductance in neocortical synaptoneurosomes as compared to the control wistar rats ( figure 6(b ) ) . the decrease in muscimol - induced cl conductivity after development of tonic - clonic kindling was in agreement with a quite some amount of data regarding the decrease in the activity of gabaa receptor during tonic - clonic kindling [ 55 , 161165 ] . the high level of muscimol - induced cl conductivity in the neocortical synaptoneurosomes of the wag / rij rats supported the concept that the swds originating from the somatosensory cortex are induced by hyperpolarization . the high level of cl conductivity during nonconvulsive ptz kindling suggests that the activity of the gabaa receptor was similar in the genetic absence epilepsy model and ptz absence seizure model . the binding of [ c]flumazenil at the bdz site of the gabaa receptor was studied in five patients with idiopathic generalized epilepsy with positron emission tomography . however , these early studies were not aimed at the cortex , the most likely location of the origin of swds . except for binding constants of labeled ligands with gabaa and gabab receptors defining efficiency of gabaergic , other parameters defining efficiency of gabaergic transmission have been used . levels of extracellular gaba and other amino acids in the ventrolateral thalamus in gaers have been monitored with in vivo microdialysis . it was shown that the basal extracellular levels of gaba and , to a lesser extent , taurine were increased when compared with values in nonepileptic controls . however , modifying gabaergic transmission with the gabab agonist ( )-baclofen , the gabab antagonist cgp-35348 , or the gaba uptake inhibitor tiagabine did not produce any further alteration in extracellular gaba levels . another study was performed to test the hypothesis that presynaptic gabab receptors in lh / lh mice inhibit [ h]gaba release to a greater degree than nonepileptic littermates ( designated + /+ ) . synaptosomes isolated from neocortex and thalamus of age - matched lh / lh and + /+ mice were similar in uptake of [ h]gaba . in the neocortical preparation , baclofen dose dependently inhibited [ h]gaba release evoked by 12 mm kcl , an effect mediated by gabab receptors . the maximal inhibition ( imax ) value was significantly greater ( 80% ) in lh / lh than in + /+ mice , whereas the ic50 ( 3 microm ) was unchanged . the effect of baclofen ( 50 microm ) was 58% less robust in thalamic preparation of the lh / lh mice . other effects mediated by gabab receptors ( inhibitions in ca uptake and camp formation ) were also significantly reduced in thalamic synaptosomes from lh / lh mice . it is possible that selective effects of presynaptic gabab receptors or gaba release in neocortex and thalamic nuclei of lh / lh mice may contribute to mechanisms underlying absence seizures . whole - cell voltage - clamp recordings were made from thalamic ventrobasal neurons of age - matched lethargic ( lh / lh ) and wildtype ( + /+ ) mice . gaba release and uptake were examined in gaers and in nonepileptic control animals , using crude synaptosomes prepared from the cerebral cortex and thalamus . uptake of [ h]gaba over time was reduced in thalamic synaptosomes from epileptic rats , compared to controls . nnc-711 , a ligand for the gat-1 uptake protein , reduced synaptosomal uptake by more than 95% ; -alanine , an inhibitor selective for the uptake proteins gat-2 and gat-3 , did not significantly reduce synaptosomal uptake . these results indicate that gaba uptake in the thalamus of gaers rats was reduced compared to control animals . the lower level of uptake in the epileptic animals corresponds to higher efficiency of gaba system in these rats . progesterone and estradiol serum levels were investigated in wag / rij rats before , during , and after pregnancy following parturition . progesterone is kept high until the 18th day of pregnancy and drastically decreased before the parturition . common duration of absences - spontaneous swd , frequency , and the duration of every swd decreased from 3rd to 19th days of pregnancy before parturition . on the basis of these data and regulation of gabaa receptor efficiency by neurosteroids , it can be assumed that the changes in the parameters of swd are possibly correlated with changes in progesterone in serum during pregnancy [ 171 , 172 ] . a time course study that examined the effects of the female estrous cycle on the more slow atypical swds , gabab receptor binding , and gabab receptor protein expression was conducted in the ay9944 model in long - evans rats . there was a significant increase in both the duration of swds and gabab receptor binding during the proestrus stage of the estrus cycle , the stage during which the levels of progesterone are at their highest . these data suggest an important role for steroid hormones and its interaction with gaba receptors in the regulation and maintenance of ay9944-induced atypical absence seizures . brain cholesterol synthesis inhibition ( csi ) at a young age in rats has been shown to be a faithful model of the symptomatic generalized epilepsies , in this case acquired absence epilepsy , a devastating condition for which few therapies or models exist . patch - clamp and whole - cell recordings were compared from neurons acutely dissociated from the rtn in long - evans hooded rats treated and untreated with the cholesterol synthesis inhibitor u18666a . in u18666a - treated animals , 91% of rats developed swds . relevant is also that mutations of the 2 subunit of the gabaa receptor abolish natural sensitivity to bdz . patch - clamp results revealed that although there was no remarkable change in gabaa receptor affinity , both a loss of ability of benzodiazepines to enhance gabaa - receptor responses and an increase of zn inhibition of gabaa - receptor responses of rtn neurons occurred in this model . this change was specific , since no significant changes were found in neurons exposed to the gaba allosteric modulator pentobarbital . taken collectively , these findings provide evidence for abnormalities in bdz and zn modulation of gabaa receptors in the cholesterol synthesis inhibition model and suggest that decreased 2 subunit expression may underlie important aspects of generation of tcswds in atypical absence seizures . the present results are also consistent with recent findings that mutation of the 2 subunit of the gabaa receptor changes benzodiazepine modulation in families with generalized epilepsy syndromes . it is known that basket cells typically express two calcium - binding proteins , parvalbumin ( pv ) and calbindin . pv is co - expressed with gaba in 90% of the gabaergic neurons [ 175 , 176 ] , and pv - immunostaining is an appropriate way to mark basket cells . quantification of pv - positive cells showed clear reductions in the parietal and forelimb area of the somatosensory cortex in wag / rij rats compared to nonepileptic control aci rats . these results are interpreted as deficiency of ca - binding protein in corresponding structures of the brain of wag / rij rats , or as deficiency of gabaergic cells . thus , the measurement of efficiency of the gabaergic system in various absence models leads to the conclusion that a huge variety of experiments demonstrate a large role for deficiencies in the gabaergic system . however , there are also diverse results , among others with the role of gabab . it is possible that different deficiencies and different mechanisms may all lead to the same phenotype : swds . the second reason of the diverse results is the so - called process of uncertainty of the nervous system . the analysis of several works has shown that increase or decrease of general activity of the neurotransmission system at different animals can be carried out by different ways and depends on specific features of these animals . the third reason for the occurrence of diverse results can be processes of indemnification ( protection against injury ) . to illustrate , in our study of synaptoneurosomes , samples from cortex of wistar and wag / rij rats were leveled on protein and on concentration of gabaa receptors . in these conditions conductivity of chlorine was higher in wag / rij rats . in brain tissue , in which gaba systems have been reduced , a reduction of the quantity of gabaa receptor can be found and a reduction of inhibition . in this case strengthening of conductivity of chlorine through gabaa receptors can be considered as compensation for the reduced functioning of gaba systems . five subunits of gabaa receptor surround the cl channel [ 105107 , 181 , 182 ] . gabaa receptor subunits are a long polypeptide which penetrates a membrane in four places , that is why subunits possess four transmembrane domains . the so - called n- and c - terminals of the polypeptide are located on the extracellular surface . it is supposed that the second domain of gabaa receptor subunit covers a wall of the chloride channel . between the third and fourth domains , there is a large intracellular fragment to which sites of protein kinase phosphorilation are concentrated and with which the basic differences in primary structure between various subunits are connected [ 181 , 184 ] . on the n - terminal of the polypeptide leaving the first domain is the active center of a receptor or the sites connecting with ligands [ 105 , 185187 ] . some specific binding sites are located on the gabaa receptor [ 105 , 182 ] . the first site is the gaba site where binding to gaba and its specific agonists such as gaboxadol , ibotenic acid , muscimol , and progabide takes place and to antagonists such as bicuculline and gabazine . next there is the site of positive allosteric modulators such as barbiturates , benzodiazepines , carisoprodol , ethanol ( alcohol ) , etomidate , glutethimide , kavalactones , meprobamate , methaqualone , neuroactive steroids [ 189 , 190 ] , niacin / niacinamide , nonbenzodiazepines , propofol , theamine , valerenic acid , and volatile / inhalation anesthetics . there is also the site of negative allosteric modulators such as flumazenil , ro15 - 4513 and sarmazenil [ 191193 ] and the site of noncompetitive channel blockers such as cicutoxin , oenanthotoxin , pentylenetetrazol , picrotoxin , and thujone . according to the definition , allosteric modulators which bind with gabaa receptors in places which do not overlap with a binding site of an agonist , after interaction of neurosteroids with the gabaa receptor , an intracellular process , oxygenation , is started ; it transforms some intracellular metabolites in steroid receptors ligands . after binding of these ligands with intracellular steroid receptors , as the gaba site is an allosteric modulator of neurosteroid site , the interaction of gaba with its own site , allosterically modified by the neurosteroid site , initiates a cascade of events leading to the start of signal transduction . the transduction signal started through receptors processes of intracellular phosphorilation which terminate in updating the expression of genes . it means that the gabaa receptor is an ionotropic receptor , but that it also initiates simultaneously signal transduction . why is signal transduction so important ? the modification of genes expression is a unique mechanism of an encoding process called memory consolidation , that is the transfer of information from short - term into long - term memory . hence , signal transduction plays an important role at the long - term storage of information , in learning and memory , in training , in neuroplasticity in general , and in pathological states . the gabaa receptor has 22 subunits : -6 subtypes , -4 subtypes , -3 subtypes , -1 subtype , -3 subtype , -1 subtype , -3 subtype , and -1 subtype [ 181 , 195 , 196 ] . it is necessary to notice that some isoforms of gabaa receptor subunits have various variants . so , for 2 subunit , two variants were revealed [ 197 , 198 ] , differing in length of an intracellular fragment between 3 and 4 domains . similar distinctions are known for 2 , 4 subunits for which the presence of two variants has also been shown [ 199 , 200 ] . each of such sequences ( 2 , 2 and 4 ) has in the designation an additional letter l or s ( accordingly , long and short ) . it is supposed , those three splays variants exist for the 5 subunit . a product of alternative splicing is the 6 subunit , truncated with an n - terminal . the 18 types of gabaa receptor subunits are expressed in a mammalian brain : subunits ( 16 ) , subunits ( 13 ) , subunits ( 13 ) , subunits ( 13 ) and on 1 subunits , and . different confirmations of the gabaa receptor are found throughout the brain , and the most common mammalian subunits composition is two 1 subunits , two 2 subunits , and one 2 subunit assembling in a pentahedron-(1)2(2)2(2 ) . two molecules of gaba interact with two sites , each of which is located on two n - terminuses from the first domain of 1 and 2 subunits . the benzodiazepine site is located on two n - terminus from the first domain of 1 and 2 subunits . for example , 80% of brain gabaa receptors are highly sensitive for diazepam , 10% of receptors have a low sensitivity for diazepam . the latter is the case if the subunit compositions of gabaa receptor contain 1 or 3 subunits . the 10% of receptors not sensitive for diazepam have subunit compositions of gabaa receptor containing 4 or 6 subunits . from the 80% of receptors that are highly sensitive for diazepam , this is the case if the subunit composition of gabaa receptor contains a 5 subunit . the composition 122 possesses high affinity for zolpidem , and compositions 232 or 332 demonstrate low affinity . neonatal treated rats with ay9944 show an increased occurrence of swds which correlated significantly with a decreased protein expression of the gabaa 2 subunit in rtn and vb nuclei of the thalamus , but not in somatosensory cortex . conversely , 1 subunit expression decreased in somatosensory cortex in the ay9944 model , but not in the thalamus . the decreased gabaa receptor 2 subunits in thalamus and increased in cortex are gender and age specific . these results coincide with data that in human absence epilepsy is more widespread among women . the synaptic physiology and network property of mice lacking gabaa receptor 3 , a subunit that is uniquely expressed in the thalamus by inhibitory neurons of the rtn , have been investigated . deletion of this subunit produced a powerful compensatory gain in inhibitory postsynaptic response in the rtn . although other forms of inhibitory and excitatory synaptic transmission in the circuit were unchanged , evoked thalamic oscillations were strongly dampened in 3 knockout mice . furthermore , pharmacologically induced tc absence seizures displayed a reduction in length and power in 3 knockout mice . these studies highlight the role of gabaergic inhibitory strength within the rtn in maintenance of thalamic oscillations and demonstrate that inhibitory intra - rtn synapses are a critical control point for regulating higher order corticothalamocortical network activity . the combination of immunocytochemistry and high - resolution immunogold electron microscopy was used to study cellular and subcellular localization of gabaa receptors 1 , 3 , and 2/3 subunits in ventral posterior nucleus ( vp ) and rtn of wag / rij and control wistar rats . in control rats , 1 subunits were prominent at inhibitory synapses in vp and much less prominent in rtn ; in contrast , the 3 subunit was highly evident at inhibitory synapses in rtn . immunogold particles representing all subunits were concentrated at postsynaptic densities with no extra synaptic localization . calculated mean number of particles for 1 subunit per postsynaptic density in nonepileptic vp was 6.1 3.7 , for 3 subunit in rtn , it was 6.6 3.4 , and for 2/3 subunits in vp and rtn , the numbers were 3.7 1.3 and 3.5 1.2 , respectively . in wag / rij rats , there was a specific loss of 3 subunit immunoreactivity at inhibitory synapses in rtn , without reduction in 3 subunit mrna or significant change in immunostaining for other markers of rtn cell identity such as gaba or parvalbumin . these combined results obtained in mice and rats allow us to assume that different combinations of subunit compositions of gabaa receptor in the rtn may contribute to or are the reason for decreased strength of gabaergic inhibition . the functional consequence is that when the rtn is stimulated by the cortex , the thalamic resonance might be larger and swds might last longer . importantly , the changes in subunit composition were already present in presymptomatic rats , suggesting that the changes in the rtn are not caused by the seizures but that they are a property of wag / rij rats . the possibility that absence seizures cause alterations in gabaa receptor ( 1 , 4 , 2 , and 2 ) subunit gene expression in thalamic relay nuclei was also explored , in this case in an acute absence seizure model , the ghb model . a marked increase in 1 mrna and a corresponding decrease in 4 mrna in thalamic relay nuclei 24 h after the onset of ghb - induced absence seizures ( when the seizures were terminating ) were detected . these changes were selective to these isoforms as neither 2 nor 2 mrna changed following seizures and occurred only in thalamic relay nuclei but not in hippocampus . the alterations in 1 and 4 mrna persisted until about 12 h , and by 24 h after the seizure - onset the mrna levels normalized . blocking ghb - seizures produced no change in the levels of 1 and 4 mrna in thalamic relay nuclei , suggesting that seizures themselves were responsible for mrna alterations . the expression of mrna of gabab receptor subunit was studied in wag / rij and gaers . by using real - time pcr , the researchers from the avoli group have found that mrna levels for most gabab1 subunits are diminished in epileptic wag / rij neocortex as compared with age - matched nonepileptic controls , whereas gabab2 mrna is unchanged . next , it was investigated the cellular distribution of gabab1 and gabab2 subunits by confocal microscopy and it was discovered that gabab1 subunits fail to localize in the distal dendrites of wag / rij neocortical pyramidal cells . the authors propose that these alterations may contribute to neocortical hyperexcitability and thus to swd generation in absence epilepsy . gabab receptor expression in the somatosensory cortex , vb , and rtn in gaers was also investigated . in situ hybridization results showed a significant increase in mrna for gabab1 in the somatosensory cortex and a decrease in the ventrobasal thalamic nucleus but not in the rtn . by contrast , the immunocytochemical data revealed an increased expression of both gabab1 and gabab2 receptor subunits in all regions examined , somatosensory cortex , ventrobasal thalamic nucleus and rtn . the main finding was an upregulation of gabab receptor protein in the corticothalamic circuit in gaers compared to controls . the results , as obtained in gaers and wag / rij rats seem rather opposite to each other , the hyperexcitability in wag / rij rats strain and a wide spread upregulation of gabab receptors . we have proposed ( see sections 3 and 4 ) , that both of these processes cause the same reaction , increase of swd . perhaps the two opposing processes provide the same response in different strain of animals . mutations in inhibitory gabaa receptor subunit genes ( gabra1 , gabrb3 , gabrg2 , and gabrd ) of human have been associated with genetic epilepsy syndromes including childhood absence epilepsy , juvenile myoclonic epilepsy , pure febrile seizures , generalized epilepsy with febrile seizures plus , and dravet syndrome ( ds)/severe myoclonic epilepsy in infancy . these mutations are found in both translated and untranslated gene regions and have been shown to affect the gabaa receptors by altering receptor function and/or by impairing receptor biogenesis by multiple mechanisms including reducing subunit mrna transcription or stability , impairing subunit folding , stability , or oligomerization and by inhibiting receptor trafficking [ 48 , 49 , 212222 ] . besides , it is supposed that the chloride channel gene clcn2 may be a susceptibility locus in a subset of cases of childhood absence epilepsy . to understand the effect of these mutations , surface targeting of gabaa receptors this may have some consequences to the effect of the heterozygous 2(r43q ) mutation leading to childhood absence epilepsy and febrile seizure . stargazer ( stg ) mutant mice fail to express stargazin ( transmembrane ampa receptor regulatory protein 2 ( tarp2 ) ) and consequently experience absence seizure - like tcswds that pervade the hippocampal formation via the dentate gyrus . as in other seizure models , the dentate granule cells of stg develop elaborate reentrant axon collaterals and transiently overexpress brain - derived neurotrophic factor . it was investigated whether gabaergic parameters were affected by the stg mutation in this brain region . gabaa receptor 4 and 3 subunits were consistently upregulated , gabaa receptor delta expression appeared to be variably reduced , whereas gabaa receptor 1 , 2 , and 2 subunits and the gabaa receptor synaptic anchoring protein gephyrin were essentially unaffected . it was established that the 4 2 subunit - containing , flunitrazepam - insensitive subtype of gabaa receptors , not normally a significant gabaa receptor in dentate gyrus neurons , was strongly upregulated in stg gyrus neurons , apparently arising at the expense of extra synaptic 4 -containing receptors . this change was associated with a reduction in neurosteroid - sensitive gabaa receptor - mediated tonic current . this switch in gabaa receptor subtypes was not reciprocated in the tottering mouse model of absence epilepsy implicating a unique , intrinsic adaptation of gabaergic networks in stg . contrary to previous reports that suggested that tarp2 is expressed in the dentate , it was found that tarp2 was neither detected in stg nor in control dentate gyrus . it was reported that tarp8 is the principal tarp isoform found in the dentate gyrus and that its expression is compromised by the stg mutation . these effects on gabaergic parameters and tarp8 expression are likely to arise as a consequence of failed expression of tarp2 elsewhere in the brain , resulting in hyperexcitable inputs to the dentate . the expression of one gabaa receptor gene , gababr1 , is distinguished by the expression of multiple splice variants that encode different isoforms of the receptor . two novel gababr1 variants , gababr1h ( r1h ) and gababr1i ( r1i ) , which appear to arise from alternative splicing of the gababr1 gene , were identified . the expression of r1h and r1i is differentially regulated in brain and peripheral tissues , but expression is not altered in the brain of gaers . both the r1h and r1i variants exhibit a novel 80-bp insert downstream of exon 4 that is flanked by consensus splice sites , and both encode c - terminal - truncated proteins . the new insight into the family of gababr1 variants gained from this study identifies exon 4 as a preferred locus , or as a hot spot for regulated splicing in the gababr1 gene . this finding correlates with the microexonic nature of exon 4 ( 21 bp ) . bioinformatic analysis of micro - exon 4 and its flanking pre - mrna sequences has revealed multiple , potentially competitive , exonic splicing enhancers that provide a mechanistic basis for the preponderance of alternative splicing events at this locus . conservation of gababr1 micro - exon 4 across species suggests a conserved functional role , facilitating either n - terminal protein production or post - transcriptional gene regulation through regulated splicing coupled to transcript decay . mutations in the genes encoding the subunits of the low - threshold t - type ca channel ( ica , t ) in patients [ 4446 , 227231 ] and also in gaers but not in wag / rij rats , are probably associated with a dysfunction of pacemaker ih channels in cortex and thalamus . first , it has been shown that the if pacemaker channel in the atrial sinus node functions in pair with the ( ica , t ) channel [ 232 , 233 ] . the if pacemaker channel in the sinus node opens at 80 mv , and the low - threshold ( ica , t ) channel is activated at 90 mv and fully inactivated at + 40 mv . blockage of the ( ica , t ) channels by low concentrations of nicl2 diminishes the spontaneous activity of the if channel but does not affect the ( ica , t ) channel ( the l - type ca channel ) . participation of the low - threshold ( ica , t ) channel in the regulation of swds has been shown in both wag / rij rats and gaers rats . there are two centers of bursting neurons which are activated by hyperpolarization : the rtn [ 1922 ] as the source for spindle oscillations and the other is localized in the somatosensory cortex , the site of origin of swds [ 6 , 9 , 13 , 14 ] . the somatosensory cortex is reciprocally connected with the vpm , vpl , and posterior nucleus of the thalamus , both the ascending and descending pathways from vpm and vpl give collaterals to the rtn , which inhibits thalamic neurons . interestingly , exactly the thalamus and pyramidal neurons of cortical layers ii , iv , and v of the somatosensory cortex [ 73 , 74 , 81 ] contain hyperpolarization - activated pacemaker ih channels . the presence of hyperpolarization - activated pacemaker ih channels in the rtn makes them an interesting object in the discussion and analysis of normal and pathological behavioral and neurophysiologic processes and mechanisms . thus , one of the reasons of the occurrence absence epilepsy is the mutation of several genes coding of gabaa receptors which causes abnormal functioning of the gabaergic system . this abnormal functioning of the gabaergic system and mutations of ( ica , t ) channel , which functions in a tandem with ih channel , can lead to the occurrence of spontaneous swds in an eeg . it had been shown some time ago that the well - known noncompetitive nmda antagonist mk-801 reduced the number and mean duration of swd ; the drug caused also large behavioural abnormalities such as head waving and agitation jeopardizing the interpretation whether the swds were primarily or secondarily ( via behavior ) affected by this nmda antagonist . intracerebroventricular ( i.c.v . ) injection of nmda induced a dose dependently increase in the number of swds . the competitive nmda antagonist 2-amino-7-phosphonoheptanoic acid ( aph ) caused a dose - dependent reduction in the number and mean duration of swds . finally , the anticonvulsant remacemide , a noncompetitive , low - affinity nmda receptor antagonist and its major metabolite fpl 12495 after oral administration suppressed the number of swds in wag / rij rats . the involvement of other glutamate receptors such as ampa and kainate receptors was also studied by i.c.v . injections of ampa , gdee ( glutamate diethyl ester , a non - nmda receptor antagonists ) , kainic acid , and kynurenic acid in wag / rij rats . eeg registrations showed that ampa dose dependently increased absences while gdee caused a dose - dependent decrease . kynurenic acid decreased dose dependently the incidence of swds ; this effect of kynurenic acid could be blocked by a nonconvulsive dosage of kainic acid . compounds acting on nmda ( nmda , aph ) and non - nmda ( ampa , gdee , kainic acid , kynurenic acid ) receptors were coinjected i.c.v . it appeared that the epilepsy increase , induced by the non - nmda receptor agonist ampa , and in a less obvious way , by kainic acid , could be blocked by the nmda receptor antagonist aph . the effects of nmda were completely blocked by the non - nmda receptor antagonists gdee and kynurenic acid . kain , the kainate agonist , had no effect on the number of swds , but the antagonist kynurenic acid decreased the discharges . cnqx , another non - nmda antagonist , also reduces the number of swds . the glutamate release inhibitor and anticonvulsant lamotrigine thought to inhibit the excitatory neurotransmitter release was indicated for nonconvulsive seizures . only in a high dose lamotrigine reduced swd in wag / rij rats , but in that dose side - effect such as abnormal locomotion , were also seen . interestingly , a recent double blind eeg multicentered controlled study confirmed the poor absence suppressive effects of lamotrigine in childhood absence epilepsy . in other series of experiments , two noncompetitive ampa receptor antagonists , cfm-2 and thiq-10c demonstrated to antagonize generalized tonic - clonic seizures in different animal models , were evaluated in wag / rij rats . animals were focally microinjected into specific brain areas of the corticothalamic circuit and eegs were recorded . the focal microinjection of the two ampa antagonists into thalamic nuclei ( ventralis posteromedialis , rtn , ventralis posterolateralis ) and forelimb region was , generally , not able to modify the occurrence of swds . however , both compounds were able to reduce the number and duration of swds dose dependently when microinjected into the perioral region of the primary somatosensory cortex . these findings suggest that ampa plays a role in absence epilepsy and that it depends on the involvement of specific neuronal areas . in the next series of experiments the authors demonstrated that noncompetitive ampa receptor antagonists alone might not be useful in the treatment of absence epilepsy because of their low therapeutic index . a similar low therapeutic index was earlier also found with the ampa antagonist 7,8-methylenedioxy-1-(4-aminophenyl)-4-methyl-3-acetyl-4,5-dihydro-2,3-benzodiazepine ( ly 300164 ) . nevertheless , the results from citraro et al . showed that the center in the perioral region of the primary somatosensory cortex takes the lead or has a more considerable position in generation swds and that the local strengthening of gabaa receptor activity in rtn strengthens swds . in some studies , the effects of glutamatergic receptors ligands can not be explained by a simple interaction at the receptor . to illustrate , the effects of ketamine , a noncompetitive antagonist at the nmda receptor , were studied on the eeg and in the open field in the wag / rij rat strain . ketamine was systemically administered in a dose range from 3 to 30 mg / kg . biphasic effects of ketamine were observed in the eeg . the first phase was a dose dependent suppression of swds , followed by a second phase characterized by the facilitation of swds . this increase was expressed first as an increased number of swds , and later on as a significant prolongation of individual discharges and decrease in frequency of swds . ketamine also produced a dose - related initial behavioral excitation , a decrease of muscle tone in hind quarters , followed by front quarters and head , and an absence of locomotor activity . however , the time course of the behavioral changes was not in parallel with the complex eeg effects . it can be concluded that ketamine has more effects on the eeg than previously assumed which can not be explained by a simple blockade of the nmda receptor . it is proposed that the obtained specific dynamics of swds ' frequency may be caused by changes in the activity of the corticothalamocortical pacemakers that are generating swds . the following study was conducted to investigate the effects of two noncompetitive ampa receptor antagonists , gyki 52466 and gyki 53405 ( the racemate of talampanel , the latter drug is a potent and selective ampa - receptor antagonist ) on the generation of swd parallel with the vigilance and behavioral changes in wag / rij rats . intraperitoneal administration of gyki 52466 ( 1-[4-aminophenyl]-4-methyl-7,8-methylenedioxy-5h-2,3-benzodiazepine ) , the prototypic compound of the 2,3-benzodiazepine family acting as an ionotropic glutamate receptor antagonist , and which unlike the conventional 4,5-benzodiazepines , does not act on gabaa receptors , caused a fast dose - dependent increase in the number and total duration of swd . these changes were accompanied by dose - dependent increase in duration of light slow wave sleep and passive awake , vigilance states associated with the presence of swd . in addition a short , transient behavioral activation occurred that was followed by strong ataxia and immobility , decrease of active wakefulness and increase in deep slow wave sleep . gyki 53405 ( 7-acetyl-5-(4-aminophenyl)-8-methyl-8,9-dihydro-7h-1,3-dioxolo[4,5-b]benzodiazepine ) , the racemate of talampanel , 16 mg / kg , i.p . failed to affect any measure of swd and vigilance . when used as a pretreatment , gyki 52466 ( 10 mg / kg ) slightly attenuated the swd - promoting effects of the 5-ht1a receptor agonist 8-oh - dpat , and it decreased the cumulative duration and mean duration of the paroxysms . interest of researchers has turned into metabotropic glutamate receptors ( mglur 's ) since their effects are more subtle and allow for a more refined intervention than would be the case with the ionotropic glutamate receptors . mglurs are positioned at synapses of the corticothalamocortical network crucial for the occurrence of swds . mglurs form a family of eight subtypes subdivided into three groups on the basis of amino acid sequence , pharmacologic profile , and g - protein coupling [ 247249 ] . the receptors of this group also regulate the activity of different types of ca and k channels . group ii includes mglu2 and mglu3 receptors and they are coupled to gi / go proteins . they negatively modulate the activity of adenyl cyclase and voltage - sensitive ca channels ( vsccs ) . group iii comprises mglu4 , mglu6 , mglu7 , and mglu8 receptors , which are also coupled to gi / go proteins . the individual receptor subtypes have a modulatory role on excitatory and inhibitory synaptic transmission in the corticothalamocortical circuitry . until now , receptors from all three groups have been studied in wag / rij rats . group i mglurs are preferentially localized in the peripheral portion of the postsynaptic density of both tcand corticothalamic cells , where they generate excitatory responses and regulate mechanisms of synaptic plasticity . group ii and iii mglurs are found preferentially ( albeit not exclusively ) in presynaptic terminals , where they negatively regulate neurotransmitter release . group i. mglu1a receptor expression is reduced in the vb nucleus of the thalamus and pharmacologic potentiation of mglu1 receptors with the selective pam , syn119 ( corresponding to ro0711401 ) , reduces the incidence of absence seizures , whereas treatment with the mglu1 receptor nam ( jnj16259685 ) exacerbates swds [ 248 , 249 ] . the expression of mglu5 receptors was increased in the somatosensory cortex and decreased in the vb . swds in symptomatic wag / rij rats were not influenced by pharmacological blockade of mglu5 receptors with mtep , but were significantly decreased by mglu5 receptor potentiation with the novel enhancer , vu0360172 - 6 without affecting motor behaviour . it seems that pams affecting group i mglurs might be interesting new targets against absence epilepsy . the outcomes do contrast with data obtained in the lethargic ( lh / hl ) mice model , where mglu1 receptor blockade with the orthosteric antagonists , aida or ly367385 , reduces the incidence of swds . therefore , it appears that the role of mglu1 receptors in the regulation of pathologic swds is species / model dependent and may also depend on the genetic determinants of absence epilepsy . symptomatic wag / rij rats showed an increased expression of mglu2/3 receptors in the ventrolateral regions of the somatosensory cortex , ventrobasal thalamic nuclei , and hippocampus , but not in the rtn and in the corpus striatum , as assessed by immunohistochemistry and western blotting . in contrast , mglu2/3 receptor signalling was reduced in slices prepared from the somatosensory cortex of these symptomatic rats , as assessed by the ability of the agonist , ly379268 , to inhibit forskolin - stimulated camp formation . it therefore seems that mglu2/3 receptors are involved in the generation of swds and that an upregulation of these receptors in the somatosensory cortex might be involved in the pathogenesis of absence epilepsy . studies towards the role of receptors of this category have been limited because of lack of specific agonists . they are , however , interesting for absence epilepsy considering that activation of these receptors reduces gabaergic inhibitory responses within the thalamus , possibly via a presynaptic mechanism [ 255257 ] . in addition , they have a profound effect in reducing the corticothalamic input onto thalamic relay cells . interestingly , and accordingly , an increased incidence of swds has been found in wag / rij rats when treated with the selective mglu4 receptor enhancer , n - phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a - carboxamide ( phccc ) . in humans , the mglu4 receptor gene is localized at a susceptibility locus for juvenile myoclonic epilepsy [ 260 , 261 ] , which is characterized by absence seizures in addition to myoclonus and tonic - clonic seizures . another group - iii mglu receptor , the mglu7 receptor , is also associated with the development of absence seizures . mutant mice in which mglu7 receptors failed to interact with pick1 ( protein interacting with c kinase 1 ) develop absence seizures [ 262 , 263 ] , as do mice injected with a peptide that disrupts the interaction between mglu7 receptors and pick1 . the precise mechanism whereby an interaction between mglu7 receptors and pick1 restrains swds in the corticothalamic network remains to be determined . thus , despite of the somewhat atypical features of the action of some substances on swds , it is possible to conclude that the role of the glutamatergic system in absence epilepsy corresponds to its role in idiopathic convulsive epilepsy . seizures typical for both nonconvulsive and convulsive epilepsy could be due to the strengthened functioning of the glutamatergic system , which might be considered as hyperexcitability [ 157 , 264 ] . the results suggest that the three types of ionotropic excitatory glutamate receptors enhance swds and this alleviating influence can be explained as follows . the spike in the swd appears as a rebound spike , in fact it is the low threshold ca spike ( figure 1(b ) ) . it appears after the hyperpolarization phase when the gating mechanism of an ih channel is activated . then the spike or the depolarization returns again to the hyperpolarization phase , and the process is repeated . the effects of all three types of excitatory glutamate receptors consist in the enhancement of the depolarization phase , that is , in increasing the amplitude of the spike . a larger spike increases the amplitude of the hyperpolarization , which in turn increases the subsequent spike . , it is known from classical neurophysiology that intensification of depolarization enlarges the frequency of action potentials , which are indeed crowned on the ca spike . it is well known [ 267269 ] that midbrain da neurons are comprised of three groups of cells : a8 , a9 , and a10 . the a10 da neurons are located in the ventral tegmental area ( vta ) , whereas the more lateral da cells of the substantia nigra pars compacta ( snpc ) belong to the a9 cell group ; the a8 neurons take a more caudal position in the retrorubral field ( rrf ) of the midbrain ( figure 7 ) . the terminals of these neuron groups form the nigrostriatal and mesolimbic ( or mesocorticolimbic ) daergic systems . the terminals of a10 vta neurons innervate the mesolimbic system : hypothalamus , nucleus accumbens ( nab , the nucleus of the ventral striatum ) , olfactory tubercle , central and basal nucleus of amygdala , habenula , septum , hippocampus , prefrontal , cingulate , and entorhinal cortex . the area of innervations by this system is the prefrontal , anterior cingulate , entorhinal , and piriform cortex and deep layer of the frontal cortex . there are 5 types of metabotropic da receptors associated with g proteins [ 270 , 271 ] d1 , d2 , d3 , d4 , and d5 . they belong to two basic families of receptors : the d1-like receptors including d1 and d5 da receptors , the d2-like receptors including d2 , d3 , and d4 da receptors . the d1-like receptors are associated with gs proteins ; they increase cyclic nucleotides ( camp ) synthesis and produce upregulation of da - induced intracellular events such as phosphorilation leading to an alteration in cellular activity and changes in the gene expression within the responding cells . the d2-like receptors are associated with gi proteins ; they decrease cyclic nucleotides ( camp ) synthesis and produce downregulation of da - induced intracellular reactions of dephosphorylation [ 272 , 273 ] . most of the d2-like presynaptic receptors are autoreceptors , an exception is the d2-like somatodendritic da receptors [ 272274 ] . these autoreceptors are located on da terminals and downregulation occurs due to inhibition of da synthesis and depolarization - induced release of da from terminals [ 275278 ] . as specified above , the nigrostriatal da system supervises the activity of gaba and glutamatergic synapses of the dorsal striatum ( nucleus caudatus and putamen ) , which in turn activate tc networks and regulate motor behavior in animals and humans [ 279 , 280 ] . corticostriatal transmission is essential in the regulation of voluntary movement , in addition to behavioural control . long - term potentiation ( ltp ) and long - term depression ( ltd ) , the two main forms of synaptic plasticity , are both represented at corticostriatal synapses and strongly depend on the activation of da receptors . about 95% of dorsal striatal neurons have the same morphology and are medium spiny neurons , their function is to integrate all incoming signals [ 272 , 279 , 281 ] . the medium spiny dorsal striatal neurons receive excitatory glutamatergic synaptic information through ampa , kainite , and nmda receptors practically from all areas of the neocortex [ 272 , 279 ] . postsynaptic gabaa receptors are located on dendrites and the soma of the striatal medium spiny neurons , and also on neurons of the globus pallidus , substantia nigra pars reticulate , and thalamus . the medium spiny neurons of nucleus caudatus and putamen are morphologically identical , but differ neurochemically . one population contains gaba , dynorphin , and substance p and primarily expresses dl receptors . these neurons send axons to gpi and to snpr ( figure 8) and form a direct pathway which inhibit gpi and snpr and are responsible for a reduction of an inhibitory influence on tc inputs , thereby activating tc input neurons . these neurons projects to gpe and form a indirect pathway which inhibits tc input ( figure 8) . it is important to note that gabaergic terminals form an indirect pathway that project to neurons of the rtn . that is , neurons of the rtn are controlled by d2 receptors through gabaergic projections from the ventral striatum . in detail , an induction of intracellular signals da by receptors and control of gabaergic and glutamatergic the darpp-32/pp-1 ( dopamine and cyclic adenosine 3,5-monophosphate - regulated phosphoprotein , 32 kda / phosphoprotein-1 ) pathway integrates information from a variety of neurotransmitters and produces a coordinated response involving numerous downstream physiological effectors . darpp-32 phosphorilation at thr-34 by pka is regulated by the actions of various neurotransmitters , principally da acting at d1 receptors , but also adenosine acting at a2a receptors and vip acting at vip receptors . pkg is activated in response to no , also phosphorylates thr-34 of darpp-32 . in medium spiny neurons that coexpress d1 and d2 classes of da receptors , activation of d2 receptors acts to decrease camp levels . in medium spiny neurons that coexpress adenosine a2a receptors and d2 receptors , activation of d2 receptors also acts to decrease camp levels . opiates acting at either or receptors decrease camp levels in cells stimulated with d1 agonists or adenosine a2a agonists , respectively . darpp-32 phosphorylated at thr-34 is dephosphorylated by pp-2b , a ca / calmodulin - dependent protein phosphatase . pp-2b activity is activated by a number of different neurotransmitter receptors , principally following ca influx mediated by glutamate acting at nmda receptors . glutamate acting at ampa receptors also stimulates darpp-32 dephosphorilation by pp-2b , by an indirect mechanism that involves depolarization of the neuron and influx of ca . activation of d2 receptors also leads to an increase in ca levels , via an unidentified mechanism , and increased activity of pp-2b . in contrast , gaba acting at gabaa receptors stimulates darpp-32 phosphorilation by hyperpolarization of the neuron , decreased influx of ca , and inactivation of pp-2b . all antipsychotic drugs achieve some of their clinical effects via antagonism of the d2 class of da , an action leading to an increase in the state of phosphorilation of darpp-32 . phospho - darpp-32 , by inhibiting the activity of pp-1 , acts in a synergistic manner with different protein kinases to increase the level of phosphorilation of various downstream effector proteins . the resulting increase in phosphorilation is associated with increased activity of nmda and ampa glutamate receptors , l- , n- , and p - type ca channels , and early gene creb and with decreased activity of gabaa receptors , na channels , and the na / k - atpase . it is important to note from scheme that antagonists of the d2 class of da receptors increase gabaa receptors activity , and agonists of the d2 class of da receptors decrease gabaa receptors activity . modification of gaba and glutamate ionotropic receptors activity will lead to updating of synaptic neuronal inputs and outputs , defining participation of neuron in functioning of a neural network . modification of voltage - dependent ionic channels activity modifies properties of the electrogenic neural membrane . integrating transduction signal possesses the ability to modify not only the activity of ionotropic , but also of metabotropic receptors of neurons localized on membranes and supervising transduction signal . such updating of all parameters of neurons , synaptic , electric , and chemical , switches neurons to another functionally active state or on other levels of functional activity . depending on the brain structure and of the modulatory system inducing modification of transduction signal , a particular neuronal state can be formed which can switch into another functional state . for example , activation of the mesocorticolimbic da system of ventral tegmental area ( figure 7 , a10 ) lateral hypothalamus [ 283285 ] , nacb [ 286 , 287 ] , prefrontal cortex [ 288291 ] , and anterior cingulate cortex [ 292 , 293 ] , induces an emotional positive state ( pleasure and happiness [ 294297 ] ) , a state referring to its own prolongation and/or intensification ( intra - cranial - self - stimulation [ 283 , 285 , 286 , 288 , 290 , 298 ] , self - administration of drugs [ 290 , 292 , 299 ] , self - control of drug administration , or place - preference paradigm [ 299301 ] ) . switching of neuron activity in other functional state , through intracellular integration with the help of transduction signal and modification of ionotropic and metabotropic receptors and potential sensitive ionic channels activity , it is possible to explain the role of the opioid [ 285 , 298 ] , gabaergic [ 284 , 287 ] , acetylcholinergic , serotoninergic [ 286 , 287 ] , and other systems in the control of the mesocorticolimbic daergic reinforcement system . we define reinforcement as an emotional positive state arising as a consequence of a reward and of an avoidance or escape of an aversive stimulus , it induces purposeful motivated behavior . as it has been shown above , one of the important structures of the mesocorticolimbic da system is the nacb , which is also called the ventral striatum . the nacb consists of two groups of cells which differ anatomically : the shell and core . the degree of participation of these two parts in reinforcement ( see above ) depends on features of the experimental model . it is supposed that the core of the nacb together with the dorsal striatum participates in the realization and control of purposeful behavior . the nigrostriatal da system participates in regulation of motor behavior by regulation of tc neural networks ( it is described in more details above ) . the shell receives innervations from a10 da cellular groups ( figure 7 ) , it is associated with the limbic system . the nacb core receives glutamatergic inputs from prefrontal cortices ( another important structure of reinforcement reaction ) , thalamus , hippocampus , amygdala , and habenula . direct and indirect projections from the core of the nacb to the snpr and vp in rats are described [ 303 , 304 ] . thus , the da system of the ventral striatum ( mesocorticolimbic ) cooperates with that of the dorsal striatum ( nigrostriatal ) at the level of snpr and vp which regulate tc networks through the gabaergic system . it is the structural basis for the realization of purposeful motivated motor behavior , in particular when a choice for the correct response can be made in order to get reinforced . behavioral experiments suggest that learning is driven by changes in the expectations about future salient events such as rewards and punishments . physiological work has recently complemented these studies by identifying da neurons in the primate whose fluctuating output signals changes or errors in the predictions of future salient and rewarding events [ 305307 ] . it is known that swds are controlled by the brain 's dopaminergic ( daergic ) system . antagonists of d2 da receptors increase and agonists decrease of swds [ 32 , 304 , 308310 ] . the wag / rij rats can be regarded as a valid genetic animal model of absence epilepsy with co morbidity of depression [ 5052 ] . behavioral studies indicate that wag / rij rats exhibit depression - like symptoms : decreased explorative activity in the open field test , increased immobility in the forced swimming test , and decreased sucrose consumption and preference ( anhedonia ) . in addition , wag / rij rats adopt passive strategies in stressful situations , express some cognitive disturbances ( reduced long - term memory ) , helplessness , and submissiveness , inability to make choices , and overcome obstacles , which are typical for depressed patients . elevated anxiety is not a characteristic ( specific ) feature of wag / rij rats ; it is a characteristic for only a substrain of wag / rij rats susceptible to audiogenic seizures . interestingly , wag / rij rats display a hyperresponse to amphetamine similar to anhedonic depressed patients . wag / rij rats are sensitive only to chronic , but not acute , antidepressant treatments , suggesting that wag / rij rats fulfill a criterion of predictive validity for a putative animal model of depression . however , more and different antidepressant drugs still await evaluation . depression - like behavioral symptoms in this model are evident at baseline conditions , not exclusively after exposure to stress . experiments with foot - shock stress do not point towards higher stress sensitivity at both behavioral and hormonal levels . however , freezing behavior ( coping deficits ) and blunted response of 5ht in the frontal cortex to uncontrollable sound stress , increased c - fos expression in the terminal regions of the mesocorticolimbic brain systems and larger da response of the mesolimbic system to forced swim stress suggests that wag / rij rats are vulnerable to some , but not to all types of stressors . in all , it seems that the depressive - like behavior in wag / rij rats has a da - dependent character . these results allow the assumption that the wag / rij strain possesses a deficiency of the brain 's da system activity . a low threshold for catalepsy , among others induced by sound stress , was repeatedly found in wag / rij compared to wistar rats [ 308 , 312 ] . wag / rij rats showed also both a larger novelty and amphetamine induced increase in total distance moved than aci rats in a brightly lit open field . this novelty / amphetamine - induced ambulation score was interpreted as an index of the reactivity of the da system in the mesolimbic system [ 313315 ] . index of the reactivity is in addition bound , as well as a catalepsy threshold , to a threshold of sensitivity of a ligand - receptor interaction . it means that the dissociation constant ( kd ) of wag / rij rats da receptors is lower than in nonepileptic rats . it is possible to assume that control of swds by d2-like da receptors is carried out through an indirect gabaergic pathway from the dorsal striatum which terminates in the rtn through pathways passing through the gp ( figure 8) , one of the sources of rhythmic oscillations . antagonists of d2-like da receptors enlarge activity of gabaa receptor and increase swd activity , and agonists reduce gabaa receptor activity and reduce swds ( see section 3.1.1 ) . d2-like da receptors in dorsal striatum induces phosphorilation of gabaa receptor and reduce its activity . it has been shown that d2-like da receptors selectively block n - type ca channels to reduce gaba release onto rat striatal cholinergic interneurons . da d2 receptor - mediated presynaptic inhibition of striatopallidal gabaa ipscs in vitro . there were significant increases in [ h]nemonapride binding to d2-like receptors at different time points due to treatment with haloperidol , chlorpromazine , and olanzapine in rat striatum . by contrast , treatment with clozapine and olanzapine caused a time - dependent decrease in [ h]muscimol binding to the gabaa receptor of striatum . an increase of gabaergic neurotransmission in the striatum of mice lacking da d2 receptors was found . the inhibitory effects of the da d2 receptor agonist quinpirole on gabaa - mediated spontaneous inhibitory postsynaptic currents correlate with the total number of d2 receptor sites in the striatum . l - dopa acting as d2-like agonist inhibits gaba release in the rat globus pallidus and induces turning behavior in rats with unilateral lesions of the da innervation . quinelorane , a dopamine d3/d2 receptor agonist , reduces prepulse inhibition of startle and gaba efflux in the vp . gaba release from pallidal terminals in the stn nucleus and in the rtn is inhibited by activation of d4 receptors . dopamine inhibits gaba transmission from the gp to the rtn via presynaptic d4 receptors [ 326 , 327 ] . thus , antagonists of d2-like da receptors strengthen gaba transmission into striatum , gp and rtn and raise swds by promoting burst activity in the rtn , and agonists operate in an opposite direction . based on the electrophysiological and lesion studies in wag / rij rats it is proposed that there is no direct source of swd activity in the rtn , however , bursting neurons in the rtn are a necessary condition for maintenance and spreading of swd [ 13 , 20 , 99 ] . an infringement of wag / rij rat 's behavior was shown [ 51 , 52 , 328331 ] . a decrease of memory reproduction , spontaneous catalepsy , low threshold of haloperidol - induced catalepsy , disturbances in maternal behavior , and symptoms of depression - like behavior were found in wag / rij rats . all these data can be explained by a da deficit on the wag / rij rat 's brain . the goal of the investigation [ 332 , 333 ] was to study the possibility of wag / rij rat 's behavior correction by pharmacological activation of daergic system by disulfiram and l-3,4-dihydroxyphenylalanine ( l - dopa , the precursor of da synthesis ) . higher doses of disulfiram ( 100300 mg / kg ) block the efficiency of dopamine--hydroxyls , the enzyme involved in the synthesis of noradrenalin ( na ) from da . a lower dose of 25 mg / kg of disulfiram increases the da content in the brain without change of na and transforms animals with an emotional negative reaction to animals with an emotional positive reaction in the emotional resonance behavioral reaction test . rats were tested in a passive light avoidance and in a classical two - way active avoidance task and the effects of this dose of disulfiram on memory were investigated . the index of memory trace storage ( imt ) was calculated as ( 1)imt = a2a1a1100% , where a1 is the number of avoidance 's in learning session on day 1 , a2 is the number of avoidance 's in the session on day 2 ( both 50 trials ) . there were three series of experiments . first series : disulfiram was administered to wistar and wag / rij rats of the 1st group 4 hours before the 1st day learning session . second series : l - dopa ( also 25 mg / kg i.p . ) was administered to wistar and wag / rij rats of the 2nd group 4 hours before the 1st learning session . in the 3rd series of experiment disulfiram control wag / rij rats showed an amnesic reaction compared to control wistar rats at day 2 of the passive avoidance conditioning procedure ( figures 9(a ) and 9(b ) ; in 2.65 and 3.89 times in ( a ) and ( b ) , resp . ) . the administration of disulfiram before as well as after passive avoidance conditioning improved memory as measured the next day both in wistar and wag / rij rats , although the extent of increase was higher in wag / rij than in wistar rats . the reproduction of memory , as established in the passive avoidance test , was increased 1.34 and 1.41 times in wistar rats and 4.21 and 4.89 times in wag / rij rats accordingly . administration of disulfiram before learning did not change the latency to enter the dark compartment in the first day nor induced a difference between the strains later . the administration of disulfiram 4 hours before establishment of active avoidance conditioning changed the learning processes in the first day ( figure 9(c ) ) . control wag / rij rats realized 2.23 times more avoidance responses than control wistar rats in the first day of learning ( figure 9(c ) ) . disulfiram administration ( dis ) before learning decreased the number of avoidance responses in the first day : in wistar rats 1.47 times and in wag / rij rats 6.45 times ( figure 9(c ) ) . an amnesic effect in control wag / rij versus control wistar rats was found in the second day of learning : the memory index of wag / rij rats was 2.11 times lower than in wistar rats ( figure 9(d ) ) . the administration of disulfiram increased memory index of wistar rats 1.44 times and in wag / rij rats 7.33 times ( figure 9(d ) ) . the other inductor of da system activation , l - dopa , was used for comparison . synergic effects of disulfiram and l - dopa administered 4 hours before learning were found ( figure 10 ) . the administration of l - dopa 4 hours before learning decreased the number of avoidances in the first day of learning in wistar rats 1.76 times and in wag / rij rats 7.65 times ( figure 10(a ) ) . herewith , the index of memory trace storage was increased in wistar rats 1.82 times and in wag / rij rats 7.70 times ( figure 8(b ) ) . a higher number of active avoidance responses in the first day of learning and an amnesic effect in the second day of learning in wag / rij versus wistar rats ( figures 9(c ) , 10(a ) , and 10(c ) ) was reproduced three times in our experiments . administration of disulfiram immediately after active avoidance conditioning in the first day significantly increased the number of avoidance responses in the second day ; the reaction of wag / rij rats was more intense than in wistar rats ( figure 10(c ) ) . in the memory trace storage the relation wistar ( dis ) to wistar is 1.8 and relation wag / rij ( dis ) to wag / rij is 2.48 ( figure 10(d ) ) . the amnesic effect in wag / rij versus wistar rats in the passive avoidance reflex was found earlier [ 328 , 335 ] . the amnesic effect can be explained by wag / rij rat 's epileptic activity , the low efficiency of da system and as a consequence , a reduced sensitivity for reward ( see above ) . the improvement in the passive avoidance , ( the dark was longer avoided ) in both wag / rij and wistar rats after disulfiram administration ( figure 7 ) can be explained by the same processes , which is an agreement with the changes in the emotional resonance behavioral reaction test . it can be supposed that a low dose of disulfiram before as well as after learning increases the da content in the brain , increase the sensitivity for reward , and , as a consequence , the consolidation process , the latter as expressed by a better performance in the passive avoidance test the next day . it is proposed here that the larger effects of disulfiram in wag / rij rats reflect their deficit in the da system . besides , the increase of da system efficiency decreases in wag / rij rat 's amount of epileptic activity [ 32 , 304 , 308 , 309 ] . activation of the da system can be considered as an anti - amnesic effect . as it was specified above , the mesocorticolimbic daergic system is a reinforcement system , not a reward system . for some people one kind of food can be reward , and for other people the same food can be aversive . hence , the relation to what is considered as reward is developed in the history of an individual 's life . as in the somatosensory and motor projections to dorsal striatum remains somatotopy inherent to the cortex [ 279 , 280 ] it is natural that the reward involves nigrostriatal da system [ 307 , 336340 ] . it is necessary to notice that a subset of striatal neurons ( 10% ) is tonically active with discharge rates of 2 to 10 hz . these neurons are distributed throughout the striatum and their discharge bears no specific relation to movement . these tonically active neurons ( tans ) fire in relation to certain sensory stimuli that are associated with reward . for example , a tan will fire in relation to a clicking sound if the click precedes a fruit juice reward but will not fire to the click alone or to the reward alone [ 279 , 341 ] . it has been suggested that these neurons signal aspects of tasks that are related to learning and reinforcement . tans are not activated by electrical stimulation of the gp and thus are probably not projection neurons . it is thought that tans are cholinergic large aspiny interneurons that innervate the medium spiny neurons ( see earlier discussions ) , which would place them in a position to modify the sensitivity of the medium spiny neurons to cortical input in relation to specific behavioral contexts . we think that reinforcing properties of reward cause emotionally positive states which induce emotionally positive reactions , such as pleasure . the purest kind of implication of the emotionally positive reaction refers to the prolongation of the emotionally positive state is the reaction of self - stimulation . self - stimulation reaction can be observed only at stimulation of the mesocorticolimbic da systems [ 291 , 343349 ] , but not by stimulation of nigrostriatal da systems . but prolongation of emotionally positive state is motivation , hence mesocorticolimbic da system executes also function of motivation [ 348 , 350 ] . thus , the mesocorticolimbic da system organizes behavior via modulation of emotional and motivation states . in this case , it is conceivable that tonically activated neurons of dorsal striatum play a complementary role , due to their small numbers . it is possible that these neurons are involved in the interaction processes of reinforcement and reward . we consider that in our investigation the avoidance of electrical current causes an emotionally positive state and reinforces an avoidance reaction . the augmentation of da concentration in the brain by disulfiram or l - dopa strengthens the reaction to a reinforcer . against the prospect of a deficiency of da and hyper reactivity of da systems , the investigated substances provoke much more intensive effect in wag / rij than in wistar rats . different agonists of kappa and antagonists of mu opioid receptors decrease swds , and mu receptors agonists increase swds [ 351353 ] . it was also shown that a specific peptide of the proenkephalin system was increased in the striatum , the frontal cortex , and the mesencephalon [ 354 , 355 ] , structures directly or indirectly controlling inhibitory processes in corticothalamocortical neuronal networks [ 21 , 279 , 280 ] . also from in situ hybridization studies , it appeared that the level of the proenkephalin mrna was enhanced in the striatum of wag / rij rats . the opioid system controls the threshold of pain sensitivity and actualizes the motivation of escape and avoidance of pain [ 299 , 357 , 358 ] . a high number of avoidance responses were found earlier in wag / rij rats on the first day of active avoidance conditioning ( figures 9 and 10 ) [ 328 , 335 ] . we explain this reaction by the low efficiency of opioid system in wag / rij strain [ 351356 ] and as a consequence a low pain threshold and a high level of escape and avoidance motivation . it has been shown [ 359361 ] that activation of the daergic system evokes analgesic reaction including activation of the opioid system [ 362366 ] . it is important that in the analgesic reaction of daergic system the nacb is included [ 359 , 360 ] and also that the interaction of daergic and opioid systems takes place in the striatum [ 272 , 364 ] , a structure controlling swds . it is supposed that disulfiram or l - dopa administration before learning increases the content of da in the brain and via an enhancement of the opioid system activity increases the pain threshold and decreases pain avoidance motivation . at the same time da - induced reinforcement ( modification of mesocorticolimbic da system ) is increased , as well as memory consolidation and memory reproduction on the next day ( figures 9 and 10 ) . it has been shown ( kuznetsova , not published data ) that l - dopa ( 25 mg / kg ) administration decreases swds activity in wag / rij rats and that the same has been found for the da agonists apomorphine and cocaine [ 32 , 367 ] . disulfiram administration immediately after learning increased da - induced reinforcement ( see above ) , increased the process of memory consolidation , and increased active avoidance memory reproduction on the next day ( figure 10 ) . it is possible that the larger effect in wag / rij rats is a reflection of the da and opioid systems efficiency deficit in this strain of rats . in all , a deficit of da system in wag / rij rats and an increase in the sensitivity to a da agent as the response on the deficit is proposed to be the causative factor for the behavioural differences in wistar and wag / rij rats . a stable memory trace was found in the passive avoidance task in wistar rats ; however , wag / rijs performed less well . disulfiram administration before and after learning increased memory reproduction in this task in both strains of rats . the enhancement of memory reproduction was more intense for wag / rij than for wistar rats . wag / rij rats realized more correct avoidances than wistar rats in the first day of active avoidance learning . but in the next day of learning a memory deficit was found in wag / rij rats . low dose of disulfiram and l - dopa administration before learning decreased learning in the first day , but increased active avoidance memory reproduction in the next day in both strains . this decrease of learning and enhancement of memory was more intense in wag / rij than in wistar rats . it was suggested that a deficit of da system in wag / rij rats is the biological correlate of these behavioural deficits and that an enhanced sensitivity to daergic agents is the consequence of this deficit . some parameters of da activity in wag / rij rats were studied in order to establish whether there was a deficiency . da synthesis and its degradation the limitation enzyme tyrosine - hydroxylase synthesizes l - dopa ( l-3,4-dihydroxyphenylanine ) from tyrosine . da is taken up by vesicles of varicosity and stored there [ 368 , 369 ] . during depolarization , da is released into the intercellular space and interacts with da receptors . at this state a higher concentration of released da , receptors activity and da efficiency are observed . further da is retaken up in terminals and glia cells by assistance of da - transporter . da is inactivated by monoamine oxidase enzyme ( mao ) and through a number of enzymatic transformations turns onto metabolite dopac ( 3,4-dihydroxyphenylacetic acid ) measured experimentally . by the help of another enzyme of inactivationcatechol - o - methyltransferase ( comt ) da passes through another enzymatic transformations and turns into the metabolite hva ( homovanillic acid ) also measured experimentally . the inhibition of the da - transporter , mao , and comt activity reduces an inactivation of da , reduces the concentration of dopac and hva , raises the duration of da interaction with receptors , and enhances the efficiency of the da system [ 370374 ] . the goal of our first experiment was to investigate da and its metabolites , dopac and hva concentration in the following brain structures of wistar and wag / rij rats : frontal cortex , parietal cortex , medulla , striatum , thalamus , and cerebellum . concentrations of da and its metabolites have been defined by method of high performance liquid chromatography ( hplc ) . there was no difference in da concentration in wag / rij compared to wistar rats . however , the concentrations of metabolites and the hva / da ratio were substantially different in some structures between wag / rij and wistar rats . dopac concentration was reduced in striatum and hva concentration in thalamus in wag / rij rats . as noted above , the dorsal striatum is innervated by nigrostriatal da system controlling activity of tc networks and modulates the occurrence of swds [ 21 , 55 , 279 ] . reduction of the concentration of metabolites in thalamus and striatum is related to enhanced da activity in these structures . the strengthening of da activity may occur as compensation for da deficiency at behavioural level . the deficiency of da activity is likely to be linked with changes of da receptors . in order to test such probability , we compared [ 376 , 377 ] d1 and d2/d3 da receptors binding sites with autoradiography in brain areas of wag / rij while aci rats were used as controls ; the latter strain is nearly free of swds . analyses of the optical densities of the autoradiograms showed a significant reduction of [ h ] sch 23390 binding sites ( d1 da receptors ) of wag / rij rats compared to aci rats in the shell of nacb and in the head of caudate nucleus . the results of [ h ] spiperone binding for d2/d3 da receptors are given in table 2 . an increase of these binding sites was seen in wag / rij compared to aci rats in motor , somatosensory , and parietal cortex . the number of binding sites was substantially lower than in the same structures of aci rat ; in the head of caudate nucleus and in the hippocampal ca3 area , a higher number was found in wag / rij rats . our results , a decrease of d1 da receptors number in nacb shell and in the head of caudate nucleus ( table 1 ) , might be related to a reduction of an upregulating effect of daergic transmission induced by phosphorilation . the increase in the number of d2/d3 da receptors in motor , somatosensory , and parietal cortex was due to an amplification of downregulating effect of daergic transmission , induced by dephosphorilation . the reduction of d2/d3 da receptors number in the head caudate nucleus and in the ca3 area of hippocampus ( table 2 ) corresponds to reduction of downregulating effect of daergic transmission , inducing the processes of dephosphorilation and enhancement of gabaa receptors activity . the d1 da receptors are somatodendritic receptors ; they have not been found on da terminals . at the same time , d2/d3 da receptors are both placed on soma and dendrites of target cells , and on da terminals [ 272 , 274278 ] . as shown above , the autoreceptors modify da release affecting a synthesis measured by da concentration in tissue samples . in our previous study , we did not reveal significant differences in da concentration in various brain structures of wag / rij compared to nonepileptic rats . it is possible that changes of d2/d3 da receptors number revealed in this work were mainly related with somatodendritic receptors . the low number of somatodendric d2-like da receptors in the caudate nucleus induces endogenous processes of dephosphorilation and enhancement of gabaa receptors activity in striatum , gp , and rtn ( figure 8) and is an endogenous cause for the increase of swd activity in the corticothalamocortical network . the high number of somatodendric d2-like da receptors in somatosensory cortex induce the endogenous processes of phosphorilation and decrease of gabaa receptors activity in somatosensory cortex , forming an endogenous cause for decreasing swds in the cortical focus , which supervises an oscillatory drive in the rtn . possibly , it assumes compensatory reaction from outside somatosensory cortex in relation to caudate nucleus . that is why in wag / rij a compensated deficiency of d2-like da receptors but d2-like da receptors antagonists ' administration both in cortex and caudate nucleus enhances processes of dephosphorilation and gabaa receptors activity amplify , enlarging activity of swds . a deficiency of mesolimbic ( nacb shell ) daergic activity at the level of somatodendritic d1-like da receptors in wag / rij versus to aci rats was found . at the same time a deficiency of nigrostriatal daergic system in the head of caudate nucleus was caused by a reduction of d1-like and d2-like da receptors number . the deficiency of nigrostriatal daergic system by reduction of d2-like da receptors is at the bottom of an additional rising activity of swds . as d1-like da receptors supervise a direct pathway from striatum which terminates on non epileptic nucleus of thalamus ( figure 8) , to us , there is no reason to assume that the deficiency d1-like da receptors has an impact on the occurrence of activity of swds . the deficiency of mesocorticolimbic da systems corresponds to behavioral features of wag / rij rats . during active and passive avoidance a deficit of reinforcement in wag / rij rats has been revealed [ 332 , 333 ] which was eliminated by administration of the da precursor , or a low dose of disulfiram ( figures 9 and 10 ) , inhibitor of dopamine--hydroxylase , by increase of da concentration in brain . besides , it was shown that wag / rij rats have a higher level of depression than control wistar rats without absence epilepsy [ 5052 , 379 ] . depression of wag / rij rats has a daergic nature and can be explained by a deficiency of the da mesolimbic system . behavioural changes and activity of the mesolimbic daergic systems in wag / rij rats are influenced by activation of d1 da receptors in the shell of the nacb [ 380 , 381 ] and prefrontal cortex [ 382 , 383 ] , the main sources of positive reinforcement . d2-like da receptors are also involved in reinforcement [ 380 , 381 , 384 , 385 ] . a link between modification of behavior and alternative splicing of d3 da it is likely that d1- and d2-like da receptors in the nacb may have more general influence on reinforced behavior . the nigrostriatal da system mediates behavioral reactions through the control of tc neuronal networks . the nacb shell receives daergic cellular innervations from the vta ; this part of the nacb is connected with limbic system [ 268 , 279 ] . the nacb core receives glutamatergic inputs from the prefrontal cortex and thalamus , as well as from hippocampus and amygdala . there are direct and indirect projections from nacb core to snpr and vp in rats . thus , interaction of mesocorticolimbic and striatonigral da systems at level of snpr and vp controls thalamocortical networks through gabaergic system , behavioral reactions , and absence epileptic activity [ 21 , 304 ] . despite a compensated deficit of da system of wag / rij rats da agonists and antagonists increase and decrease swds , respectively . intra - accumbal injections of da agonists and antagonists produced increase and decrease of swds respectively . herewith the significant changes in the nacb ( upregulation of mrna d3 da receptor ) were revealed , although the levels of mrna coding transporters or enzyme of da synthesis did not differ from controls . the changes of daergic system in nacb core in gaers rats were slightly different than those received in wag / rij rats at present work . but the general orientation of the regulation of swds by daergic in gaers and wag / rij rats was the same . therefore it is possible to admit that the mechanisms of daergic regulation are also similar . it can be that upregulation of mrna d3 da receptor in the nacb also is a process of compensation . thus a deficiency in da systems in wag / rij rats is described and is mediated by d1- and d2-like da receptors . reduction of d1-like da receptors number in the shell of the nacb and in nucleus caudatus , reduction of d2-like da receptors number in nucleus caudatus and increase of d2-like da receptors number in motor , somatosensory , and parietal cortex . reduction of d2-like da receptors number in nucleus caudatus enhances the activity of gabaa receptors in striatum , gp , and rtn , causing a rise in swd - activity in corticothalamocortical networks . high number of somatodendric d2-like da receptors in somatosensory cortex inducing the endogenous processes of phosphorilation and decrease of gabaa receptors activity in somatosensory cortex , causing endogenous decrease of swds in cortical focus , which supervises an oscillatory source in the rtn . we assume that it reflects compensated deficiency of d2-like da receptors of wag / rij rats . despite process of compensation antagonists of d2-like da receptors can raise activity gabaa receptors both in a somatosensory cortex and in the rtn , raising swds . deficiency of wag / rij rats mesolimbic da systems , caused by reduction d1-like da receptors in the shell of the nacb causes depression - like behavior of animals , weakens formation of emotional positive state , worsens processes of reinforcement , and counteracts learning and memory processes . pharmacological intensification of da systems takes out negative reaction of deficiency of mesolimbic da system . the question arises , if absence epilepsy is related to disturbances or mutations of gabaa receptor and low - threshold t - type ca channel , then what is the role of da receptors in it . we suggest two mechanisms of infringement of the function of the da system during absence epilepsy.gabaa receptors mediate trophic effectson embryonic brainstem monoamine neurons . gaba is present in the mammalian brain during early stages of development , where it may act as a trophic signal for developing neurons . in the embryonic rat , gaba axons project through the brainstem this raises the possibility that gaba exert trophic influences on developing monoamine neurons , if they express appropriate receptors . in adult rat brain , the initial disruption of gabaa receptor disrupts trophic influences on developing monoamine neurons and induces pathological changes in monoaminergic systems in adult animals.intraneuronal integration triggered by a transduction signal . we believe that infringement of the function of the da system by intraneuronal integration triggered by transduction signal occurs in the brain , especially in structures in which there are no da neurons , but da terminals , such as cortex , striatum , gp , thalamus , nucleus nacb , and so on . gaba is present in the mammalian brain during early stages of development , where it may act as a trophic signal for developing neurons . in the embryonic rat , gaba axons project through the brainstem this raises the possibility that gaba exert trophic influences on developing monoamine neurons , if they express appropriate receptors . in adult rat brain , the initial disruption of gabaa receptor disrupts trophic influences on developing monoamine neurons and induces pathological changes in monoaminergic systems in adult animals . we believe that infringement of the function of the da system by intraneuronal integration triggered by transduction signal occurs in the brain , especially in structures in which there are no da neurons , but da terminals , such as cortex , striatum , gp , thalamus , nucleus nacb , and so on . earlier it has been shown that the development of pentylenetetrazole ( ptz ) kindling ( 4-month - old animals ) is accompanied by 20% reduction of the density ( bmax ) of gabaa receptor benzodiazepine ( bdz ) sites . in 10-month animals , kindling reduced densities of the bdz site of gabaa receptor by 50% . acute administration of a subconvulsive dose of ptz in the rats at 10 months of age that were kindled at 4 month induces convulsive seizures and densities of gabaa receptor bdz site ( bmax ) to 20% of control rats . the results suggest that the density of the bdz site at the gabaa receptors is reproduced at a level of receptor molecule density independent of their current status and age . the increased density of bdz site of gabaa receptors in kindled versus control rats can be interpreted as an enhancement of gabaergic inhibition , while it is thought that seizures are based on the process of neuronal hyperactivation accompanied by a reduction in the bdz site density both in kindling - induced and single - dose ptz - induced seizures . therefore , it is likely that at 10 months , when seizures are retrieved by a ptz challenge and the level of gabaergic inhibition is restored , the level of glutamate receptors may also be restored , assuming that they were modified and consolidated in the process of kindling 6 months ago . the level of glutamate receptors may only be restored if neuronal gaba and glutamate receptors interact within a single integrated system interconnected through an intracellular transduction signal . the interaction and integration of neuronal gaba and glutamate receptors have been shown in several studies . a reduction in gabaergic functions in ptz - induced kindling nmda receptors are also involved in the process of kindling induced by fg 7142 , an inverse agonist of the bdz receptor . kindling produced by high - frequency hippocampal stimulation induces an enhancement of nmda receptors as well as modification of gabaa receptors . also , the nmda - induced ltp is found to be controlled by the intercellular metabolic systems of the gabaa receptor complex , being inhibited by agonists acting at the bdz site [ 401 , 402 ] and facilitated by its antagonists [ 403 , 404 ] . conversely , the nmda antagonist cpp can modulate the long - term modification ( tolerance ) of bdz receptors . positive allosteric activation of gabaa receptors bi - directionally modulates hippocampal glutamate plasticity and behavior . finally , bdz withdrawal anxiety is associated with potentiation of ampa receptor currents in hippocampal ca1 pyramidal neurons attributable to increased synaptic incorporation of glua1-containing ampa receptors . the bdz site is one of the metabolic sites of the gabaa supramolecular receptor complex [ 408412 ] which is subject to long - term modifications [ 165 , 400 , 413 ] . the next of the gabaa receptor metabolic site is the neuroactive steroids site [ 116 , 414 ] . ltp and ltd , like any long - term processes , are maintained by modifications of gene expression [ 399 , 400 , 415418 ] . bdz site , acting via the second messengers system and protein kinase c , can induce slow metabolic reactions in the cell and modify gene expression in various structures of rat brain [ 105 , 408 , 410 , 419 ] . gabaa receptors are known to be regulated by phosphorilation mediated by protein kinase c and g [ 272 , 273 , 420422 ] . substrate for phosphorilation and dephosphorilation of gabaa receptor is the intracellular loop between the 3rd and 4th domain [ 204 , 423 , 424 ] . as indicated above , changes in cellular phosphorilation levels by protein kinases can modify glutamatergic receptors [ 162 , 398 , 399 , 401405 ] , augmenting their responses to endogenous excitatory amino acids . the metabolic regulation of glutamatergic synapses ( figure 11 ) is described as a regulation of ampa receptors by nmda receptors . control of ampa receptor by nmda receptor is described by plasticity in glutamatergic synapses of hippocampus , striatum , and cortex [ 273 , 425 ] . we added a feedback loop for the regulation of ampa receptors by nmda receptors as it is repeatedly described in studies of the synthesis and trafficking of ampa receptors subunit to the plasmatic membrane [ 426428 ] . the regulation of ampa receptors and autoregulation of nmda receptors in the hippocampus , striatum and cortex have been studied experimentally [ 427 , 429 , 430 ] . the autoregulation of gabaa receptors is also well described in investigations of synthesis and trafficking of gabaa receptors subunit to the plasmatic membrane [ 204 , 431433 ] . thus , one can assume that plasticity is a result of cooperative activity of gaba and glutamatergic receptors integrated into interrelated system . furthermore , a new level of activity , produced by secondary intranuclear signals , modifies the gene expression and consolidates the newly developed activity of receptors . functional interactions of the striatal glutamate and da receptors in learning and memory [ 279 , 305 , 306 , 434 , 435 ] , and postsynaptic integration of glutamatergic and da signals in the striatum based on interactions of intracellular second messengers [ 272 , 273 , 279 , 436438 ] have been reported . the d4 receptor activation increases cofilin activity , causing a decrease in the myosin - based transport of gabaa receptors clusters to the surface in the prefrontal cortex . it has been shown that the induction of ltd by low frequency stimulation in hippocampal ca1 neurons is under the influence of both nmda and gaba receptors ; both d1 and d2 receptors are involved in the modulation of ltd ( activation of d1 receptors enhances ltd , activation of d2 receptors inhibits it ) and the induction of ltd is blocked by picrotoxin , this effect is superseded by d1 receptors agonist skf-38393 . our results also point to synergistic interactions of gabaa and da receptors in memory retrieval . the intracellular integration by transduction signal of glutamate , gabaa and da receptors is schematically shown in figure 11 . da receptors can undergo automodification by the metabolic feedback loop and then modify the activity of glutamate and gabaa receptors by intracellular phosphorilation [ 272 , 273 ] . via the same reactions of intracellular phosphorilation , glutamate and gabaa receptors can control the efficiency of da receptors . at the second stage , the modifications established at the first stage are consolidated through the modification of expression of the respective genes . the ability of da receptors to undergo automodifications has been demonstrated both at the level of radioligand binding and at the level of gene expression in various brain structures and various experimental procedures [ 319 , 442447 ] . we suggest that diminished activity of da receptors and da system deficit occur due to disruption of intracellular integration triggered by a transduction signal ( figure 11 ) . in the first place the initial disruption of gabaa receptor activity disrupts the transduction signal from this receptor . disruption of transduction signal shapes the modification of other receptors and their activity . in the second place the disrupted activity of receptors is consolidated and stored by expression of genes . it should be noted that we did not see changes of da concentrations in the structures that we investigated , but we could see changes in receptor activity and consequences of receptors activity . so , a process of intracellular integration may disrupt activity of other neurotransmitter and neuromodulatory systems , for instance the activity of the opioid system that is disrupted in wag / rij rats [ 351356 ] . if the first possible mechanism of infringement of the function of the da system , mediation of trophic factor by gabaa receptors , operates during a limited time period corresponding to the period of embryonic development , the second mechanism , intraneuronal integration triggered by transduction signal , operates during the whole life and , possibly , collects as a snow clot . the hyperpolarization - activated and camp - modulated hcn channels contribute to direct rhythmic activation in the brain . hcn channels resemble voltage - dependent k channels ; they are tetrameric and consist of monomeric subunits , each containing six transmembrane domains ( s1s6 ) with a p - loop that forms a pore between the s5 and s6 domains . different hcn subunits may be connected and form a heteromultimeric complex , thus significantly increasing the diversity of ih channels . the hcn1 channel has a minimal response to camp binding ( + 4 mv ) with a camp - binding domain , whereas the hcn2 channel has a clear response ( + 17 mv ) . the coexpression of hcn1 and hcn2 with heteromultimeric channel formation leads to ih currents , which tend to possess functional properties intermediate between hcn1 and hcn2 homomeric channels . experimental data suggest that the ih channel and the low - threshold t - type ca channel ( ica , t ) work in tandem . hyperpolarization opens the ih channel and a cationic current depolarizes the membrane to the threshold and induces a spike . the ca ions that enter the cell induce ca - dependent camp synthesis and camp dramatically increases channel activity through binding to the cnbd locus of hcn subunits . the hcn1 subunit reacts weakly to camp binding ; therefore , the decrease in the proportion of hcn1 subunits in the channel increases pacemaker activity and an increase in the proportion of hcn1 subunits in the channel decreases pacemaker activity . analysis of the experimental data shows that one of the basic mechanisms for the long - term regulation of ih pacemaker activity is the modification of the number of hcn1 subunits in the pacemaker channel . it has been assumed that the source of swds is the ih pacemaker channel that is localized at pyramidal cells in the perioral region of the somatosensory cortex ( in wag / rij rats ) , more specifically layers iii - vi . this part of the cortex is fully integrated in a corticothalamocortical circuit , which intactness is obligatory for the full expression of the bilateral generalized swds . the rtn is part of this circuitry , it has also pacemaker properties , and it generates sleep spindles . the density of a subtype of hcn channels ( hcn1 ) in the apical dendrites of the subgranular layers in this onset zone determines the somatodendritic excitability and a decrease of these dendritic hcn1 channels neurons increases the occurrence of absences . also in the thalamus hcn channels are involved in burst firing typical for delta activity . burst firing of tc - cells is , however , rare during swds ; it occurs in only 7% of the tc cells although it can not be excluded that this a necessary condition for swd occurrence . burst firing is caused by tandem hyperpolarization of ih and ( ica , t ) channels . pharmacological observations supported this point of view since activation of gabaergic system through gabaa , gabab receptors and inhibition of gaba inactivation in the synaptic cleft all leads to unequivocal result , intensification of swd activity . however , the measurements of the efficiency of gabaergic system lead to diverse results first , different models of absence epilepsy such as gaers and wag / rij rats , various types of mice with spontaneous mutations , and knock - out lines have been used . it is possible that in these different models , various mechanisms of gabaergic system deficiency are present . the second reason of occurrence of the variety of results is the so - called process of uncertainty of the central nervous system . the analysis of several works has shown that an increase or decrease of the general activity of the transmission system in different animals can be achieved in different ways and depends on specific features of these animals . strengthening of conductivity of chlorine through gabaa receptors as found in wag / rij rats can be considered as a compensation for the failure of inhibition of gabaergic systems . one of the reasons for the occurrence of absence epilepsy is the mutation of several genes coding for gabaa receptors which cause abnormal functioning of gabaergic system . such abnormal functioning of the gabaergic system and mutations of ( ica , t ) channel , which functions in tandem with ih channel , can contribute to the occurrence of swds in the eeg . however , swds during absence epilepsy may also be enhanced by the activation of the glutamate ionotropic and metabotropic excitatory system . agonists of three type 's ionotropic excitatory glutamate receptors ampa , kainate and nmda enhance swds ; antagonists of these receptors reduce swds . it is known from classical neurophysiology that intensification of depolarization enlarges the frequency of action potential discharges . the metabotropic glutamatergic receptors also control swds , strengthening or controlling them , dependent on their location within the corticothalamocortical network . indeed some mglur 's enhance swds , while mglu1 and mglu5 receptor agonists and especially the positive allosteric modulators of group i receptors might be good candidates to control absence seizures . reduction of the number of d1-like da receptors in the shell of the nucleus nacb and in the nucleus caudatus , and a reduction of d2-like da receptors number in nucleus caudatus and increase of d2-like da receptors number in motor , somatosensory , and parietal cortex . reduction of d2-like da receptors number in nucleus caudatus causes an enhancement of gabaa receptor activity in the striatum , gp , and rtn ; they are responsible for sleep spindle generation and other rhythmic activity in tc networks . high number of somatodendric d2-like da receptors in somatosensory cortex inducing the endogenous processes of phosphorilation and decrease of gabaa receptors activity in somatosensory cortex , causing endogenous decreasing of swd activity in the cortical focus , which might trigger and entrain thalamic rhythmic firing and oscillations in the form of swds . we assume that the enhanced d2-like da receptors reflect a compensated deficiency . despite process of compensation , antagonists of d2-like da receptors can raise activity gabaa receptors both in a somatosensory cortex and in the rtn , raising swds . deficiency of wag / rij rats mesolimbic da systems , caused by reduction d1-like da receptors in the shell of the nucleus nacb , causes depression - like behavior in animals , weakens the formation of emotional positive state , worsens processes of reinforcement and counteracts learning and memory processes . a pharmacological increase of the da system removes the negative consequences of the deficiency of mesolimbic da system . the question arises , if absence epilepsy is due to disturbances or mutations of gabaa receptors and low - threshold t - type ca channel what is the role of da receptors in it . we propose two mechanisms of infringement of the function of the da system during absence epilepsy . we believe that the infringement of the function of the da system by intraneuronal integration triggered by transduction signal is carried out in the brain , especially in structures in which there are no da neurons , but da terminals are present such as cortex , striatum , gp , thalamus , and nucleus nacb . we believe that the infringement of the function of the da system by intraneuronal integration triggered by transduction signal is carried out in the brain , especially in structures in which there are no da neurons , but da terminals are present such as cortex , striatum , gp , thalamus , and nucleus nacb . we suggest that diminished activity of da receptors and da system deficits occur due to disruption of intracellular integration triggered by a transduction signal . the initial disruption of gabaa receptor activity disrupts the transduction signal of this receptor in the first stage . disruption of the transduction signal changes and modifies other receptors and their activity . on the second stage it should be noted that we did not see changes of da concentrations in structures investigated , but we could see the changes of receptors activity . so , a process of intracellular integration may disrupt activity of other neurotransmitter and neuromodulatory systems , for instance the activity of the opioid system is disrupted in wag / rij rats.(2)gabaa receptors mediate trophic effectson embryonic brainstem monoamine neurons . gaba is present in the mammalian brain during early stages of development , where it may act as a trophic signal for developing neurons . in the embryonic rat , gaba axons project through the brainstem this raises the possibility that gaba could exert trophic influences on developing monoamine neurons , if they express the appropriate receptors . in adult rat brain , the initial disruption of gabaa receptor disrupts the trophic influences on developing monoamine neurons and induces pathological changes in monoaminergic systems of adult animals . gaba is present in the mammalian brain during early stages of development , where it may act as a trophic signal for developing neurons . in the embryonic rat , gaba axons project through the brainstem this raises the possibility that gaba could exert trophic influences on developing monoamine neurons , if they express the appropriate receptors . in adult rat brain , the initial disruption of gabaa receptor disrupts the trophic influences on developing monoamine neurons and induces pathological changes in monoaminergic systems of adult animals . the first possible mechanism , intraneuronal integration triggered by transduction signal , operates during the whole life and , possibly , collects as a snow clod . the second mechanism , the infringement of the da system 's function on absence epilepsy and the mediation of trophic factor by gabaa receptors , operates during a limited period of time corresponding to the period of embryonic development .

the absence epilepsy typical electroencephalographic pattern of sharp spikes and slow waves ( swds ) is considered to be due to an interaction of an initiation site in the cortex and a resonant circuit in the thalamus . the hyperpolarization - activated cyclic nucleotide - gated cationic ih pacemaker channels ( hcn ) play an important role in the enhanced cortical excitability . the role of thalamic hcn in swd occurrence is less clear . absence epilepsy in the wag / rij strain is accompanied by deficiency of the activity of dopaminergic system , which weakens the formation of an emotional positive state , causes depression - like symptoms , and counteracts learning and memory processes . it also enhances gabaa receptor activity in the striatum , globus pallidus , and reticular thalamic nucleus , causing a rise of swd activity in the cortico - thalamo - cortical networks . one of the reasons for the occurrence of absences is that several genes coding of gabaa receptors are mutated . the question arises : what the role of da receptors is . two mechanisms that cause an infringement of the function of da receptors in this genetic absence epilepsy model are proposed .

1. Introduction 2. Hyperpolarization-Activated 3. GABAergic Inhibitory System 4. Excitatory Glutamatergic System 5. Dopaminergic System of the Brain 6. Conclusions

swds , as can be found in wag / rij rats , have a local cortical origin in the perioral region of the somatosensory cortex [ 612 ] , which is now confirmed in gaers [ 13 , 14 ] , another well validated and often used strain of rats with absence epilepsy [ 15 , 16 ] . at the age of six months , the model has some features , which further increase the interest.wag/rij have a changed expression of genes coding for the low threshold t - type ca channel ( ica , t ) compared to aci control rats and absence epileptic patients have a mutation of genes coding for ica , t [ 4446].reduction of expression of the 3 subunit protein in gabaa receptors in the rtn of wag / rij rats and mutation of genes coding for subunits of gabaa receptors in people with absence epilepsy were found [ 48 , 49].wag / rij rats have complex phenotype : they are comorbid for depression [ 5052 ] . reduction of expression of the 3 subunit protein in gabaa receptors in the rtn of wag / rij rats and mutation of genes coding for subunits of gabaa receptors in people with absence epilepsy were found [ 48 , 49 ] . we assume that the ih pacemaker channels localized in the somatosensory cortex contribute to the initiation of the occurrence of swds and that a second set of pacemaker channels can be found in the thalamus . the latter is responsible for the occurrence of thalamic delta activity and sleep spindles , as can be assumed from the classical studies of the firing pattern in thalamic relay cells , tonic and burst firing modes [ 53 , 54 ] . hyperpolarization - activated cyclic nucleotide - gated cationic if or ih pacemaker channels maintain spontaneous periodic activation , and they were discovered in heart ( if ) and brain ( ih ) . studies aimed at elucidating the role of ih channel activity in absence epilepsy compared ih channels in cortical neurons of epileptic and control strains of rats : wag / rij , wistar ( of the same genetic background as wag / rij but less prone to absence epilepsy ) and aci rats ( an inbred strain practically devoid of epileptic activity ) [ 73 , 79 , 80 ] . this loss of neocortical hcn1 function may contribute to an increased cortical excitability since there are substantially fewer hcn1 subunits in the combined complex of the ih channel in the cortical zone containing the focal region in wag / rij strain compared to the two control strains . thus , loss of function of dendritic hcn1 channels in layer 5 pyramidal neurons provides a somatodendritic mechanism for increasing the synchronization of cortical output , and is therefore likely to play an important role in the generation of swds accompanying absence seizures . it has been shown that neonatal handling and maternal deprivation postnatally ( 121 days ) resulted in reduced number of absences , decreased interspike interval , and changes in power of the frequency spectrum of swds in adult wag / rij rats . whole cell patch - clamp recordings from the cells of the fifth pyramidal layer , in situ hybridization , and western blot analysis of the cortex of adult wag / rij rats showed an increase in the hcn1 protein level in the somatosensory cortex of handled and mother - deprived rats as compared to control rats . an increased number of hcn1 subunits in the channel due to their extensive expression ( increased hcn1 protein level without changes in hcn2 , hcn3 , and hcn4 mrna levels ) , and consequently , a decreased number of hcn2 , hcn3 , and hcn4 subunits in the channel complex , as mentioned above [ 68 , 69 ] , result in impaired activity of the ih channel , reduced cortical excitability , and less swds , as it has been demonstrated experimentally [ 73 , 74 , 81 ] . the in vitro studies done in the late eighties and in the nineties of the last century were influential for the ideas than have been developed on the role of the interaction between ih and ica , t . the weakening of the binding of camp to hcn channels in the thalamus proceeds , and likely promotes , epileptogenesis in gaers rats , whereas compensatory mechanisms stabilizing the function of ih channels contribute to the completion of the swds in chronic absence epilepsy . our hypothesis that burst firing in tc neurons is caused by ( tandem ) hyperpolarization - activated ih channel and hyperpolarization - activated ( ica , t ) channel and that burst firing of at least some tc cells plays a role in swd occurrence , is supported by pharmacological studies indicating that increased gabaergic inhibition in the ventral basal ( vb ) complex of the thalamus , as induced by local injections of gaba - agonists , enhances swds . since progesterone is rapidly metabolized in the brain to the positive modulator of gabaa receptor allopregnanolone , which is also known to increase swds in wag / rij rats , it is rather likely that the epileptoformic effects of progesterone are mediated through this metabolite . allopregnanolone and ganaxolone ( the two positive allosteric modulators of the gabaa receptor complex acting on the steroid recognition site ) and pregnenolone sulphate , ( the negative allosteric modulator of gabaa receptors and a positive modulator of nmda receptors ) were investigated . it is obvious that their results voted against a predominant or even exclusive contribution of gabab receptors to spontaneous swds in thalamic relay nuclei in the wag / rij strain , but rather point to a critical role of gabaa receptor activation . these clinical cases were predicted based on results in the genetic absence models : effects of tiagabine in various doses were investigated on eeg , two types of swds , and behavior of wag / rij rats . the increase of swds by gaba agonists confirms our assumption that the hyperpolarization - induced ih pacemaker in the thalamus contributes to the occurrence of swds . the effect of ghba - induced swds on the function of various components of the gabaa receptor complex in the cortex of the rat was determined in a series of in vitro experiments . the high level of cl conductivity during nonconvulsive ptz kindling suggests that the activity of the gabaa receptor was similar in the genetic absence epilepsy model and ptz absence seizure model . hence , signal transduction plays an important role at the long - term storage of information , in learning and memory , in training , in neuroplasticity in general , and in pathological states . the combination of immunocytochemistry and high - resolution immunogold electron microscopy was used to study cellular and subcellular localization of gabaa receptors 1 , 3 , and 2/3 subunits in ventral posterior nucleus ( vp ) and rtn of wag / rij and control wistar rats . thus , one of the reasons of the occurrence absence epilepsy is the mutation of several genes coding of gabaa receptors which causes abnormal functioning of the gabaergic system . showed that the center in the perioral region of the primary somatosensory cortex takes the lead or has a more considerable position in generation swds and that the local strengthening of gabaa receptor activity in rtn strengthens swds . symptomatic wag / rij rats showed an increased expression of mglu2/3 receptors in the ventrolateral regions of the somatosensory cortex , ventrobasal thalamic nuclei , and hippocampus , but not in the rtn and in the corpus striatum , as assessed by immunohistochemistry and western blotting . postsynaptic gabaa receptors are located on dendrites and the soma of the striatal medium spiny neurons , and also on neurons of the globus pallidus , substantia nigra pars reticulate , and thalamus . for example , activation of the mesocorticolimbic da system of ventral tegmental area ( figure 7 , a10 ) lateral hypothalamus [ 283285 ] , nacb [ 286 , 287 ] , prefrontal cortex [ 288291 ] , and anterior cingulate cortex [ 292 , 293 ] , induces an emotional positive state ( pleasure and happiness [ 294297 ] ) , a state referring to its own prolongation and/or intensification ( intra - cranial - self - stimulation [ 283 , 285 , 286 , 288 , 290 , 298 ] , self - administration of drugs [ 290 , 292 , 299 ] , self - control of drug administration , or place - preference paradigm [ 299301 ] ) . switching of neuron activity in other functional state , through intracellular integration with the help of transduction signal and modification of ionotropic and metabotropic receptors and potential sensitive ionic channels activity , it is possible to explain the role of the opioid [ 285 , 298 ] , gabaergic [ 284 , 287 ] , acetylcholinergic , serotoninergic [ 286 , 287 ] , and other systems in the control of the mesocorticolimbic daergic reinforcement system . behavioral studies indicate that wag / rij rats exhibit depression - like symptoms : decreased explorative activity in the open field test , increased immobility in the forced swimming test , and decreased sucrose consumption and preference ( anhedonia ) . these results allow the assumption that the wag / rij strain possesses a deficiency of the brain 's da system activity . it is possible to assume that control of swds by d2-like da receptors is carried out through an indirect gabaergic pathway from the dorsal striatum which terminates in the rtn through pathways passing through the gp ( figure 8) , one of the sources of rhythmic oscillations . antagonists of d2-like da receptors enlarge activity of gabaa receptor and increase swd activity , and agonists reduce gabaa receptor activity and reduce swds ( see section 3.1.1 ) . based on the electrophysiological and lesion studies in wag / rij rats it is proposed that there is no direct source of swd activity in the rtn , however , bursting neurons in the rtn are a necessary condition for maintenance and spreading of swd [ 13 , 20 , 99 ] . a higher number of active avoidance responses in the first day of learning and an amnesic effect in the second day of learning in wag / rij versus wistar rats ( figures 9(c ) , 10(a ) , and 10(c ) ) was reproduced three times in our experiments . it was also shown that a specific peptide of the proenkephalin system was increased in the striatum , the frontal cortex , and the mesencephalon [ 354 , 355 ] , structures directly or indirectly controlling inhibitory processes in corticothalamocortical neuronal networks [ 21 , 279 , 280 ] . in all , a deficit of da system in wag / rij rats and an increase in the sensitivity to a da agent as the response on the deficit is proposed to be the causative factor for the behavioural differences in wistar and wag / rij rats . the increase in the number of d2/d3 da receptors in motor , somatosensory , and parietal cortex was due to an amplification of downregulating effect of daergic transmission , induced by dephosphorilation . the low number of somatodendric d2-like da receptors in the caudate nucleus induces endogenous processes of dephosphorilation and enhancement of gabaa receptors activity in striatum , gp , and rtn ( figure 8) and is an endogenous cause for the increase of swd activity in the corticothalamocortical network . the high number of somatodendric d2-like da receptors in somatosensory cortex induce the endogenous processes of phosphorilation and decrease of gabaa receptors activity in somatosensory cortex , forming an endogenous cause for decreasing swds in the cortical focus , which supervises an oscillatory drive in the rtn . that is why in wag / rij a compensated deficiency of d2-like da receptors but d2-like da receptors antagonists ' administration both in cortex and caudate nucleus enhances processes of dephosphorilation and gabaa receptors activity amplify , enlarging activity of swds . a deficiency of mesolimbic ( nacb shell ) daergic activity at the level of somatodendritic d1-like da receptors in wag / rij versus to aci rats was found . the deficiency of nigrostriatal daergic system by reduction of d2-like da receptors is at the bottom of an additional rising activity of swds . behavioural changes and activity of the mesolimbic daergic systems in wag / rij rats are influenced by activation of d1 da receptors in the shell of the nacb [ 380 , 381 ] and prefrontal cortex [ 382 , 383 ] , the main sources of positive reinforcement . reduction of d2-like da receptors number in nucleus caudatus enhances the activity of gabaa receptors in striatum , gp , and rtn , causing a rise in swd - activity in corticothalamocortical networks . high number of somatodendric d2-like da receptors in somatosensory cortex inducing the endogenous processes of phosphorilation and decrease of gabaa receptors activity in somatosensory cortex , causing endogenous decrease of swds in cortical focus , which supervises an oscillatory source in the rtn . deficiency of wag / rij rats mesolimbic da systems , caused by reduction d1-like da receptors in the shell of the nacb causes depression - like behavior of animals , weakens formation of emotional positive state , worsens processes of reinforcement , and counteracts learning and memory processes . the question arises , if absence epilepsy is related to disturbances or mutations of gabaa receptor and low - threshold t - type ca channel , then what is the role of da receptors in it . we suggest two mechanisms of infringement of the function of the da system during absence epilepsy.gabaa receptors mediate trophic effectson embryonic brainstem monoamine neurons . we believe that infringement of the function of the da system by intraneuronal integration triggered by transduction signal occurs in the brain , especially in structures in which there are no da neurons , but da terminals , such as cortex , striatum , gp , thalamus , nucleus nacb , and so on . we believe that infringement of the function of the da system by intraneuronal integration triggered by transduction signal occurs in the brain , especially in structures in which there are no da neurons , but da terminals , such as cortex , striatum , gp , thalamus , nucleus nacb , and so on . earlier it has been shown that the development of pentylenetetrazole ( ptz ) kindling ( 4-month - old animals ) is accompanied by 20% reduction of the density ( bmax ) of gabaa receptor benzodiazepine ( bdz ) sites . the increased density of bdz site of gabaa receptors in kindled versus control rats can be interpreted as an enhancement of gabaergic inhibition , while it is thought that seizures are based on the process of neuronal hyperactivation accompanied by a reduction in the bdz site density both in kindling - induced and single - dose ptz - induced seizures . functional interactions of the striatal glutamate and da receptors in learning and memory [ 279 , 305 , 306 , 434 , 435 ] , and postsynaptic integration of glutamatergic and da signals in the striatum based on interactions of intracellular second messengers [ 272 , 273 , 279 , 436438 ] have been reported . if the first possible mechanism of infringement of the function of the da system , mediation of trophic factor by gabaa receptors , operates during a limited time period corresponding to the period of embryonic development , the second mechanism , intraneuronal integration triggered by transduction signal , operates during the whole life and , possibly , collects as a snow clot . analysis of the experimental data shows that one of the basic mechanisms for the long - term regulation of ih pacemaker activity is the modification of the number of hcn1 subunits in the pacemaker channel . it has been assumed that the source of swds is the ih pacemaker channel that is localized at pyramidal cells in the perioral region of the somatosensory cortex ( in wag / rij rats ) , more specifically layers iii - vi . the density of a subtype of hcn channels ( hcn1 ) in the apical dendrites of the subgranular layers in this onset zone determines the somatodendritic excitability and a decrease of these dendritic hcn1 channels neurons increases the occurrence of absences . however , the measurements of the efficiency of gabaergic system lead to diverse results first , different models of absence epilepsy such as gaers and wag / rij rats , various types of mice with spontaneous mutations , and knock - out lines have been used . one of the reasons for the occurrence of absence epilepsy is the mutation of several genes coding for gabaa receptors which cause abnormal functioning of gabaergic system . reduction of the number of d1-like da receptors in the shell of the nucleus nacb and in the nucleus caudatus , and a reduction of d2-like da receptors number in nucleus caudatus and increase of d2-like da receptors number in motor , somatosensory , and parietal cortex . reduction of d2-like da receptors number in nucleus caudatus causes an enhancement of gabaa receptor activity in the striatum , gp , and rtn ; they are responsible for sleep spindle generation and other rhythmic activity in tc networks . high number of somatodendric d2-like da receptors in somatosensory cortex inducing the endogenous processes of phosphorilation and decrease of gabaa receptors activity in somatosensory cortex , causing endogenous decreasing of swd activity in the cortical focus , which might trigger and entrain thalamic rhythmic firing and oscillations in the form of swds . deficiency of wag / rij rats mesolimbic da systems , caused by reduction d1-like da receptors in the shell of the nucleus nacb , causes depression - like behavior in animals , weakens the formation of emotional positive state , worsens processes of reinforcement and counteracts learning and memory processes . the question arises , if absence epilepsy is due to disturbances or mutations of gabaa receptors and low - threshold t - type ca channel what is the role of da receptors in it . we propose two mechanisms of infringement of the function of the da system during absence epilepsy . we believe that the infringement of the function of the da system by intraneuronal integration triggered by transduction signal is carried out in the brain , especially in structures in which there are no da neurons , but da terminals are present such as cortex , striatum , gp , thalamus , and nucleus nacb . we believe that the infringement of the function of the da system by intraneuronal integration triggered by transduction signal is carried out in the brain , especially in structures in which there are no da neurons , but da terminals are present such as cortex , striatum , gp , thalamus , and nucleus nacb .

cancer is a major health problem worldwide and imposes a great economic and psychological burden in addition to loss of life and fertility . colorectal cancer ( crc ) is one of the most common types of cancer affecting 1.23 million individuals per year ( 9.7% of overall cancers ) and is the fourth most common cause of death from cancer worldwide ( 608,000 cases , 8% of overall cancer deaths ) . the incidence of crc has been estimated to be 3050 cases ( per 100,000 ) in northern america and europe and 37 cases ( per 100,000 ) in most middle east countries . although epidemiology data show a marked variability around the world , and almost 60% of cases occur in developed countries , its overall incidence rate shows a slow but steady decrease ( about 2% per year ) in developed countries . conversely , in developing countries and most of asian ones , the annual incidence is unfortunately expected to increase over the next two decades . however , the proportion of crc cases occurring among younger age group ( 40 years ) is 28 percent in western countries where it is about 1535 percent in the middle east region [ 13 ] . a large number of epidemiologic studies have been performed to investigate specific hypotheses about risk factors of colorectal cancer , particularly for diet , as western diet ( high meat and energy and less fruit , vegetable , and fiber intake ) , which may have a role in g e ( gene and environmental ) interaction . some approaches specifically reveal a correlation between folate and the mthfr ( methylenetetrahydrofolate reductase ) gene in red meat with a series of metabolic genes . according to who classification of tumors of the digestive system , the following factors may play an etiologic role in the development of crc , including sedentary lifestyle and obesity , high body and abdominal fatness , hormone replacement therapy , tobacco smoking , alcohol , and nonsteroidal anti - inflammatory drugs [ 46 ] . however , polyps and high risk bowel syndromes such as inflammatory bowel diseases , especially ulcerative colitis , must be noted as a risk factor for crc in general population . it has been proved that about 25% of crc patients have some degrees of familial background , and a strong family history as a first or second degree relation is seen in about 15% of other patients with colorectal cancer . sporadic or nonhereditary colorectal cancer occurs in people who have no ( or very little ) family history of crc and in patients who have no identifiable genetic risk . the majority of crcs ( 7085% ) are sporadic and they are believed to be influenced by diet , lifestyle , environmental factors , and acquired somatic mutations [ 2 , 6 ] . its characteristics are the stepwise progression from normal epithelium to carcinoma , associated with sequential molecular abnormalities in each step . the origin of the disease may be attributed to the presence of a large number of genetic variants that may act according to a polygenic model . however it is now recognized that there are two distinct forms of familial colorectal cancer syndrome , whose inheritance is diagnosed as autosomal dominant trait . in familial adenomatous polyposis ( fap ) and its subspecies , gardner syndrome , at risk individuals develop thousands of polyps over the course of their lives . the reported incidence of familial polyposis varies from 1 in 7,000 to 1 in 22,000 individuals . virtually all patients with fap will develop crc at a medium age of about 40 , and , because of the diffuse nature of the polyposis , the best way for prevention of crc is surgical therapy [ 3 , 911 ] . in contrast , hereditary nonpolyposis colorectal cancer ( hnpcc ) could be defined in terms of the pattern of familial aggregation . its frequency is about one in 10,000 , and it includes almost 24% of colorectal cancers . hnpcc is a group of five familial cancer syndromes ( hnpcc 1 to hnpcc 5 ) , which occur due to mutation in 5 independent genes involved in dna mismatch repair . the same genes might be involved in the etiology of both hereditary and sporadic cancers , with the difference that , in hereditary cancers , one copy of a mutant allele is inherited in the germline and the other mutation occurs somatically , while in sporadic cancers , both mutations are acquired somatically [ 3 , 912 ] . over the past two decades , molecular studies have led to a tremendous increase in our knowledge of genetic changes that affect malignancy in crc . this has enabled us to better characterize tumors individually and classify them according to certain molecular or genetic features . however , mutation detection methods with high sensitivity will increase the possibility of choosing the correct individual therapy and reduce the risk of metastasis in low stage diseases . a better understanding of the causality of crc can be established by combining epidemiology and research on molecular mechanisms . the pathogenesis of colorectal cancer is complex and diverse , with different molecular pathways , and in fact leading to different phenotypes . crc develops through a series of events that lead to the transformation of normal epithelium to adenoma and then to carcinoma . it is the most common type of crc pathways , which accounts for 85% of all crcs and 6570% of sporadic colorectal cancer . this pathway includes the following : activation of protooncogenes by mutation , including kras ( kirsten rat sarcoma viral oncogene homolog 0 ) , c - src ( tyrosine - protein kinase src ) , and c - myc ( avian myelocytomatosis viral oncogene homolog),inactivation of at least three tumor suppressor genes , as apc ( adenomatous polyposis coli ) , tumor suppressor tp53 gene , and subsequent loh ( loss of heterozygosity ) of chromosome 18q . activation of protooncogenes by mutation , including kras ( kirsten rat sarcoma viral oncogene homolog 0 ) , c - src ( tyrosine - protein kinase src ) , and c - myc ( avian myelocytomatosis viral oncogene homolog ) , inactivation of at least three tumor suppressor genes , as apc ( adenomatous polyposis coli ) , tumor suppressor tp53 gene , and subsequent loh ( loss of heterozygosity ) of chromosome 18q . this pathway is driven by hypermethylation of genes and is characterized by the presence of protooncogene braf ( protein kinase b - raf ) , which causes increased mapks / erks ( mitogen activated protein kinases / extracellular signal - regulated kinases ) signaling , leading to increased cell proliferation , cell division , and secretion . cimp is found in about 2030% of colorectal cancers , based on the number of methylator markers , and can be divided into five subgroups ( figure 1 ) . this pathway is involved in the genesis of about 1015% of sporadic and > 90% of hnpcc syndrome . instability of microsatellites is a reflection of genetic hypermutability or somatic inactivation that results from inactivation of dna mismatch repair ( mmr ) genes , including hmlh1 ( human mutl homolog 1 ) , hmsh2 ( human muts homologue 2 ) , hmsh6 , and hpms2 ( human postmeiotic segregation 2 ) [ 2 , 14 , 15 ] . clinically , crcs are classified as msi - h ( high level msi , cancers with instability at around 3040% of markers ) , msi - l ( low level msi , cancers with instability less than 3040% of markers ) , and mss ( microsatellite stable , cancers with no apparent instability ) based on the extent of msi . braf mutation is frequently seen in sporadic msi - h crc , but not in hnpcc . indeed , in sporadic settings , msi - h crcs are not mostly due to epigenetic silencing of the hmlh1 gene promoter [ 14 , 15 ] . msi pathway can be considered an early event in the adenoma carcinoma sequence and in ulcerative colitis associated neoplasia . targeted cancer therapy is becoming a powerful strategy for the treatment of patients selected on the basis of their molecular characteristics . a mutant kras gene has been associated with resistance to immunotherapy using monoclonal anti - egfr ( epidermal growth factor receptor ) antibodies ( i.e. , cetuximab and panitumumab ) . the clinical diagnosis of metastatic crc currently involves screening of tumor tissue for kras mutations so that patients with wild - type kras - containing tumors are only given anti - egfr treatment . in fact , patients with wild - type kras often have better response with cetuximab while those with kras mutation or with tumors of unknown kras gene orientations are often responsive to bevacizumab . targeted anti - egfr treatment has led to a considerable improvement in disease control and survival rates . information pertaining to kras mutation status in patients appears to be effective in choosing target factors for therapy . a survey conducted in 14 european , latin american , and asian countries revealed increasing use of kras test from 3 percent in 2008 to 69 percent in 2010 . screening strategies include the diagnosis of precancerous polyps and cancer in asymptomatic individuals which may help the prevention of malignancy [ 19 , 20 ] . previous studies have shown that use of screening strategies can reduce about 60 percent of the deaths resulting from crc [ 20 , 21 ] . moreover , the administration of crc screening programs in the united states has led to considerable reduction in cancer - associated death rates . current regulations suggest that screening for crc should be initiated in the general population for 50 years old and more , and for the high risk group from a younger age . the regulations for prevention of diseases in the united states require that every man or woman aged 50 years or above should be screened for crc using one of the screening tests including occult blood test annually , sigmoidoscopy every 5 years , barium enema every 5 years , or colonoscopy every 10 years . screening is performed using the colonoscopy as a gold standard to determine the prognosis of adenoma of the colon and crc . previous studies have shown that the use of colonoscopy for screening for crc results in reduced danger of cancer - related mortality due to the removal of precancerous polyps . given that family members of crc patients are at increased risk for the disease , screening through colonoscopy is usually recommended for the closest relatives ( father , mother , sister , brother , and children ) of patients with crc or adenomatous polyps . the new regulations propose that screening methods should be initiated from the age of 40 years or 10 years younger than the age of the first patient diagnosed in a family . according to the iranian national cancer registry report , the incidence of crc has increased during the last 25 years showing that the distribution of crc has shifted towards lower age groups in the country . in addition , iran still faces challenges regarding the access to anticancer medicines , especially costly anti - egfr - targeted cancer therapy protocols . so despite high cost of molecular techniques , it is obvious that screening of crcs who are candidates for these therapies by detecting of mutations in kras and braf genes is recommended , particularly to those who are not likely to benefit for avoiding unnecessary costs . in general , it is now clear that routine molecular approaches of crc including kras and braf testing are cost saving . to set up an efficient screening procedure , the prevalence of disease , demographic characteristics of the population , possibility of early diagnosis , availability of new treatment modalities for patients with positive screening tests , and the cost - benefit of the whole procedure will have to be determined if risk assessment tool needs to be conducted this review study was conducted to document the epidemiologic features of crc in iran : age standardized incidence , survival rates , prognostic factors , burden of disease , heredity pattern , occurrence of sporadic cases in relatives , and so forth . we also give a report on the molecular routes and cellular metabolism , mutation in kras and braf genes , and other tumor markers in the country . to retrieve published studies and to identify the studies of interest , we conducted a comprehensive literature search . the databases medline , embase , sid ( scientific information database ) , magiran , iran medex , and google scholar were searched for english and persian articles from year 2000 to recently published studies . as for search criterions , we used medical subject headings including colorectal cancer , molecular genetics , kras mutation , braf mutation , screening , survival , epidemiologic study , and iran to retrieve the relevant evidence . we did a careful review on the full text of the articles retrieved for this study . the reference lists of articles were also reviewed to identify any additional relevant resources on the same search area . recent figures published by the iran ministry of health showed that cancer is the third common cause of death in the country . it is now ranked in the third place after heart diseases and accidents with an increasing incidence over the past few decades in the population [ 24 , 25 ] . the latest data by the iran national cancer registry ( incr ) reported an annual number of 51,000 cases of cancer with about 35,000 cancer - related deaths in the country . this is the second highest in the eastern mediterranean region according to the world health organization reports . the same report shows that crc was the fourth most common cancer in iran between 2000 and 2009 [ 26 , 27 ] , with a five - year survival rate between 43 and 49 percent [ 2830 ] . the annual report of the incr shows that crc is the fourth common cancer in men after stomach , bladder , and prostate . it is however the second one among women after breast cancer [ 13 , 26 , 31 , 32 ] . a research study in 2008 reported an asr of 7 - 8 ( per 100,000 ) for both sexes this is higher than that of previous reports in the country while closer to the statistics reported from other middle east areas and less than the similar rates in western countries [ 1 , 32 ] . reported that the overall asr during a four - year period ( 20082012 ) was 38 ( per 100,000 ) with higher rates in men ( 39.96 ) compared to women ( 36.16 ) . asr among individuals younger than 50 years was also estimated 8.26 ( per 100,000 ) in the same report . adenocarcinoma was the most frequently detected type of colorectal carcinoma and most common tumor diagnosed in the colon . an asr of 5.9 and 10.7 has been reported from female population of ardebil and males in golestan , respectively . in each province , crc is ranked second to fourth representing 68 percent of all types of cancers . according to somi et al . from northwest of the country in 2014 , annual asr was estimated 11.2 and 8.93 in men and women , respectively . the crude rates for men and women were reported 11.5 and 9.22 , respectively , in the same study . the occurrence of crc has recently shown a slightly decreasing trend compared to the 2008 data [ 31 , 35 , 36 ] . crc is normally a disease of aged people occurring in individuals over 65 years . however , as indicated before , several epidemiological studies in iran have shown that the proportion of crc among the younger population is considerably higher compared to western populations . early crc ( in less than 40 years ) accounts for almost one - fifth of all cases of crc in iran , compared with high risk countries , with rates ranging from 2 to 8% [ 1 , 13 , 3744 ] . this might be attributed to two factors : a high proportion of young population in iran and a relatively low rate of crc in aged people in the country . almost all economic indices show a considerable improvement in living conditions in iran over the past few decades ( statistic center of iran , 2007 , world bank report , 2006 ) . this has been accompanied by considerable changes in lifestyle including sedentary lifestyle and a diet rich in fat and meat and poor in grains and fiber . the recent trends of crc in youth in iran could be a result of these changes in the diet pattern in this generation towards western lifestyle in recent decades . it is therefore expected that the occurrence of crc in iran will probably be similar to those of western countries in the forthcoming decades . some studies showed that genetic factors may probably play a role in the development of crc among young population in iran , as well . this suggests that crc in iran could be caused by different factors both environmentally and genetically [ 33 , 45 ] . the prognosis of crc depends on various factors associated with tumor characteristics , patient condition , diagnosis , and treatment of the disease . advanced stages of cancer , rectal localization of tumor , pathology grade , and patient 's performance , vascular invasion , older age , and genetic instability such as amplification of certain genes are the factors that correlate with the worst prognosis of crc patients at diagnosis . the surgical technique and proper use of adjuvant and neoadjuvant treatments considerably affect the overall survival of patients [ 30 , 46 , 47 ] . the results of study mentioned by haghighi et al . showed that overall 5-year survival of hnpcc patients was higher than sporadic ones ( p = 0.044 ) , and they compared their data with other similar studies . even patients with sporadic crc had a risk of death about threefold of those with hnpcc . these differences are probably due to different clinical pathological characteristics of neoplasia and genetic alteration [ 48 , 49 ] . survival also varies greatly with the geographical and economic status , host response , the accuracy and availability of diagnostic procedures , and availability of treatment . poor survival with advancing age was observed in a few studies from iran , which may be attributed to poor general health condition and limitations of cancer therapies in the elderly [ 49 , 50 ] . however , the results of a population - based cohort study from golestan in iran , where more than 42% of crcs were under 50 years of age , revealed a worse survival in young crc patients , more likely because their cancer is diagnosed in advanced stages . finally , race and ethnicity as measures of socioeconomic status have a major impact on crc incidence rates , tumor characteristics , mortality rates , and survival rates . a research study showed that the one- , two- , three- , four- , and five - year survival rates of crc were 84 , 68 , 54 , 43 , and 41 percent , respectively . men had poor prognosis compared to women where the five - year survival rate was 45 and 88 percent in men and women , respectively . the poorest prognosis was observed in those younger than 20 years and older than 80 years . the overall five - year survival rate for crc patients in iran ( 41 percent ) was similar to those from other developing countries while it was lower compared to developed world where advanced health care systems and population - based screening programs exist . the epidermal growth factor receptor ( egfr ) plays a key role as a receptor of tyrosine kinase ( tk ) . egfr mutations have been detected in several types of cancer . a study in iran on locally advanced rectal cancer has shown the expression of egfr functions as a predictor of response to treatment in these tumors . they can thus be used in the recognition of a subgroup of patients who are likely to be unresponsive to this method of treatment . mutations in kras and braf genes are considered important primary incidents in the development of crc . it shows strong association with the incidence of methylguanine in promoter regions of various genes in crc . while kras has been investigated as a unique marker , simple or multivariate studies did not indicate any effects on progression - free survival ( pfs ) . however , existing evidence indicates that patients with metastatic tumors as well as kras mutation show worse prognosis than patients with wild - type kras . similarly , existing evidence failed to implicate braf mutation as the sole predetermining factor for pfs . there is , however , sufficient evidence showing that mutation in braf gene is a negative prognostic marker for overall survival of patients . mutation in kras gene is one of the most common oncogenic changes in various types of human cancer . mutation in kras gene occurs early in the development of cancer and may therefore play an important role in several stages of cancer progression and development of malignancy including the initiation of neoplasia and metastasis and prediction of prognosis [ 50 , 51 ] . mutation in codons 12 , 13 , and 61 of kras is common in crc . this produces an active kras protein resulting in the activation of the mapk ( mitogen activated protein kinase ) cascade independently of egfr activation . recent reports have shown that mutation in codon 12 is observed at frequencies of 1230 percent and 3550 percent among crc patients in asian and western countries , respectively . various frequencies of mutations in different populations may be attributable to the effect of environmental factors and food habits as well as genetic factors . kras mutation is mostly observed in right - side tumors , low - grade or msi - low ( microsatellite instability - low ) and mss ( microsatellite stable ) tumors . it does not seem to have an impact on the prognosis or progression - free and overall survival rates . although kras mutations have been widely studied in crcs in western countries , there are few data on kras mutation and spectrum in crcs from iran . a study in iran by bishehsari et al . reported 37.4 percent of crc patients with kras mutations . this rate is within the range of 2550 percent reported in studies of other crc series . codon 12 was the most frequently mutated ( 66 percent ) , followed by codon 13 ( 32.5 percent ) , while codon 61 was only mutated in one of 182 tumors in this investigation . this confirms that codons 12 and 13 are preferentially involved in crc progression in iranian population , as well . they found an overall rate of kras mutations in the italian series ( 46.3 percent ) which is higher than that found in iranian crcs . although mutations in codon 12 were similar in both series , codon 13 was more frequently detected in crcs from italy . another study from iran in 2010 reported that 20.3 percent ( 12 cases ) of crcs ( 10 in codon 12 and 2 in codon 13 ) have shown a point mutation . braf is a cytoplasmic serine - threonine kinase that mediates cellular response to growth signals of egf through the ras / raf / mapk pathway . the activation of braf gene through mutation has been identified in 515 percent of sporadic cases of crc . more than 80 percent of the mutations are in the hot spot of exon 15 resulting in the v600e mutation in codon 600 . efforts to identify other mutations in tumors that would facilitate the identification of patients who would be more responsive to anti - egfr immunotherapy have revealed that an activating mutation in braf gene among patients with wild - type kras is likely to result in a weak response . crc on the right side along with braf mutation and microsatellite stability leads to a definite weak prognosis for overall as well as progression - free survival . braf mutation is often observed in tumors on the right side in patients of 60 years and more , in women , and also in msi - high or highly malignant tumors . a study in iran by naghibalhossaini et al . showed the absence of any braf mutation among the iranian population supporting the observations of brim et al . that minimum mutation rates of this gene are found among patients in iran ( 2 percent ) compared to those in amman ( 19 percent ) and african - americans ( 10 percent ) genetic or environmental factors are believed to be responsible for the lack of braf mutation in crc patients in iran [ 5 , 51 , 56 ] . p53 is a tumor suppressor gene which is believed to play a role in the regulation of cell proliferation , involved in dna repair , cell cycle gene transcription , and apoptosis . when the dna damage can not be successfully repaired , p53 initiates the apoptosis , and cell dies with the help of cell death genes . mutations in tp53 are major genetic alterations involved in crc progression . actually , mutation in p53 is the most frequently detected genetic alteration in most human cancers . p53 also interacts with cytooxygenase-2 ( cox-2 ) , indicating that cox-2 could be an independent prognostic factor in crc . the most frequently mutated region of p53 gene is exons 5 to 8 of the human tp53 . also it is believed that some mutations in this gene lead to resistance to common treatment protocol chemotherapy drugs in crc patients , so an effective treatment can be planned after p53 mutation testing . previous studies showed overexpression of mutant p53 in 5963% of patients with colorectal cancer using ihc method ( immunohistochemical ) , while sequencing of amplified dna samples revealed the rate as 2344% [ 5860 ] . according to results of malekzadeh et al . , p53 gene mutation in iranian crcs occurs as frequent as in other series , but proximal and distal side of colon show different p53 mutation patterns , which may suggest different tumorogenesis pathways of proximal and distal colon [ 13 , 58 ] . however , it is believed that p53 alterations are more frequent in distal than in proximal crcs , and a recent meta - analysis of case series from 17 countries showed that sites and types of p53 mutations were comparable in proximal and distal colorectal cancers [ 58 , 61 ] . adenomatous polyposis coli ( apc ) gene is a tumor suppressor gene and encodes a cytoplasmic protein which binds to -catenin and it is widely accepted that the apc tumor suppressor gene inactivation is the earliest and key event for mutation initiation in more than 80% of early colorectal cancers , and consequently it is named as gate keeper for adenoma development [ 2 , 7 , 14 ] . the majority of apc mutations in the mcr ( mutation cluster region ) introduce a stop codon , resulting in a truncated protein which lacks the binding site for two important interactants , -catenin and axin , which act together in the wnt- ( wingless type- ) signaling pathway . the location of mutation seems to be related to disease severity and presence of extracolonic manifestations in fap patients . there are a few reports about apc gene mutation in iranian fap patients , which revealed that the mutation pattern is the same as other reports from other countries . in a case series report , kashfi et al . reported frame shift mutations in exon 15 and two siblings with germline mutation at codon 849 and two faps had frame shift mutation at codon 1309 . on ten unrelated iranian fap patients identified 5 mutations at exon 15 of apc gene . it has been reported that about 30% of fap patients do not have any identifiable germline apc mutations . depending on ethnicity , geographical region , dietary , and genetic predisposition and mostly because of heterogeneous nature of colorectal cancer , apc mutation genes are considerably diverse and distinct , as well as in sporadic crcs . it occurs in 3480% of sporadic colorectal cancers , and mutation in exon 15 covers more than 75% of coding sequence . to date , some hundred mutations have been reported around the world , but some studies in iran reported a unique profile of apc mutations in iranian crcs , and the overall frequency of apc mutation was about 2530% in sporadic colorectal patients . the failure of mmr genes subsequently leads to mutations in specific target genes involved in proliferation and cellular differentiation . mmr genes lead to failure of dna mismatches repair during replication and generation bases and also encode proteins for correcting dna nucleotide base mispairs . microsatellites ( msi ) are simple dna sequences which consist of repeating unit of 15 bp , with 2560 bp in length , which are distributed throughout the genome . msi is detected on an average of 1015% of sporadic crcs and is the important underlying event in 8590% of hnpccs . the five suitable markers include bat25 , bat26 , d2s123 , d17s250 , and d5s346 . several studies showed high frequency of msi in sporadic colorectal cancer patients in iran , and bat25 , bat26 , and nr-21 were showed to be the most sensitive markers for diagnosis sporadic crcs and hnpccs [ 13 , 6669 ] . msi status in iran was observed in 23% of crc cases , and it was more frequent in females , in early - onset colorectal cancers , and in tumors located in the proximal colon ( 40% ) , with the majority of msi - h tumors , compared to the common trends in the world . moghbeli et al . detected msi in about 43% of their patients with 27% of msi - h , while bishehsari et al . analyzed 170 sporadic crcs , with 19.4% msi - h [ 53 , 67 ] . these different frequencies suggest that the molecular epidemiology of genetic alterations involved in the crc carcinogenesis has varied in the iranian population . compared with sporadic crcs , about 59% of hnpccs have shown msi in bat26 and bat25 from one report by galehdari et al . , which was considered lower than other previous findings . there was no significant relation to the stage and location in comparison with both markers , and all of them were msi - low phenotype . in another survey on 32 hnpcc iranian families , mutation of mlh1 and msh2 genes was identified in about 63% of their patients . despite increasing knowledge about genetics and biochemistry of the mmr genes , a few data about mutation screening of mmr in iranian crcs , as well as hnpcc patients , are available . indeed , while the detection of msi is simple and economical and has high correlation with the clinicopathologic feature of colorectal carcinoma , large and more executive molecular research studies are needed to know about factors that contributed to msi - associated carcinogenesis in iranian crcs . carcinoembryonic antigen ( cea ) , a serum biomarker , is routinely used in clinical practice and is considered a major tool for the control of cancer metastasis to the liver . since its identification in colon adenocarcinoma in 1965 , cea has been shown to play an important role in the attachment of cancer cells . the marker shows association with disease stage , and the levels of cea and ca19 - 9 before surgery are correlated with the disease prognosis . after the removal of the primary tumor , serum levels of cea and ca19 - 9 are serially measured every 2 - 3 months for at least 2 years for obtaining information about the cancer or its metastasis . however , no consensus has been reached regarding the extent of changes in cea levels in the assessment of higher probability of cancer onset or progression . also other findings showed that the cea level may be a suitable biomarker for predicting tumor response to specific chemotherapy in colorectal cancer . the high occurrence of crc in records of relatives of crc patients in iran shows that a considerable number of these crc patients are from families whose members or relatives have been suffering from crc in the past . showed that family history of crc is more common among young crc patients and with tumors on the right side . moreover , the incidence of crc in a close family member during their youth leads to considerably increased risk of acquiring crc ( 1530 percent ) . familial risk of colorectal cancer has been described in a number of publications . a cohort study of 32,085 men and 87,031 women conducted by fuchs et al . ( 1994 ) provided baseline data on first degree relative with crc , diet , and other risk factors . they revealed that the familial relative risk in first degree relatives of crcs was about 1.7 , rising to 2.8 if two or more first degree relatives were affected or / and to 5.4 if both relatives were less than 45 years old at diagnosis . studying the colonoscopy results , in a few studies in iran , indicated that having a family member with a history of crc , 32.7-fold , increases the risk of colorectal polyps and crc [ 7476 ] . also the incidence of adenoma among the relatives of crc patients aged 5060 years is approximately 1734 percent and lower among younger relatives [ 32 , 77 ] . another study 's results showed that the prevalence of colorectal adenoma and precancerous lesions in first degree relatives of crc patients is significantly higher than in the average risk population . according to different studies , one in every 6 ( 16 percent ) and one in every 5 ( 20 percent ) crc patients have a first degree relative ( either a parent or sibling ) suffering from crc . studies have shown that the incidence of crc in a close member of one 's family almost doubles the risk of crc for an individual and the risk of developing crc is 2.53 times greater among the relatives of crc patients . these observations highlight the need to improve efforts for enhancing public awareness and screening strategies in families with a crc patient , particularly a young patient , or with proximal tumors . screening programs may therefore begin with family members of crc patients from a young age using colonoscopy as the preferred method of screening . the increased asr observed beyond 50 years of age suggests that screening from 50 years of age onward might be appropriate for high risk groups . however , all characteristics of crc and differences unique to the given region should also be taken into account . several studies have assessed the efficiency of crc screening in individuals aged 50 years or below . a timely diagnosis and the removal of adenomatous polyps may help the prevention of crc . the diagnosis of local crc may also improve the survival rate of crc patients , and the five - year survival rate of crc diagnosed early was reported to be around 90 percent . often , crc is caused by adenomatous polyps , whose sizes vary from small ( less than 5 mm ) to large ( more than 1 cm ) and also vary between dysplasia and cancer . this development route of cancer from dysplasia probably lasts at least 10 years for a large number of individuals [ 32 , 77 ] . although the early diagnosis of crc reduces the associated death burden , administration of screening programs is not a common practice even in developed countries . previous studies showed the absence of any national screening programs for crc in iran , with very little information available on screening strategies for cancer in general in the country . in fact , a few screening programs were tested among the high risk groups for crc , which include family members of crc patients , by the center for liver and digestive system studies in the medical science and medical services department of shahid beheshti university [ 77 , 79 ] . however , some educational programs on crc screening may increase overall knowledge and motivation about colorectal cancer risk factors and screening modalities . on the whole , screening of even average risk groups for crc , who are exposed to less than acceptable risk levels , is economical . the decision making involved in crc screening in iran has proven valuable for cost saving with respect to local resources , personal preferences of the patients , or the choice of screening tests , including the highly sensitive stool occult blood test conducted annually ( such as stool immunochemistry test ) or colonoscopy conducted every 10 years . colonoscopy is both the most effective and sensitive and the most costly strategy according to latest trials in iran [ 25 , 77 , 80 , 81 ] , and many studies recommend it as golden standard tool for early detection of precancerous as well as crc [ 77 , 82 ] . as well as pathology findings , genetic history and information content on pedigree play a significant role in planning and management of colorectal cancer screening . in fact , because of lack of comprehensive studies about screening programs in iran , we can only recommend screening program and colonoscopy as a golden standard every 5 to 10 years , for people with increased risk and high risk syndromes . colorectal cancer increases significantly beyond the 5th decade of life ; therefore , it is often thought of as a disease of the elderly , and crc screening is usually not recommended for individuals at average risk younger than 50 years . increased risk patients include as above the following : personal history of adenoma , crc , and inflammatory bowel disease ( ulcerative colitis , crohn 's disease),positive family history ( particularly first degree relative with crc ) . personal history of adenoma , crc , and inflammatory bowel disease ( ulcerative colitis , crohn 's disease ) , positive family history ( particularly first degree relative with crc ) . high risk syndromes include the following : lynch syndrome ( hereditary nonpolyposis colorectal cancer ( hnpcc)),polyposis syndromes ( classical familial adenomatous polyposis ( fap ) , attenuated familial adenomatous polyposis ( afap ) , mutyh - associated polyposis ( map ) , peutz - jeghers syndrome , juvenile polyposis syndrome , and serrated polyposis syndrome)cowden syndrome , li - fraumeni syndrome . lynch syndrome ( hereditary nonpolyposis colorectal cancer ( hnpcc ) ) , polyposis syndromes ( classical familial adenomatous polyposis ( fap ) , attenuated familial adenomatous polyposis ( afap ) , mutyh - associated polyposis ( map ) , peutz - jeghers syndrome , juvenile polyposis syndrome , and serrated polyposis syndrome ) li - fraumeni syndrome . similar to other screening programs , the strategy for screening for crc should be assessed with respect to its effectiveness , sensitivity , the number of false positive results , safety , and comfort . furthermore , the cost and economic factors pertaining to the screening programs should be observed in order to help patients with decision making , and the prevailing clinical policies should be taken into consideration . however , the devising and administration of screening programs in every country requires basic epidemiological information , including assessments of the severity of cancer prevalence , the average- and high risk groups for crc , the most common tumor localization , age distribution of the patients at the time of diagnosis , and calculation of the attributed risk of crc and a high performance risk assessment tool . whereas the efficacy of screening is clearly useful for people with increased and average risk and high risk syndromes , there is no evidence that specific mass screening programs on adolescent and young adults in iran would increase early detection and impact on survival , so more comprehensive and general studies are required to prove this claim . besides , it should be determined which method of screening gives better outcomes . the development of an executive plan to identify the most appropriate screening method and the best age group to be screened for crc is highly recommended in iran .

purpose . the increasing incidence of colorectal cancer ( crc ) in the past three decades in iran has made it a major public health burden . this study aimed to report its epidemiologic features , molecular genetic aspects , survival , heredity , and screening pattern in iran . methods . a comprehensive literature review was conducted to identify the relevant published articles . we used medical subject headings , including colorectal cancer , molecular genetics , kras and braf mutations , screening , survival , epidemiologic study , and iran . results . age standardized incidence rate of iranian crcs was 11.6 and 10.5 for men and women , respectively . overall five - year survival rate was 41% , and the proportion of crc among the younger age group was higher than that of western countries . depending on ethnicity , geographical region , dietary , and genetic predisposition , mutation genes were considerably diverse and distinct among crcs across iran . the high occurrence of crc in records of relatives of crc patients showed that family history of crc was more common among young crcs . conclusion . appropriate screening strategies for crc which is amenable to early detection through screening , especially in relatives of crcs , should be considered as the first step in crc screening programs .

1. Introduction and Background 2. Materials and Methods 3. Results 4. Conclusion

colorectal cancer ( crc ) is one of the most common types of cancer affecting 1.23 million individuals per year ( 9.7% of overall cancers ) and is the fourth most common cause of death from cancer worldwide ( 608,000 cases , 8% of overall cancer deaths ) . the incidence of crc has been estimated to be 3050 cases ( per 100,000 ) in northern america and europe and 37 cases ( per 100,000 ) in most middle east countries . although epidemiology data show a marked variability around the world , and almost 60% of cases occur in developed countries , its overall incidence rate shows a slow but steady decrease ( about 2% per year ) in developed countries . however , the proportion of crc cases occurring among younger age group ( 40 years ) is 28 percent in western countries where it is about 1535 percent in the middle east region [ 13 ] . a large number of epidemiologic studies have been performed to investigate specific hypotheses about risk factors of colorectal cancer , particularly for diet , as western diet ( high meat and energy and less fruit , vegetable , and fiber intake ) , which may have a role in g e ( gene and environmental ) interaction . according to who classification of tumors of the digestive system , the following factors may play an etiologic role in the development of crc , including sedentary lifestyle and obesity , high body and abdominal fatness , hormone replacement therapy , tobacco smoking , alcohol , and nonsteroidal anti - inflammatory drugs [ 46 ] . however , polyps and high risk bowel syndromes such as inflammatory bowel diseases , especially ulcerative colitis , must be noted as a risk factor for crc in general population . it has been proved that about 25% of crc patients have some degrees of familial background , and a strong family history as a first or second degree relation is seen in about 15% of other patients with colorectal cancer . sporadic or nonhereditary colorectal cancer occurs in people who have no ( or very little ) family history of crc and in patients who have no identifiable genetic risk . the majority of crcs ( 7085% ) are sporadic and they are believed to be influenced by diet , lifestyle , environmental factors , and acquired somatic mutations [ 2 , 6 ] . its frequency is about one in 10,000 , and it includes almost 24% of colorectal cancers . the same genes might be involved in the etiology of both hereditary and sporadic cancers , with the difference that , in hereditary cancers , one copy of a mutant allele is inherited in the germline and the other mutation occurs somatically , while in sporadic cancers , both mutations are acquired somatically [ 3 , 912 ] . over the past two decades , molecular studies have led to a tremendous increase in our knowledge of genetic changes that affect malignancy in crc . the pathogenesis of colorectal cancer is complex and diverse , with different molecular pathways , and in fact leading to different phenotypes . this pathway includes the following : activation of protooncogenes by mutation , including kras ( kirsten rat sarcoma viral oncogene homolog 0 ) , c - src ( tyrosine - protein kinase src ) , and c - myc ( avian myelocytomatosis viral oncogene homolog),inactivation of at least three tumor suppressor genes , as apc ( adenomatous polyposis coli ) , tumor suppressor tp53 gene , and subsequent loh ( loss of heterozygosity ) of chromosome 18q . activation of protooncogenes by mutation , including kras ( kirsten rat sarcoma viral oncogene homolog 0 ) , c - src ( tyrosine - protein kinase src ) , and c - myc ( avian myelocytomatosis viral oncogene homolog ) , inactivation of at least three tumor suppressor genes , as apc ( adenomatous polyposis coli ) , tumor suppressor tp53 gene , and subsequent loh ( loss of heterozygosity ) of chromosome 18q . moreover , the administration of crc screening programs in the united states has led to considerable reduction in cancer - associated death rates . current regulations suggest that screening for crc should be initiated in the general population for 50 years old and more , and for the high risk group from a younger age . the regulations for prevention of diseases in the united states require that every man or woman aged 50 years or above should be screened for crc using one of the screening tests including occult blood test annually , sigmoidoscopy every 5 years , barium enema every 5 years , or colonoscopy every 10 years . given that family members of crc patients are at increased risk for the disease , screening through colonoscopy is usually recommended for the closest relatives ( father , mother , sister , brother , and children ) of patients with crc or adenomatous polyps . according to the iranian national cancer registry report , the incidence of crc has increased during the last 25 years showing that the distribution of crc has shifted towards lower age groups in the country . so despite high cost of molecular techniques , it is obvious that screening of crcs who are candidates for these therapies by detecting of mutations in kras and braf genes is recommended , particularly to those who are not likely to benefit for avoiding unnecessary costs . in general , it is now clear that routine molecular approaches of crc including kras and braf testing are cost saving . to set up an efficient screening procedure , the prevalence of disease , demographic characteristics of the population , possibility of early diagnosis , availability of new treatment modalities for patients with positive screening tests , and the cost - benefit of the whole procedure will have to be determined if risk assessment tool needs to be conducted this review study was conducted to document the epidemiologic features of crc in iran : age standardized incidence , survival rates , prognostic factors , burden of disease , heredity pattern , occurrence of sporadic cases in relatives , and so forth . we also give a report on the molecular routes and cellular metabolism , mutation in kras and braf genes , and other tumor markers in the country . to retrieve published studies and to identify the studies of interest , we conducted a comprehensive literature search . as for search criterions , we used medical subject headings including colorectal cancer , molecular genetics , kras mutation , braf mutation , screening , survival , epidemiologic study , and iran to retrieve the relevant evidence . recent figures published by the iran ministry of health showed that cancer is the third common cause of death in the country . it is now ranked in the third place after heart diseases and accidents with an increasing incidence over the past few decades in the population [ 24 , 25 ] . the same report shows that crc was the fourth most common cancer in iran between 2000 and 2009 [ 26 , 27 ] , with a five - year survival rate between 43 and 49 percent [ 2830 ] . a research study in 2008 reported an asr of 7 - 8 ( per 100,000 ) for both sexes this is higher than that of previous reports in the country while closer to the statistics reported from other middle east areas and less than the similar rates in western countries [ 1 , 32 ] . asr among individuals younger than 50 years was also estimated 8.26 ( per 100,000 ) in the same report . adenocarcinoma was the most frequently detected type of colorectal carcinoma and most common tumor diagnosed in the colon . an asr of 5.9 and 10.7 has been reported from female population of ardebil and males in golestan , respectively . from northwest of the country in 2014 , annual asr was estimated 11.2 and 8.93 in men and women , respectively . the crude rates for men and women were reported 11.5 and 9.22 , respectively , in the same study . the occurrence of crc has recently shown a slightly decreasing trend compared to the 2008 data [ 31 , 35 , 36 ] . however , as indicated before , several epidemiological studies in iran have shown that the proportion of crc among the younger population is considerably higher compared to western populations . early crc ( in less than 40 years ) accounts for almost one - fifth of all cases of crc in iran , compared with high risk countries , with rates ranging from 2 to 8% [ 1 , 13 , 3744 ] . this might be attributed to two factors : a high proportion of young population in iran and a relatively low rate of crc in aged people in the country . almost all economic indices show a considerable improvement in living conditions in iran over the past few decades ( statistic center of iran , 2007 , world bank report , 2006 ) . the recent trends of crc in youth in iran could be a result of these changes in the diet pattern in this generation towards western lifestyle in recent decades . it is therefore expected that the occurrence of crc in iran will probably be similar to those of western countries in the forthcoming decades . some studies showed that genetic factors may probably play a role in the development of crc among young population in iran , as well . this suggests that crc in iran could be caused by different factors both environmentally and genetically [ 33 , 45 ] . advanced stages of cancer , rectal localization of tumor , pathology grade , and patient 's performance , vascular invasion , older age , and genetic instability such as amplification of certain genes are the factors that correlate with the worst prognosis of crc patients at diagnosis . showed that overall 5-year survival of hnpcc patients was higher than sporadic ones ( p = 0.044 ) , and they compared their data with other similar studies . however , the results of a population - based cohort study from golestan in iran , where more than 42% of crcs were under 50 years of age , revealed a worse survival in young crc patients , more likely because their cancer is diagnosed in advanced stages . a research study showed that the one- , two- , three- , four- , and five - year survival rates of crc were 84 , 68 , 54 , 43 , and 41 percent , respectively . men had poor prognosis compared to women where the five - year survival rate was 45 and 88 percent in men and women , respectively . the overall five - year survival rate for crc patients in iran ( 41 percent ) was similar to those from other developing countries while it was lower compared to developed world where advanced health care systems and population - based screening programs exist . mutations in kras and braf genes are considered important primary incidents in the development of crc . it shows strong association with the incidence of methylguanine in promoter regions of various genes in crc . mutation in codons 12 , 13 , and 61 of kras is common in crc . recent reports have shown that mutation in codon 12 is observed at frequencies of 1230 percent and 3550 percent among crc patients in asian and western countries , respectively . reported 37.4 percent of crc patients with kras mutations . they found an overall rate of kras mutations in the italian series ( 46.3 percent ) which is higher than that found in iranian crcs . that minimum mutation rates of this gene are found among patients in iran ( 2 percent ) compared to those in amman ( 19 percent ) and african - americans ( 10 percent ) genetic or environmental factors are believed to be responsible for the lack of braf mutation in crc patients in iran [ 5 , 51 , 56 ] . p53 is a tumor suppressor gene which is believed to play a role in the regulation of cell proliferation , involved in dna repair , cell cycle gene transcription , and apoptosis . however , it is believed that p53 alterations are more frequent in distal than in proximal crcs , and a recent meta - analysis of case series from 17 countries showed that sites and types of p53 mutations were comparable in proximal and distal colorectal cancers [ 58 , 61 ] . depending on ethnicity , geographical region , dietary , and genetic predisposition and mostly because of heterogeneous nature of colorectal cancer , apc mutation genes are considerably diverse and distinct , as well as in sporadic crcs . to date , some hundred mutations have been reported around the world , but some studies in iran reported a unique profile of apc mutations in iranian crcs , and the overall frequency of apc mutation was about 2530% in sporadic colorectal patients . several studies showed high frequency of msi in sporadic colorectal cancer patients in iran , and bat25 , bat26 , and nr-21 were showed to be the most sensitive markers for diagnosis sporadic crcs and hnpccs [ 13 , 6669 ] . msi status in iran was observed in 23% of crc cases , and it was more frequent in females , in early - onset colorectal cancers , and in tumors located in the proximal colon ( 40% ) , with the majority of msi - h tumors , compared to the common trends in the world . despite increasing knowledge about genetics and biochemistry of the mmr genes , a few data about mutation screening of mmr in iranian crcs , as well as hnpcc patients , are available . indeed , while the detection of msi is simple and economical and has high correlation with the clinicopathologic feature of colorectal carcinoma , large and more executive molecular research studies are needed to know about factors that contributed to msi - associated carcinogenesis in iranian crcs . the marker shows association with disease stage , and the levels of cea and ca19 - 9 before surgery are correlated with the disease prognosis . the high occurrence of crc in records of relatives of crc patients in iran shows that a considerable number of these crc patients are from families whose members or relatives have been suffering from crc in the past . showed that family history of crc is more common among young crc patients and with tumors on the right side . moreover , the incidence of crc in a close family member during their youth leads to considerably increased risk of acquiring crc ( 1530 percent ) . they revealed that the familial relative risk in first degree relatives of crcs was about 1.7 , rising to 2.8 if two or more first degree relatives were affected or / and to 5.4 if both relatives were less than 45 years old at diagnosis . studying the colonoscopy results , in a few studies in iran , indicated that having a family member with a history of crc , 32.7-fold , increases the risk of colorectal polyps and crc [ 7476 ] . also the incidence of adenoma among the relatives of crc patients aged 5060 years is approximately 1734 percent and lower among younger relatives [ 32 , 77 ] . another study 's results showed that the prevalence of colorectal adenoma and precancerous lesions in first degree relatives of crc patients is significantly higher than in the average risk population . studies have shown that the incidence of crc in a close member of one 's family almost doubles the risk of crc for an individual and the risk of developing crc is 2.53 times greater among the relatives of crc patients . these observations highlight the need to improve efforts for enhancing public awareness and screening strategies in families with a crc patient , particularly a young patient , or with proximal tumors . screening programs may therefore begin with family members of crc patients from a young age using colonoscopy as the preferred method of screening . several studies have assessed the efficiency of crc screening in individuals aged 50 years or below . the diagnosis of local crc may also improve the survival rate of crc patients , and the five - year survival rate of crc diagnosed early was reported to be around 90 percent . although the early diagnosis of crc reduces the associated death burden , administration of screening programs is not a common practice even in developed countries . previous studies showed the absence of any national screening programs for crc in iran , with very little information available on screening strategies for cancer in general in the country . in fact , a few screening programs were tested among the high risk groups for crc , which include family members of crc patients , by the center for liver and digestive system studies in the medical science and medical services department of shahid beheshti university [ 77 , 79 ] . however , some educational programs on crc screening may increase overall knowledge and motivation about colorectal cancer risk factors and screening modalities . the decision making involved in crc screening in iran has proven valuable for cost saving with respect to local resources , personal preferences of the patients , or the choice of screening tests , including the highly sensitive stool occult blood test conducted annually ( such as stool immunochemistry test ) or colonoscopy conducted every 10 years . colonoscopy is both the most effective and sensitive and the most costly strategy according to latest trials in iran [ 25 , 77 , 80 , 81 ] , and many studies recommend it as golden standard tool for early detection of precancerous as well as crc [ 77 , 82 ] . colorectal cancer increases significantly beyond the 5th decade of life ; therefore , it is often thought of as a disease of the elderly , and crc screening is usually not recommended for individuals at average risk younger than 50 years . increased risk patients include as above the following : personal history of adenoma , crc , and inflammatory bowel disease ( ulcerative colitis , crohn 's disease),positive family history ( particularly first degree relative with crc ) . personal history of adenoma , crc , and inflammatory bowel disease ( ulcerative colitis , crohn 's disease ) , positive family history ( particularly first degree relative with crc ) . high risk syndromes include the following : lynch syndrome ( hereditary nonpolyposis colorectal cancer ( hnpcc)),polyposis syndromes ( classical familial adenomatous polyposis ( fap ) , attenuated familial adenomatous polyposis ( afap ) , mutyh - associated polyposis ( map ) , peutz - jeghers syndrome , juvenile polyposis syndrome , and serrated polyposis syndrome)cowden syndrome , li - fraumeni syndrome . lynch syndrome ( hereditary nonpolyposis colorectal cancer ( hnpcc ) ) , polyposis syndromes ( classical familial adenomatous polyposis ( fap ) , attenuated familial adenomatous polyposis ( afap ) , mutyh - associated polyposis ( map ) , peutz - jeghers syndrome , juvenile polyposis syndrome , and serrated polyposis syndrome ) li - fraumeni syndrome . similar to other screening programs , the strategy for screening for crc should be assessed with respect to its effectiveness , sensitivity , the number of false positive results , safety , and comfort . furthermore , the cost and economic factors pertaining to the screening programs should be observed in order to help patients with decision making , and the prevailing clinical policies should be taken into consideration . however , the devising and administration of screening programs in every country requires basic epidemiological information , including assessments of the severity of cancer prevalence , the average- and high risk groups for crc , the most common tumor localization , age distribution of the patients at the time of diagnosis , and calculation of the attributed risk of crc and a high performance risk assessment tool . whereas the efficacy of screening is clearly useful for people with increased and average risk and high risk syndromes , there is no evidence that specific mass screening programs on adolescent and young adults in iran would increase early detection and impact on survival , so more comprehensive and general studies are required to prove this claim . the development of an executive plan to identify the most appropriate screening method and the best age group to be screened for crc is highly recommended in iran .

this study was conducted at a large midwestern academic institution . the pediatric residency program at our institution trains over 40 residents per year , with modules in general pediatrics and combined medicine - pediatric pathways . the hospital also supports one of the largest nicus in the country , with 168 neonatal beds and 15 board - certified neonatology faculty . prior to 2009 , resident teaching responsibilities in the nicu were placed solely on faculty during their clinical care ( ward ) month , with no formal involvement from neonatology fellows . in light of poor resident satisfaction with their nicu experiences in the 2007 and 2008 academic years , the nicu fellows were invited by faculty to design and implement an education program at the beginning of the 2009 academic year . the basic tenet of the fellow - led program was to improve the resident educational experience in the nicu . additional goals , as outlined in collaboration with participating faculty , were to provide fellows with an opportunity to develop and enhance their teaching skills and promote interest in a career in academic medicine and teaching . faculty believed that participation in this program would provide a practical introduction for fellows in creating an interactive learning environment for medical education . although all fellows in our program had successfully graduated from pediatric residency programs and expressed interest in teaching residents , none had prior experience in formal classroom instruction . prior to the start of the program , the fellows met with faculty to develop learning objectives for the educational series ( fig . the objectives were based on topics outlined by the american academy of pediatrics ( aap ) on the care of the newborn and fetus . in addition , the fellows created an institutional database of nicu - related questions to be used during teaching sessions ( 10 ) . the questions were developed to guide the content of discussion and stimulate the learning environment . for a given month , individual fellows typically were assigned two to four learning topics . at each session , fellows were expected to review current literature on their respective topic , with the goal of promoting active discussion and exchange of information with the residents . although each session had specific teaching content to be addressed , the lectures were designed to be interactive , with the goal of providing residents with enough time to ask questions . the daily sessions were conducted before morning nicu rounds and typically lasted 45 to 60 minutes . faculty were available to review learning objectives , clarify conflicting evidence in the literature on a particular topic and provide insight on strategies to teach the residents more effectively in a classroom setting . the education program was a natural by - product of the growing fellowship presence at our institution , with the total number of neonatology fellows increasing from four in 2007 to eight in 2009 . source : adapted from aap 2008 guidelines on the care of the newborn and fetus . we employed four strategies , itemized here , to evaluate the potential value of the fellow - led education program on resident and fellow satisfaction and experience . following institutional requirements for resident graduation , and consistent with recommendations provided by the acgme , residents completed a rotation evaluation at the end of their nicu rotation . this generalized evaluation is completed online ( e - value , minneapolis , mn ) . the questions included the following items.did the organization of the rotation facilitate your learning?were the rotation 's expectations clear to you during the month?was the rotation well organized?was the rotation valuable for your development as a general pediatrician?rate the overall teaching excellence in the rotation . did the organization of the rotation facilitate your learning ? were the rotation 's expectations clear to you during the month ? was the rotation well organized ? responses were reported using a categorical variable scale ranging from one to five ( 1=strongly disagree ; 2=somewhat disagree ; 3=neutral ; 4=somewhat agree ; 5=strongly agree ) . scores on the evaluation were assessed over three consecutive academic years ( 20072009 ) to evaluate the potential impact of the 2009 fellow - led program on resident responses . residents are assigned to this rotation only once during their training , thus minimizing the chance of repeat responses before and after program implementation . the e - value online tool we used in the assessment of resident satisfaction with their nicu rotation represents a standardized method for evaluating resident experience that has been in place at our institution since 2005 . data were also obtained on age and practice plans following completion of pediatric training over the 20072009 academic years . fellows completed an anonymous survey that addressed the usefulness of their participation in the program . fellows respondents rated the educational value across four areas ( understanding of basic neonatal pathophysiology , preparation for specialty board examinations , enhancement of teaching skills and enhancement of clinical skills ) using a five - point scale ( 1=little or no value , 3=moderate value , 5=great value ) . an assessment of the overall educational experience of participation in the program was conducted by asking fellows to rate the following statements : education program should continue next year ; time devoted to course was acceptable given benefits ; greater interest in neonatal pathophysiology ; increased interest in career in academic medicine ; faculty interaction was viewed positively ; resident feedback was viewed positively . responses were evaluated using a five - point scale ( 1=strongly agree , 3=unsure / neutral , 5=strongly agree ) . a separate and unique voluntary survey was designed to assess the relationship between pediatric residents and fellows in the setting of a growing fellow presence at our institution . the survey consisted of the following statements.the nicu fellows were very effective teachers.the nicu fellows were interested in your learning and development.the nicu fellows did not compete or limit your ability to perform procedures.the distinction of clinical responsibilities between you and the nicu fellows was very clear.overall , the nicu fellows were very important to your nicu training . responses were reported using a categorical variable ranging from one to five ( as described above ) . the survey we used was adapted from a previously validated survey on fellow - resident interactions ( 12 ) . in line with acgme requirements , our institution maintains a database of all procedures performed by residents throughout their pediatric training . a review of the resident procedural case - logs during the neonatology rotation from 2007 to 2009 was performed . we calculated the total number of procedures performed by residents during their neonatology rotation in five primary areas of procedural competency : endotracheal intubation ; umbilical arterial catheter ( uac ) ; umbilical venous catheter ( uvc ) ; arterial puncture ; and lumbar puncture . the total number of procedures was divided by the number of residents , which provided an index of the average number of procedures per resident for any given year . responses to the fellow survey on the usefulness of their participation in the education program were described by their mean and standard deviation ( fig . responses to the resident survey on resident - fellow interactions were described by their overall percentile ( fig . 4 ) . the sample size was one of convenience and represented all residents who completed the nicu rotation over the years 20072009 . note : at the end of their nicu rotation , residents ( n=105 ; 69 before education program and 36 after program ) completed an online evaluation ( e - value ) . scores 1=strongly disagree ; 2=somewhat disagree ; 3=neutral ; 4=somewhat agree ; and 5=strongly agree . unpaired t - test with mann - whitney was performed on average score for each of five possible questions . note : all eight nicu fellows in the education program completed the online survey at the end of the 2009 academic year following one year of teaching responsibilities ( 100% response rate ) . responses to questions on the educational value of the program were reported using a categorical variable ranging from 1 to 5 ( 1=little or no value , 3=moderate value , 5=great value ) . responses to questions on the overall experience of participating in the program were evaluated using a five - point scale ( 1=strongly disagree , 3=unsure / neutral , 5=strongly agree ) . survey of resident - fellow interaction : positive responses from residents regarding the resident - fellow relationship in the nicu . note : thirty - six residents completed the online survey at the end of the 2009 academic year ( 36 out of a possible 40 , 90% response rate ) . responses were reported using a categorical variable ranging from one to five ( 1=strongly disagree ; 2=somewhat disagree ; 3=neutral ; 4=somewhat agree ; 5=strongly agree ) . prior to the start of the program , the fellows met with faculty to develop learning objectives for the educational series ( fig . the objectives were based on topics outlined by the american academy of pediatrics ( aap ) on the care of the newborn and fetus . in addition , the fellows created an institutional database of nicu - related questions to be used during teaching sessions ( 10 ) . the questions were developed to guide the content of discussion and stimulate the learning environment . for a given month , individual fellows typically were assigned two to four learning topics . at each session , fellows were expected to review current literature on their respective topic , with the goal of promoting active discussion and exchange of information with the residents . although each session had specific teaching content to be addressed , the lectures were designed to be interactive , with the goal of providing residents with enough time to ask questions . the daily sessions were conducted before morning nicu rounds and typically lasted 45 to 60 minutes . faculty were available to review learning objectives , clarify conflicting evidence in the literature on a particular topic and provide insight on strategies to teach the residents more effectively in a classroom setting . the education program was a natural by - product of the growing fellowship presence at our institution , with the total number of neonatology fellows increasing from four in 2007 to eight in 2009 . source : adapted from aap 2008 guidelines on the care of the newborn and fetus . we employed four strategies , itemized here , to evaluate the potential value of the fellow - led education program on resident and fellow satisfaction and experience . following institutional requirements for resident graduation , and consistent with recommendations provided by the acgme , residents completed a rotation evaluation at the end of their nicu rotation . this generalized evaluation is completed online ( e - value , minneapolis , mn ) . the questions included the following items.did the organization of the rotation facilitate your learning?were the rotation 's expectations clear to you during the month?was the rotation well organized?was the rotation valuable for your development as a general pediatrician?rate the overall teaching excellence in the rotation . did the organization of the rotation facilitate your learning ? were the rotation 's expectations clear to you during the month ? responses were reported using a categorical variable scale ranging from one to five ( 1=strongly disagree ; 2=somewhat disagree ; 3=neutral ; 4=somewhat agree ; 5=strongly agree ) . scores on the evaluation were assessed over three consecutive academic years ( 20072009 ) to evaluate the potential impact of the 2009 fellow - led program on resident responses . residents are assigned to this rotation only once during their training , thus minimizing the chance of repeat responses before and after program implementation . the e - value online tool we used in the assessment of resident satisfaction with their nicu rotation represents a standardized method for evaluating resident experience that has been in place at our institution since 2005 . data were also obtained on age and practice plans following completion of pediatric training over the 20072009 academic years . fellows completed an anonymous survey that addressed the usefulness of their participation in the program . fellows respondents rated the educational value across four areas ( understanding of basic neonatal pathophysiology , preparation for specialty board examinations , enhancement of teaching skills and enhancement of clinical skills ) using a five - point scale ( 1=little or no value , 3=moderate value , 5=great value ) . an assessment of the overall educational experience of participation in the program was conducted by asking fellows to rate the following statements : education program should continue next year ; time devoted to course was acceptable given benefits ; greater interest in neonatal pathophysiology ; increased interest in career in academic medicine ; faculty interaction was viewed positively ; resident feedback was viewed positively . responses were evaluated using a five - point scale ( 1=strongly agree , 3=unsure / neutral , 5=strongly agree ) . a separate and unique voluntary survey was designed to assess the relationship between pediatric residents and fellows in the setting of a growing fellow presence at our institution . the survey consisted of the following statements.the nicu fellows were very effective teachers.the nicu fellows were interested in your learning and development.the nicu fellows did not compete or limit your ability to perform procedures.the distinction of clinical responsibilities between you and the nicu fellows was very clear.overall , the nicu fellows were very important to your nicu training . responses were reported using a categorical variable ranging from one to five ( as described above ) . the survey we used was adapted from a previously validated survey on fellow - resident interactions ( 12 ) . in line with acgme requirements , our institution maintains a database of all procedures performed by residents throughout their pediatric training . a review of the resident procedural case - logs during the neonatology rotation from 2007 to 2009 was performed . we calculated the total number of procedures performed by residents during their neonatology rotation in five primary areas of procedural competency : endotracheal intubation ; umbilical arterial catheter ( uac ) ; umbilical venous catheter ( uvc ) ; arterial puncture ; and lumbar puncture . the total number of procedures was divided by the number of residents , which provided an index of the average number of procedures per resident for any given year . responses to the fellow survey on the usefulness of their participation in the education program were described by their mean and standard deviation ( fig . 3 ) . responses to the resident survey on resident - fellow interactions were described by their overall percentile ( fig . the sample size was one of convenience and represented all residents who completed the nicu rotation over the years 20072009 . note : at the end of their nicu rotation , residents ( n=105 ; 69 before education program and 36 after program ) completed an online evaluation ( e - value ) . scores 1=strongly disagree ; 2=somewhat disagree ; 3=neutral ; 4=somewhat agree ; and 5=strongly agree . unpaired t - test with mann - whitney was performed on average score for each of five possible questions . note : all eight nicu fellows in the education program completed the online survey at the end of the 2009 academic year following one year of teaching responsibilities ( 100% response rate ) . responses to questions on the educational value of the program were reported using a categorical variable ranging from 1 to 5 ( 1=little or no value , 3=moderate value , 5=great value ) . responses to questions on the overall experience of participating in the program were evaluated using a five - point scale ( 1=strongly disagree , 3=unsure / neutral , 5=strongly agree ) . survey of resident - fellow interaction : positive responses from residents regarding the resident - fellow relationship in the nicu . note : thirty - six residents completed the online survey at the end of the 2009 academic year ( 36 out of a possible 40 , 90% response rate ) . responses were reported using a categorical variable ranging from one to five ( 1=strongly disagree ; 2=somewhat disagree ; 3=neutral ; 4=somewhat agree ; 5=strongly agree ) . the neonatology fellows invested over 300 hours in resident teaching during the 2009 academic year . all nicu fellows ( n=8 ) actively participated in all aspects of the program 's development and implementation . five faculty members in the field of neonatology were involved in the development of the aap - based curriculum and provided constant oversight throughout the process . a review of the program revealed that less than 5% of scheduled lessons were cancelled due to resident , fellow or faculty conflict . over the course of the three academic years in review , responses were available from 105 of 113 residents who completed the nicu rotation ( 92.9% response rate ) . demographics between the two groups of residents before and after program implementation were similar with respect to age , gender , race and future plans . specifically , a similar number of residents before and after the program planned on pursuing a nicu fellowship following graduation ( 12% before program versus 11% after program ) . fig . 2 shows the perceptions of residents on the nicu rotation before and after the fellow - led education program was initiated . on the overall rotation evaluation , mean scores between the cohorts were markedly improved after program implementation for all questions posed . specifically , when asked if the organization of the rotation facilitated their learning ( question 1 ) , residents in the nicu responded with a mean score of 3.60 out of 5 before the education program and a mean score of 4.51 out of 5 after initiation of the program ( p<0.001 ) . when asked if the rotation expectations were clear during their nicu month ( question 2 ) and the rotation was well organized ( question 3 ) , residents response improved from mean scores of 4.00 and 3.74 before implementation of the program to mean scores of 4.50 and 4.60 after its initiation ( p<0.001 and p<0.001 , respectively ) . of note , when asked about the value of the nicu rotation for their development as a general pediatrician ( question 4 ) , there was a significantly higher score among residents exposed to the education program than among those completing their nicu rotation before the program 's introduction , with the mean score improving from 3.71 to 4.44 ( p<0.001 ) . finally , when residents rated the overall teaching excellence during their nicu rotation , scores were higher after the program was in place , with mean scores improving from 4.44 to 4.67 ( p<0.05 ) . 3 shows responses from neonatology fellows on the educational value and overall benefits of their participating in the program . fellows rated the development of teaching skills and improved understanding of neonatal pathophysiology as the most valuable aspects of participation . additionally , a majority ( 88% ) believed that the program should continue next year . interestingly , despite the considerable amount of time spent in developing and implementing the program , most fellows ( 63% ) responded that they strongly agree this effort was acceptable given the mutual benefits to residents and fellows . a separate , voluntary survey was conducted to assess resident attitudes toward the growing nicu fellow presence at our institution ( fig . results on the anonymous survey were available from 36 out of a possible 40 residents ( 90% response rate ) . when asked questions about nicu fellows effectiveness as teachers and interest in resident learning and development , over 95% of residents responded positively ( strongly agree / somewhat agree ) . however , only 62.5% strongly agreed or somewhat agreed with the statement the nicu fellows did not compete or limit your ability to perform procedures. interestingly , when asked if the distinction of clinical responsibilities between resident and nicu fellow was clear , 85.5% responded positively . finally , when asked if the nicu fellows were very important to their overall nicu training and education , 87.5% responded positively , with only 5% of residents reporting a negative influence of nicu fellows on their training . we calculated the total number of procedures performed by residents during their neonatology rotation in five primary areas of procedural competency ( intubation , uac , uvc , arterial puncture , lumbar puncture ) . despite an increase in the number of neonatology fellows from four in 2007 to eight in 2009 , the nicu procedures per resident did not change in any of the core areas we reviewed . resident procedural experience in setting of increasing number of neonatology fellows total residents : n=113 . number of nicu fellows=4 ( 2007 academic year ) ; 4 ( 2008 academic year ) ; 8 ( 2009 academic year ) . the neonatology fellows invested over 300 hours in resident teaching during the 2009 academic year . all nicu fellows ( n=8 ) actively participated in all aspects of the program 's development and implementation . five faculty members in the field of neonatology were involved in the development of the aap - based curriculum and provided constant oversight throughout the process . a review of the program revealed that less than 5% of scheduled lessons were cancelled due to resident , fellow or faculty conflict . over the course of the three academic years in review , responses were available from 105 of 113 residents who completed the nicu rotation ( 92.9% response rate ) . demographics between the two groups of residents before and after program implementation were similar with respect to age , gender , race and future plans . specifically , a similar number of residents before and after the program planned on pursuing a nicu fellowship following graduation ( 12% before program versus 11% after program ) . 2 shows the perceptions of residents on the nicu rotation before and after the fellow - led education program was initiated . on the overall rotation evaluation , mean scores between the cohorts were markedly improved after program implementation for all questions posed . specifically , when asked if the organization of the rotation facilitated their learning ( question 1 ) , residents in the nicu responded with a mean score of 3.60 out of 5 before the education program and a mean score of 4.51 out of 5 after initiation of the program ( p<0.001 ) . when asked if the rotation expectations were clear during their nicu month ( question 2 ) and the rotation was well organized ( question 3 ) , residents response improved from mean scores of 4.00 and 3.74 before implementation of the program to mean scores of 4.50 and 4.60 after its initiation ( p<0.001 and p<0.001 , respectively ) . of note , when asked about the value of the nicu rotation for their development as a general pediatrician ( question 4 ) , there was a significantly higher score among residents exposed to the education program than among those completing their nicu rotation before the program 's introduction , with the mean score improving from 3.71 to 4.44 ( p<0.001 ) . finally , when residents rated the overall teaching excellence during their nicu rotation , scores were higher after the program was in place , with mean scores improving from 4.44 to 4.67 ( p<0.05 ) . fig . 3 shows responses from neonatology fellows on the educational value and overall benefits of their participating in the program . fellows rated the development of teaching skills and improved understanding of neonatal pathophysiology as the most valuable aspects of participation . great value in improving their teaching skills . additionally , a majority ( 88% ) believed that the program should continue next year . interestingly , despite the considerable amount of time spent in developing and implementing the program , most fellows ( 63% ) responded that they a separate , voluntary survey was conducted to assess resident attitudes toward the growing nicu fellow presence at our institution ( fig . 4 ) . results on the anonymous survey were available from 36 out of a possible 40 residents ( 90% response rate ) . when asked questions about nicu fellows effectiveness as teachers and interest in resident learning and development , over 95% of residents responded positively ( strongly agree / somewhat agree ) . however , only 62.5% strongly agreed or somewhat agreed with the statement the nicu fellows did not compete or limit your ability to perform procedures. interestingly , when asked if the distinction of clinical responsibilities between resident and nicu fellow was clear , 85.5% responded positively . finally , when asked if the nicu fellows were very important to their overall nicu training and education , 87.5% responded positively , with only 5% of residents reporting a negative influence of nicu fellows on their training . we calculated the total number of procedures performed by residents during their neonatology rotation in five primary areas of procedural competency ( intubation , uac , uvc , arterial puncture , lumbar puncture ) . despite an increase in the number of neonatology fellows from four in 2007 to eight in 2009 , the nicu procedures per resident did not change in any of the core areas we reviewed . resident procedural experience in setting of increasing number of neonatology fellows total residents : n=113 . number of nicu fellows=4 ( 2007 academic year ) ; 4 ( 2008 academic year ) ; 8 ( 2009 academic year ) . in light of restrictions to resident work hours potentially limiting clinical exposure , as well as increased demands on academic faculty outside their role as teachers , efforts to develop complementary models of residency education are well founded ( 35 , 13 ) . the role of fellows as a positive force in pediatric resident education may offer advantages for residents , fellows and faculty ; however , the best methods to organize and accomplish this have received little attention until now . here , we described our initial efforts to use fellows in a defined teaching role to optimize resident educational experience . at our institution , the nicu rotation in 20072008 was rated by pediatric residents as one of the least favorable during the course of their pediatric training ( 65th out of 67 possible rotations , bottom 5% ) . in an effort to improve resident satisfaction in the nicu rotation , we found that this approach , although requiring a time investment from faculty and fellows in planning , was generally easy to implement . prior to the program 's induction , meetings of fellows and faculty helped to define the content and format for the program . this effort not only provided stability of teaching content , but also gave an opportunity for fellows to interact positively with potential faculty mentors . the success of the fellow - led education program on resident educational experience is shown by improvements across multiple areas of resident satisfaction ( fig . 2 ) . not surprisingly , these results corresponded with an overall improvement in the residents review of the nicu rotation during the 2009 academic year to 26th out of 67 rotations a marked improvement from previous years . importantly , the benefits of the educational program were not limited to residents , as fellows also rated the overall experience favorably . specifically , fellows noted the acquisition of teaching skills and enhanced knowledge of neonatal pathophysiology as among the most useful aspects of their participation ( fig . 3 ) . considering that the fellow - led education program represented a new initiative in our nicu , the number of teaching hours between faculty and residents did not change with its inception . of note ( personal communication ) , many faculty reported that the program allowed them to direct their resident teaching efforts to more advanced topics , with the understanding that the basic tenets are being taught by the fellows . as such , we feel that the education program not only enhanced resident - fellow interaction , but also likely supported teaching efforts between residents and faculty . this is the first study to evaluate the potential value of utilizing pediatric fellows in resident medical education . in contrast to previous work reporting that a strong fellow presence may dilute the educational experience for residents , the present study found that nicu fellows being active in a defined teaching role results in an increased level of resident satisfaction with their nicu experience ( 14 , 15 ) . an unexpected effect was that residents almost uniformly responded that nicu fellows were very important to their overall training in the nicu ( 87.5% positive response ) . the importance of fellows accepting a teaching role in residency education is becoming more recognized . the acgme program requirements in neonatology identify teaching skills among a group of core competencies that fellows must become effective at during their training ( 8) . however , recent evidence suggests that fellows are considered the primary personnel responsible for resident supervision and education in the nicu less than 15% of the time ( 1 ) . in our study , it is notable that over 95% of residents responded positively ( strongly agree or somewhat agree ) when asked about the effectiveness of the nicu fellows as teachers and the fellows interest in their learning and education . results of the fellow survey suggest that development of a structured educational program provides fellows with the opportunity to enhance their teaching skills and develop their understanding of basic pathophysiology within their discipline . we believe the fellow - led program provides a potential model for fellows to fulfill the acgme requirement for teaching , and that broad participation by fellows in a leadership role enhances the depth and quality of the educational experience of the fellows . this suggests that the traditional model of education , one that places neonatology faculty as the sole teachers in the nicu , may be significantly enhanced by the incorporation of fellows into existing educational paradigms . the resident survey provides information on potential competition between fellows and residents for procedures , a common challenge in procedure - oriented practices such as neonatology . despite a majority of residents responding that fellows do not compete or limit their ability to perform procedures in the nicu , over 35% had a negative ( somewhat disagree / strongly disagree ) or neutral response to the question . however , despite an increase in the number of neonatology fellows from four to eight , the nicu procedures documented by residents did not change over the three - year period in review ( table 1 ) . this suggests that while the actual number of nicu procedures performed by the residents has not changed with the growth of the fellowship program , there is an underlying perception by some residents of competition with fellows for procedures . this finding is consistent with previous literature showing that residents often perceive fellows as detractors from their procedural experience ( 7 ) . therefore , program directors must clearly define the program 's expectations for resident involvement in procedures , as well as discussing the role of fellows in supporting and supervising residents in achieving procedural competency . although over 85% of residents answered positively ( strongly agree / somewhat agree ) to the question regarding distinction of clinical responsibilities between the resident and fellows , we believe that the maintenance of clear and consistent guidelines for procedural responsibilities is also warranted . first , we recognize that these data represent the opinions of resident physicians from a single academic institution , and regional and national differences may exist . however , as the first to address this issue in the pediatric literature , we hope our findings will be the springboard for future scholarly investigation into the potential role of fellows in pediatric resident education . second , the success of a fellow - led education program relies heavily on the interest , enthusiasm and commitment of fellows to teaching . given the relatively large size of our neonatology fellowship program this was not a major obstacle for implementation , although such barriers may become more apparent in smaller programs . third , we can not account for the fact that improvements in resident satisfaction on the survey can be partly explained by the increased number of nicu fellows from four in 2007 to eight in 2009 , such that more opportunities for resident - fellow interaction in the nicu improved the residents overall experience with the rotation . however , only one nicu fellow was assigned to clinical care duties in the nicu at any given time from 2007 to 2009 . to that end , while the number of total nicu fellows increased , the number assigned to clinical care duties with the pediatric residents remained unchanged . it is also important to recognize that while fellows had faculty oversight throughout the development and implementation of the educational series , there was no formal assessment of the fellows performance as teachers . we are currently developing an evaluation process ( scheduled to be implemented in july 2011 ) wherein both faculty and residents evaluate the fellows overall teaching effectiveness and provide feedback on ways to refine their teaching skills . the resident survey provides valuable information on resident - fellow interaction in the setting of a growing fellow presence at our institution . however , data from the nicu survey were only available during the 2009 academic year , thus we are not able to evaluate changes in resident opinion on the survey before and after the education program was in place . additionally , while we have shown that the residents perceptions of their nicu educational experience markedly improved with the new educational model , the ultimate measure of the efficacy of this approach will be the knowledge and competency of these residents as they become pediatricians in the community . to our knowledge this is the first study to address the impact of a fellow - led education program on pediatric resident education . in summary , our findings support the concept that utilizing fellows in a defined teaching role results in an increased level of resident satisfaction with their educational experience . also , such educational models provide fellows with an opportunity to develop teaching skills , consistent with the acgme 's practice - based iearning and improvement competencies for fellowship training programs ( 8) . while such efforts require cooperation and coordination between residency and fellowship program personnel in design and implementation , there are clear benefits for residents , fellows and faculty . further refinement and evaluation of this teaching model , an investigation of this approach in other medical subspecialties and a more rigorous assessment of educational outcomes are warranted .

objectivepressures on academic faculty to perform beyond their role as educators has stimulated interest in complementary approaches in resident medical education . while fellows are often believed to detract from resident learning and experience , we describe our preliminary investigations utilizing clinical fellows as a positive force in pediatric resident education . our objectives were to implement a practical approach to engage fellows in resident education , evaluate the impact of a fellow - led education program on pediatric resident and fellow experience , and investigate if growth of a fellowship program detracts from resident procedural experience.methodsthis study was conducted in a neonatal intensive care unit ( nicu ) where fellows designed and implemented an education program consisting of daily didactic teaching sessions before morning clinical rounds . the impact of a fellow - led education program on resident satisfaction with their nicu experience was assessed via anonymous student evaluations . the potential value of the program for participating fellows was also evaluated using an anonymous survey.resultsthe online evaluation was completed by 105 residents . scores were markedly higher after the program was implemented in areas of teaching excellence ( 4.44 out of 5 versus 4.67 , p<0.05 ) and overall resident learning ( 3.60 out of 5 versus 4.61 , p<0.001 ) . fellows rated the acquisition of teaching skills and enhanced knowledge of neonatal pathophysiology as the most valuable aspects of their participation in the education program . the anonymous survey revealed that 87.5% of participating residents believed that nicu fellows were very important to their overall training and education.conclusionswhile fellows are often believed to be a detracting factor to residency training , we found that pediatric resident attitudes toward the fellows were generally positive . in our experience , in the specialty of neonatology a fellow - led education program can positively contribute to both resident and fellow learning and satisfaction . further investigation into the value of utilizing fellows as a positive force in resident education in other medical specialties appears warranted .

Methods Development and implementation of the fellow-led educational program Assessment of resident perceptions of the NICU rotation Assessment of fellow perceptions on participating in the education program Assessment of resident-fellow relationship in the NICU Assessment of the impact of increasing number of neonatology fellows on resident procedural experience in the NICU Data analyses Results General implementation Resident perceptions Fellow perceptions Resident satisfaction with their interactions with neonatology fellows Impact of neonatology fellows on resident procedural experience in the NICU Discussion Disclosures

the hospital also supports one of the largest nicus in the country , with 168 neonatal beds and 15 board - certified neonatology faculty . in light of poor resident satisfaction with their nicu experiences in the 2007 and 2008 academic years , the nicu fellows were invited by faculty to design and implement an education program at the beginning of the 2009 academic year . the basic tenet of the fellow - led program was to improve the resident educational experience in the nicu . additional goals , as outlined in collaboration with participating faculty , were to provide fellows with an opportunity to develop and enhance their teaching skills and promote interest in a career in academic medicine and teaching . faculty believed that participation in this program would provide a practical introduction for fellows in creating an interactive learning environment for medical education . although all fellows in our program had successfully graduated from pediatric residency programs and expressed interest in teaching residents , none had prior experience in formal classroom instruction . prior to the start of the program , the fellows met with faculty to develop learning objectives for the educational series ( fig . in addition , the fellows created an institutional database of nicu - related questions to be used during teaching sessions ( 10 ) . faculty were available to review learning objectives , clarify conflicting evidence in the literature on a particular topic and provide insight on strategies to teach the residents more effectively in a classroom setting . the education program was a natural by - product of the growing fellowship presence at our institution , with the total number of neonatology fellows increasing from four in 2007 to eight in 2009 . we employed four strategies , itemized here , to evaluate the potential value of the fellow - led education program on resident and fellow satisfaction and experience . following institutional requirements for resident graduation , and consistent with recommendations provided by the acgme , residents completed a rotation evaluation at the end of their nicu rotation . the questions included the following items.did the organization of the rotation facilitate your learning?were the rotation 's expectations clear to you during the month?was the rotation well organized?was the rotation valuable for your development as a general pediatrician?rate the overall teaching excellence in the rotation . scores on the evaluation were assessed over three consecutive academic years ( 20072009 ) to evaluate the potential impact of the 2009 fellow - led program on resident responses . the e - value online tool we used in the assessment of resident satisfaction with their nicu rotation represents a standardized method for evaluating resident experience that has been in place at our institution since 2005 . fellows completed an anonymous survey that addressed the usefulness of their participation in the program . fellows respondents rated the educational value across four areas ( understanding of basic neonatal pathophysiology , preparation for specialty board examinations , enhancement of teaching skills and enhancement of clinical skills ) using a five - point scale ( 1=little or no value , 3=moderate value , 5=great value ) . an assessment of the overall educational experience of participation in the program was conducted by asking fellows to rate the following statements : education program should continue next year ; time devoted to course was acceptable given benefits ; greater interest in neonatal pathophysiology ; increased interest in career in academic medicine ; faculty interaction was viewed positively ; resident feedback was viewed positively . a separate and unique voluntary survey was designed to assess the relationship between pediatric residents and fellows in the setting of a growing fellow presence at our institution . the survey consisted of the following statements.the nicu fellows were very effective teachers.the nicu fellows were interested in your learning and development.the nicu fellows did not compete or limit your ability to perform procedures.the distinction of clinical responsibilities between you and the nicu fellows was very clear.overall , the nicu fellows were very important to your nicu training . responses to the fellow survey on the usefulness of their participation in the education program were described by their mean and standard deviation ( fig . note : at the end of their nicu rotation , residents ( n=105 ; 69 before education program and 36 after program ) completed an online evaluation ( e - value ) . note : all eight nicu fellows in the education program completed the online survey at the end of the 2009 academic year following one year of teaching responsibilities ( 100% response rate ) . responses to questions on the educational value of the program were reported using a categorical variable ranging from 1 to 5 ( 1=little or no value , 3=moderate value , 5=great value ) . responses to questions on the overall experience of participating in the program were evaluated using a five - point scale ( 1=strongly disagree , 3=unsure / neutral , 5=strongly agree ) . note : thirty - six residents completed the online survey at the end of the 2009 academic year ( 36 out of a possible 40 , 90% response rate ) . prior to the start of the program , the fellows met with faculty to develop learning objectives for the educational series ( fig . in addition , the fellows created an institutional database of nicu - related questions to be used during teaching sessions ( 10 ) . the education program was a natural by - product of the growing fellowship presence at our institution , with the total number of neonatology fellows increasing from four in 2007 to eight in 2009 . we employed four strategies , itemized here , to evaluate the potential value of the fellow - led education program on resident and fellow satisfaction and experience . following institutional requirements for resident graduation , and consistent with recommendations provided by the acgme , residents completed a rotation evaluation at the end of their nicu rotation . the questions included the following items.did the organization of the rotation facilitate your learning?were the rotation 's expectations clear to you during the month?was the rotation well organized?was the rotation valuable for your development as a general pediatrician?rate the overall teaching excellence in the rotation . scores on the evaluation were assessed over three consecutive academic years ( 20072009 ) to evaluate the potential impact of the 2009 fellow - led program on resident responses . the e - value online tool we used in the assessment of resident satisfaction with their nicu rotation represents a standardized method for evaluating resident experience that has been in place at our institution since 2005 . fellows completed an anonymous survey that addressed the usefulness of their participation in the program . fellows respondents rated the educational value across four areas ( understanding of basic neonatal pathophysiology , preparation for specialty board examinations , enhancement of teaching skills and enhancement of clinical skills ) using a five - point scale ( 1=little or no value , 3=moderate value , 5=great value ) . an assessment of the overall educational experience of participation in the program was conducted by asking fellows to rate the following statements : education program should continue next year ; time devoted to course was acceptable given benefits ; greater interest in neonatal pathophysiology ; increased interest in career in academic medicine ; faculty interaction was viewed positively ; resident feedback was viewed positively . a separate and unique voluntary survey was designed to assess the relationship between pediatric residents and fellows in the setting of a growing fellow presence at our institution . the survey consisted of the following statements.the nicu fellows were very effective teachers.the nicu fellows were interested in your learning and development.the nicu fellows did not compete or limit your ability to perform procedures.the distinction of clinical responsibilities between you and the nicu fellows was very clear.overall , the nicu fellows were very important to your nicu training . responses to the fellow survey on the usefulness of their participation in the education program were described by their mean and standard deviation ( fig . note : at the end of their nicu rotation , residents ( n=105 ; 69 before education program and 36 after program ) completed an online evaluation ( e - value ) . note : all eight nicu fellows in the education program completed the online survey at the end of the 2009 academic year following one year of teaching responsibilities ( 100% response rate ) . responses to questions on the educational value of the program were reported using a categorical variable ranging from 1 to 5 ( 1=little or no value , 3=moderate value , 5=great value ) . responses to questions on the overall experience of participating in the program were evaluated using a five - point scale ( 1=strongly disagree , 3=unsure / neutral , 5=strongly agree ) . note : thirty - six residents completed the online survey at the end of the 2009 academic year ( 36 out of a possible 40 , 90% response rate ) . all nicu fellows ( n=8 ) actively participated in all aspects of the program 's development and implementation . five faculty members in the field of neonatology were involved in the development of the aap - based curriculum and provided constant oversight throughout the process . a review of the program revealed that less than 5% of scheduled lessons were cancelled due to resident , fellow or faculty conflict . 2 shows the perceptions of residents on the nicu rotation before and after the fellow - led education program was initiated . specifically , when asked if the organization of the rotation facilitated their learning ( question 1 ) , residents in the nicu responded with a mean score of 3.60 out of 5 before the education program and a mean score of 4.51 out of 5 after initiation of the program ( p<0.001 ) . when asked if the rotation expectations were clear during their nicu month ( question 2 ) and the rotation was well organized ( question 3 ) , residents response improved from mean scores of 4.00 and 3.74 before implementation of the program to mean scores of 4.50 and 4.60 after its initiation ( p<0.001 and p<0.001 , respectively ) . of note , when asked about the value of the nicu rotation for their development as a general pediatrician ( question 4 ) , there was a significantly higher score among residents exposed to the education program than among those completing their nicu rotation before the program 's introduction , with the mean score improving from 3.71 to 4.44 ( p<0.001 ) . finally , when residents rated the overall teaching excellence during their nicu rotation , scores were higher after the program was in place , with mean scores improving from 4.44 to 4.67 ( p<0.05 ) . 3 shows responses from neonatology fellows on the educational value and overall benefits of their participating in the program . fellows rated the development of teaching skills and improved understanding of neonatal pathophysiology as the most valuable aspects of participation . a separate , voluntary survey was conducted to assess resident attitudes toward the growing nicu fellow presence at our institution ( fig . results on the anonymous survey were available from 36 out of a possible 40 residents ( 90% response rate ) . when asked questions about nicu fellows effectiveness as teachers and interest in resident learning and development , over 95% of residents responded positively ( strongly agree / somewhat agree ) . finally , when asked if the nicu fellows were very important to their overall nicu training and education , 87.5% responded positively , with only 5% of residents reporting a negative influence of nicu fellows on their training . despite an increase in the number of neonatology fellows from four in 2007 to eight in 2009 , the nicu procedures per resident did not change in any of the core areas we reviewed . resident procedural experience in setting of increasing number of neonatology fellows total residents : n=113 . all nicu fellows ( n=8 ) actively participated in all aspects of the program 's development and implementation . five faculty members in the field of neonatology were involved in the development of the aap - based curriculum and provided constant oversight throughout the process . a review of the program revealed that less than 5% of scheduled lessons were cancelled due to resident , fellow or faculty conflict . 2 shows the perceptions of residents on the nicu rotation before and after the fellow - led education program was initiated . specifically , when asked if the organization of the rotation facilitated their learning ( question 1 ) , residents in the nicu responded with a mean score of 3.60 out of 5 before the education program and a mean score of 4.51 out of 5 after initiation of the program ( p<0.001 ) . when asked if the rotation expectations were clear during their nicu month ( question 2 ) and the rotation was well organized ( question 3 ) , residents response improved from mean scores of 4.00 and 3.74 before implementation of the program to mean scores of 4.50 and 4.60 after its initiation ( p<0.001 and p<0.001 , respectively ) . of note , when asked about the value of the nicu rotation for their development as a general pediatrician ( question 4 ) , there was a significantly higher score among residents exposed to the education program than among those completing their nicu rotation before the program 's introduction , with the mean score improving from 3.71 to 4.44 ( p<0.001 ) . finally , when residents rated the overall teaching excellence during their nicu rotation , scores were higher after the program was in place , with mean scores improving from 4.44 to 4.67 ( p<0.05 ) . 3 shows responses from neonatology fellows on the educational value and overall benefits of their participating in the program . fellows rated the development of teaching skills and improved understanding of neonatal pathophysiology as the most valuable aspects of participation . interestingly , despite the considerable amount of time spent in developing and implementing the program , most fellows ( 63% ) responded that they a separate , voluntary survey was conducted to assess resident attitudes toward the growing nicu fellow presence at our institution ( fig . results on the anonymous survey were available from 36 out of a possible 40 residents ( 90% response rate ) . when asked questions about nicu fellows effectiveness as teachers and interest in resident learning and development , over 95% of residents responded positively ( strongly agree / somewhat agree ) . finally , when asked if the nicu fellows were very important to their overall nicu training and education , 87.5% responded positively , with only 5% of residents reporting a negative influence of nicu fellows on their training . despite an increase in the number of neonatology fellows from four in 2007 to eight in 2009 , the nicu procedures per resident did not change in any of the core areas we reviewed . resident procedural experience in setting of increasing number of neonatology fellows total residents : n=113 . in light of restrictions to resident work hours potentially limiting clinical exposure , as well as increased demands on academic faculty outside their role as teachers , efforts to develop complementary models of residency education are well founded ( 35 , 13 ) . the role of fellows as a positive force in pediatric resident education may offer advantages for residents , fellows and faculty ; however , the best methods to organize and accomplish this have received little attention until now . here , we described our initial efforts to use fellows in a defined teaching role to optimize resident educational experience . at our institution , the nicu rotation in 20072008 was rated by pediatric residents as one of the least favorable during the course of their pediatric training ( 65th out of 67 possible rotations , bottom 5% ) . in an effort to improve resident satisfaction in the nicu rotation , we found that this approach , although requiring a time investment from faculty and fellows in planning , was generally easy to implement . the success of the fellow - led education program on resident educational experience is shown by improvements across multiple areas of resident satisfaction ( fig . not surprisingly , these results corresponded with an overall improvement in the residents review of the nicu rotation during the 2009 academic year to 26th out of 67 rotations a marked improvement from previous years . specifically , fellows noted the acquisition of teaching skills and enhanced knowledge of neonatal pathophysiology as among the most useful aspects of their participation ( fig . considering that the fellow - led education program represented a new initiative in our nicu , the number of teaching hours between faculty and residents did not change with its inception . as such , we feel that the education program not only enhanced resident - fellow interaction , but also likely supported teaching efforts between residents and faculty . this is the first study to evaluate the potential value of utilizing pediatric fellows in resident medical education . in contrast to previous work reporting that a strong fellow presence may dilute the educational experience for residents , the present study found that nicu fellows being active in a defined teaching role results in an increased level of resident satisfaction with their nicu experience ( 14 , 15 ) . an unexpected effect was that residents almost uniformly responded that nicu fellows were very important to their overall training in the nicu ( 87.5% positive response ) . however , recent evidence suggests that fellows are considered the primary personnel responsible for resident supervision and education in the nicu less than 15% of the time ( 1 ) . in our study , it is notable that over 95% of residents responded positively ( strongly agree or somewhat agree ) when asked about the effectiveness of the nicu fellows as teachers and the fellows interest in their learning and education . results of the fellow survey suggest that development of a structured educational program provides fellows with the opportunity to enhance their teaching skills and develop their understanding of basic pathophysiology within their discipline . we believe the fellow - led program provides a potential model for fellows to fulfill the acgme requirement for teaching , and that broad participation by fellows in a leadership role enhances the depth and quality of the educational experience of the fellows . this suggests that the traditional model of education , one that places neonatology faculty as the sole teachers in the nicu , may be significantly enhanced by the incorporation of fellows into existing educational paradigms . however , despite an increase in the number of neonatology fellows from four to eight , the nicu procedures documented by residents did not change over the three - year period in review ( table 1 ) . this suggests that while the actual number of nicu procedures performed by the residents has not changed with the growth of the fellowship program , there is an underlying perception by some residents of competition with fellows for procedures . this finding is consistent with previous literature showing that residents often perceive fellows as detractors from their procedural experience ( 7 ) . although over 85% of residents answered positively ( strongly agree / somewhat agree ) to the question regarding distinction of clinical responsibilities between the resident and fellows , we believe that the maintenance of clear and consistent guidelines for procedural responsibilities is also warranted . however , as the first to address this issue in the pediatric literature , we hope our findings will be the springboard for future scholarly investigation into the potential role of fellows in pediatric resident education . second , the success of a fellow - led education program relies heavily on the interest , enthusiasm and commitment of fellows to teaching . third , we can not account for the fact that improvements in resident satisfaction on the survey can be partly explained by the increased number of nicu fellows from four in 2007 to eight in 2009 , such that more opportunities for resident - fellow interaction in the nicu improved the residents overall experience with the rotation . it is also important to recognize that while fellows had faculty oversight throughout the development and implementation of the educational series , there was no formal assessment of the fellows performance as teachers . we are currently developing an evaluation process ( scheduled to be implemented in july 2011 ) wherein both faculty and residents evaluate the fellows overall teaching effectiveness and provide feedback on ways to refine their teaching skills . the resident survey provides valuable information on resident - fellow interaction in the setting of a growing fellow presence at our institution . however , data from the nicu survey were only available during the 2009 academic year , thus we are not able to evaluate changes in resident opinion on the survey before and after the education program was in place . additionally , while we have shown that the residents perceptions of their nicu educational experience markedly improved with the new educational model , the ultimate measure of the efficacy of this approach will be the knowledge and competency of these residents as they become pediatricians in the community . to our knowledge this is the first study to address the impact of a fellow - led education program on pediatric resident education . in summary , our findings support the concept that utilizing fellows in a defined teaching role results in an increased level of resident satisfaction with their educational experience .

viral infections represent a particularly difficult type of challenge to overcome . though the field of anti - viral strategies continues to grow , success rates lag greatly behind antibacterial strategies . this is due in large part to the lack of understanding of virus interactions with their host cells . the small scale of viruses results in difficulties in the efficient imaging of virus - infected samples . to date , the microscopy forms that are able to achieve the level of magnification and resolution for imaging viruses are the scanning electron microscope ( sem ) and transmission electron microscope ( tem ) . the invention of the atomic force microscope ( afm ) by binnig and colleagues has allowed for high - resolution imaging of nanostructures in living samples [ 2 - 4 ] . a number of these studies involved the observation of purified samples of biomolecules including large viruses , for example , the tobacco mosaic virus , pox virus and human immunodeficiency virus [ 5 - 7 ] . the homogeneity in the sample appears to lend itself to better afm imaging . the afm is able to produce images in several different formats , of which three were consistently analysed : height , phase and amplitude . the height data image is obtained via recording the changes in afm scanner height as it shifts to keep the vibrational amplitude of the cantilever of the probe constant . the phase data image produces an image that provides information of the differing materials / texture in the sample by analyzing the different responses of the probe on such materials . the amplitude data image produces an emphasized view of the height data image by describing the change in the amplitude of the probe directly . while structures are not distorted in the amplitude data image , accurate quantitative measurements are more likely garnered from the height data image . the afm does not require much in sample processing : in fact , its main requirement is that the sample is well - adhered to a substrate such that it does not move around when the afm probe engages it . beyond that , additional sample preparation such as fixation or labelling is decided on a sample - specific basis . while a live sample would be an ideal specimen to study , biosafety concerns stipulated the use of mild fixation in our virus infected samples . fixation was preformed on mock - infected samples for the sake of consistency . as a result the objectives of this study are to investigate the suitability of atomic force microscopy for virus - host interaction studies using the flavivirus model . in addition , to gauge if the images obtained can reveal more information than with the known conventional ultra - structural studies . at the late stages of west nile ( sarafend ) virus infection in vero cells , several interesting aspects warranted investigation . active budding was also present at the proliferated filopodia . in conjunction with the budding of the maturing virus particles , there was a progressive lengthening and thickening of the actin filaments at the cell peripheral . the postulation was that the vectorial force of the growing length of the actin filament provided the bending force to expel the virus particles . 1a & 1b show a budding virus ( arrowhead ) extruding from the plasma membrane at 24 h p.i . 1b shows a higher resolution scan of the extruding virion , with the envelope clearly surrounding the virus nucleocapsid ( arrowhead ) . a tem image of a virus particle ( fig . the arrowheads point to the extruding progeny virions while the arrow indicates the electron dense immunogold label targeted against the wnv envelope protein . ( a ) afm height scan of west nile virus - infected vero cells at 24 h p.i . scan size is 251 nm 251 nm . a progeny virus ( arrow ) budding from the plasma membrane of the host cell . ( b ) afm height scan of west nile virus - infected vero cells at 24 h p.i . ( c ) tem image of west nile virus - infected vero cells ( arrowheads ) budding from host cell . the electron dense dots are the immunogold particles targeted against the envelope protein of the wnv . the hard tapping afm technique used provided a certain degree of " translucency " to the plasma membrane , which enabled the imaging of sub - surface structures in addition to producing surface morphological data . by adjusting the probe to engage the sample with greater force , the probe tip physically pushes against the soft sample surface to image sub - surface structures . it was through this ability that the afm was able to observe the up - regulation of actin filaments in the late infection stages . in fig . 2 the formation of actin filaments near the cell periphery in infected cells was seen over time . 2a shows some degree of enhancement of the cytoskeleton network around the cell periphery ( arrow ) in the west nile ( sarafend ) virus - infected vero cells at 12 h p.i . 2b shows an increased degree of cytoskeletal thickening ( arrow ) of similarly infected cells at 16 h p.i . it is noted that the formation of cytoskeleton clustering in the infected cells appears to be proximal to the formation of filopodia . this detailed progression of cytoskeleton enhancement can not be visualized by the sem ( as it can only provide surface morphology data ) or tem ( as it can only provide 2-dimensional data ) . a lower magnification scan of a mock - infected vero control cell is provided in fig . while the endoplasmic reticulum region is clearly observed at the perinuclear region ( arrow ) , there is the lack of the filopodia and cytoskeleton clustering that is observed in the west nile virus - infected cells at the periphery of the plasma membrane . ( a ) afm height scan of west nile virus infected vero cells at 12 h p.i . ( b ) afm height scan of west nile virus infected vero cells at 24 h p.i . arrows show greater degree of thickening and clumping of actin filaments in close proximity to the cell periphery of the infected cell at 16 h p.i . arrows show the actin filaments forming near the cell edge and the nascent filopodia ( arrowheads ) . ( d ) afm height scan of mock - infected vero cells at 24 h p.i . this control cell shows a lack of cytoskeleton thickening and clumping at the periphery of the cell plasma membrane . these structures were not seen in mock - infected vero cells at 36 h p.i . these control cells also showed a lack of cytoskeletal formation at the periphery of the plasma membrane when compared to infected cells . 3b shows the filopodia ( arrows ) formation between four infected host cells at 36 h p.i . 3c shows bags of virus particles ( thin arrows ) liberated from the cells surface as well as the filopodia at 36 h p.i . during this stage of infection when the cytopathic effects were advanced , arrowhead shows the filopodia , but these were not as pronounced as in the afm imaging due to required ultrathin sectioning of the sample . ( a ) afm height scan of mock infected vero cells at 36 h p.i . scan size is 10 m 10 m . in the mock - infected control sample , ( b ) afm amplitude scan of west nile virus infected vero cells at 36 h p.i . ( c ) afm height scan of west nile virus infected vero cells at 36 h p.i . of 6.9 m 6.9 m size . thin arrows show enveloped bags containing progeny west nile viruses as they egress from host vero cells . ( d ) the transmission electron micrograph shows similar image as in ( c ) . the bags of virus ( arrows ) are seen exiting from the cell filopodia ( filopodia ) . at the late stages of west nile ( sarafend ) virus infection in vero cells , several interesting aspects warranted investigation . active budding was also present at the proliferated filopodia . in conjunction with the budding of the maturing virus particles , there was a progressive lengthening and thickening of the actin filaments at the cell peripheral . the postulation was that the vectorial force of the growing length of the actin filament provided the bending force to expel the virus particles . 1a & 1b show a budding virus ( arrowhead ) extruding from the plasma membrane at 24 h p.i . 1b shows a higher resolution scan of the extruding virion , with the envelope clearly surrounding the virus nucleocapsid ( arrowhead ) . a tem image of a virus particle ( fig . the arrowheads point to the extruding progeny virions while the arrow indicates the electron dense immunogold label targeted against the wnv envelope protein . ( a ) afm height scan of west nile virus - infected vero cells at 24 h p.i . scan size is 251 nm 251 nm . a progeny virus ( arrow ) budding from the plasma membrane of the host cell . ( b ) afm height scan of west nile virus - infected vero cells at 24 h p.i . ( c ) tem image of west nile virus - infected vero cells ( arrowheads ) budding from host cell . the electron dense dots are the immunogold particles targeted against the envelope protein of the wnv . the hard tapping afm technique used provided a certain degree of " translucency " to the plasma membrane , which enabled the imaging of sub - surface structures in addition to producing surface morphological data . by adjusting the probe to engage the sample with greater force , the probe tip physically pushes against the soft sample surface to image sub - surface structures . it was through this ability that the afm was able to observe the up - regulation of actin filaments in the late infection stages . in fig . 2 the formation of actin filaments near the cell periphery in infected cells was seen over time . 2a shows some degree of enhancement of the cytoskeleton network around the cell periphery ( arrow ) in the west nile ( sarafend ) virus - infected vero cells at 12 h p.i . 2b shows an increased degree of cytoskeletal thickening ( arrow ) of similarly infected cells at 16 h p.i . it is noted that the formation of cytoskeleton clustering in the infected cells appears to be proximal to the formation of filopodia . this detailed progression of cytoskeleton enhancement can not be visualized by the sem ( as it can only provide surface morphology data ) or tem ( as it can only provide 2-dimensional data ) . a lower magnification scan of a mock - infected vero control cell is provided in fig . while the endoplasmic reticulum region is clearly observed at the perinuclear region ( arrow ) , there is the lack of the filopodia and cytoskeleton clustering that is observed in the west nile virus - infected cells at the periphery of the plasma membrane . ( a ) afm height scan of west nile virus infected vero cells at 12 h p.i . ( b ) afm height scan of west nile virus infected vero cells at 24 h p.i . arrows show greater degree of thickening and clumping of actin filaments in close proximity to the cell periphery of the infected cell at 16 h p.i . arrows show the actin filaments forming near the cell edge and the nascent filopodia ( arrowheads ) . ( d ) afm height scan of mock - infected vero cells at 24 h p.i . this control cell shows a lack of cytoskeleton thickening and clumping at the periphery of the cell plasma membrane . these structures were not seen in mock - infected vero cells at 36 h p.i . these control cells also showed a lack of cytoskeletal formation at the periphery of the plasma membrane when compared to infected cells . 3b shows the filopodia ( arrows ) formation between four infected host cells at 36 h p.i . 3c shows bags of virus particles ( thin arrows ) liberated from the cells surface as well as the filopodia at 36 h p.i . during this stage of infection when the cytopathic effects were advanced , arrowhead shows the filopodia , but these were not as pronounced as in the afm imaging due to required ultrathin sectioning of the sample . ( a ) afm height scan of mock infected vero cells at 36 h p.i . scan size is 10 m 10 m . in the mock - infected control sample , ( b ) afm amplitude scan of west nile virus infected vero cells at 36 h p.i . ( c ) afm height scan of west nile virus infected vero cells at 36 h p.i . of 6.9 m 6.9 m size . thin arrows show enveloped bags containing progeny west nile viruses as they egress from host vero cells . ( d ) the transmission electron micrograph shows similar image as in ( c ) . the bags of virus ( arrows ) are seen exiting from the cell filopodia ( filopodia ) . the afm successfully imaged the west nile virus - induced changes in infected vero cells . this is the first study using atomic force microscopy for flaviviruses , a more cautious approach was taken . mildly - fixed infected cells were used to optimize the afm technique for virus - host interaction studies . to gain further confidence with this technique , as these samples were fixed and dried prior to imaging , certain considerations had to be addressed . the effects of this drying procedure may cause a certain degree of flattening to the nanostructures on the sample , and this was taken into account during interpretation of the results . however , the effect of fixation on these samples brings the samples morphologically more similar to the tem images that we used for comparison , and made such comparisons more meaningful . at such an early stage of research , confirmations and corroboration from other more established microscopy forms are essential to successfully interpret the results from the afm . the alternative contact mode involves the constant contact between the probe and the sample , thus causing damage to the soft biological samples . hard tapping was applied when there was a need to image sub - surface structures . these modes proved to be very compatible for virus and cell imaging . in the case of west nile ( sarafend ) virus infections , the new information provided by the afm gave further insight into virus - induced changes during the infection . nanostructures such as the budding of individual progeny viruses ( fig.1 ) or of bags containing progeny virus were observed as enveloped sacs associated with the filopodia extending from the plasma membrane in the west nile virus - infected vero cells ( fig . 2 ) . the afm can image the virus - induced changes in the host cell as well as minute structures ( 50 nm ) such as west nile virus particles illustrates that the afm is able to provide both the macro and nano data germane to virus - host interaction studies . the usefulness of hard tapping techniques was most notable in the observation of actin filament formation near the periphery of the infected cell membrane . the time course of west nile virus infection through the vero cell has been documented in previous studies [ 8 - 10 ] using other microscopy forms , and they corroborate the observations made in this study using the afm . the virus studied here is of great medical and economical impact on the world . flaviviruses have long been a medical problem in many parts of the world , especially with the resurgence of west nile virus infections in north america . with emerging and re - emerging viruses such as this , new and pertinent data of how these viruses replicate in their host cells is becoming increasingly and the nano - biotechnological field of afm represents a new set of " eyes " in which virologists may use to meet these new challenges . vero cells derived from african green monkey kidney ( a kind gift from professor e. g. westaway , sir albert sakzewski virus research laboratory , queensland , australia ) were used . cell lines were maintained in m199 growth media supplemented with 10% foetal calf serum ( fcs ) . cells were cultured onto sterile ethanol - treated coverslips placed into 24-well cell culture plates and incubated overnight to achieve a level of 80% confluency . west nile ( sarafend ) virus , a kind gift from professor e. g. westaway , sir albert sakzewski virus research laboratory , queensland , australia ) , was propagated in vero cells throughout this study . the vero cells were infected with west nile ( sarafend ) virus at a multiplicity of infection ( m.o.i . ) = 1 . the cells grown on the coverslips were washed once with pbs , before infection with 100 l of viruses for 1 h. the coverslips were rocked gently every 15 min to ensure the uniform spreading of the virus solution over the cell monolayer on the coverslip . after 1 h of infection , 1.5 ml of the appropriate growth media supplemented with 2% fcs was added to the coverslips , and these were left to incubate in a 5% co2 incubator at 37c . the samples were fixed , and imaged at 12 , 16 , 24 and 36 h p.i . as this is a pioneering study on the flavivirus , mildly fixed samples were used . the samples were washed thrice with pbs for the duration of 5 min each time , after which they were fixed with a 2% paraformaldehyde/0.2% glutaraldehyde solution at ph 7.4 for 30 min . a final wash with deionised water was followed by gentle drying with a nitrogen gas gun . the samples were then imaged in the afm using the force modulation etched silicon probe ( fesp ) , ( veeco , usa ) which has a length of 219 m and a vibrational frequency of 69 - 88 khz . at selected times p.i . , vero cells infected with wnv were washed twice with cold pbs and fixed with 0.2% glutaraldehyde/ 4 % paraformaldehyde for 30 min . the cell pellet was resuspended in 10% gelatin at room temperature and spin , and the gelatin with the pelleted cells was solidified at 4c for 10 min . the hardened block was post - fixed with the above fixative again for 20 min . the gelatin block was then cut into 1-mm cubes and immersed into cryo - protection buffer of 2.3 m sucrose for 2 h. the block was frozen by plunging into liquid nitrogen before cryo - ultramicrotomy , using an ultracut microtome ( uct ) with a cryo - attachment ( em fcs leica , austria ) . ultrathin sections of the infected cells were picked up onto formvar - coated grids and immunolabelling was carried out at room temperature . this was followed by three washes ( at 5 min each ) with 0.05 m glycine in pbs ( to block the aldehyde groups ) before incubating for 30 min in pbs with 5% bsa ( protein block ) . sections were then washed in 0.1% bsa in pbs ( 3 5 min ) and exposed to the primary antibody ( against the wnv envelope protein ) at 1:100 dilution ( 0.1% bsa in pbs ) for 1 h. six washes ( at 5 min each ) in 1% bsa in pbs was done before exposing the cells to the protein a colloidal gold ( 10 nm at 1:20 dilution to label envelope protein ) for 1 h. samples were then postfixed in 2% glutaraldehyde in pbs for 5 min and washed ( 2 5 min ) in pbs and distilled water ( 4 5 min ) . finally , sections were embedded in 2% uranyl acetate and 1.8% methyl cellulose mixture ( 1:9 parts ) for 5 min and dried before viewing under the cm120 biotwin transmission electron microscope ( philips , the netherlands ) . vero cells derived from african green monkey kidney ( a kind gift from professor e. g. westaway , sir albert sakzewski virus research laboratory , queensland , australia ) were used . cell lines were maintained in m199 growth media supplemented with 10% foetal calf serum ( fcs ) . cells were cultured onto sterile ethanol - treated coverslips placed into 24-well cell culture plates and incubated overnight to achieve a level of 80% confluency . west nile ( sarafend ) virus , a kind gift from professor e. g. westaway , sir albert sakzewski virus research laboratory , queensland , australia ) , was propagated in vero cells throughout this study . the vero cells were infected with west nile ( sarafend ) virus at a multiplicity of infection ( m.o.i . ) = 1 . the cells grown on the coverslips were washed once with pbs , before infection with 100 l of viruses for 1 h. the coverslips were rocked gently every 15 min to ensure the uniform spreading of the virus solution over the cell monolayer on the coverslip . after 1 h of infection , 1.5 ml of the appropriate growth media supplemented with 2% fcs was added to the coverslips , and these were left to incubate in a 5% co2 incubator at 37c . the samples were fixed , and imaged at 12 , 16 , 24 and 36 h p.i . as this is a pioneering study on the flavivirus , mildly fixed samples were used . the samples were washed thrice with pbs for the duration of 5 min each time , after which they were fixed with a 2% paraformaldehyde/0.2% glutaraldehyde solution at ph 7.4 for 30 min . a final wash with deionised water was followed by gentle drying with a nitrogen gas gun . the samples were then imaged in the afm using the force modulation etched silicon probe ( fesp ) , ( veeco , usa ) which has a length of 219 m and a vibrational frequency of 69 - 88 khz . at selected times p.i . , vero cells infected with wnv were washed twice with cold pbs and fixed with 0.2% glutaraldehyde/ 4 % paraformaldehyde for 30 min . the cell pellet was resuspended in 10% gelatin at room temperature and spin , and the gelatin with the pelleted cells was solidified at 4c for 10 min . the hardened block was post - fixed with the above fixative again for 20 min . the gelatin block was then cut into 1-mm cubes and immersed into cryo - protection buffer of 2.3 m sucrose for 2 h. the block was frozen by plunging into liquid nitrogen before cryo - ultramicrotomy , using an ultracut microtome ( uct ) with a cryo - attachment ( em fcs leica , austria ) . ultrathin sections of the infected cells were picked up onto formvar - coated grids and immunolabelling was carried out at room temperature . this was followed by three washes ( at 5 min each ) with 0.05 m glycine in pbs ( to block the aldehyde groups ) before incubating for 30 min in pbs with 5% bsa ( protein block ) . sections were then washed in 0.1% bsa in pbs ( 3 5 min ) and exposed to the primary antibody ( against the wnv envelope protein ) at 1:100 dilution ( 0.1% bsa in pbs ) for 1 h. six washes ( at 5 min each ) in 1% bsa in pbs was done before exposing the cells to the protein a colloidal gold ( 10 nm at 1:20 dilution to label envelope protein ) for 1 h. samples were then postfixed in 2% glutaraldehyde in pbs for 5 min and washed ( 2 5 min ) in pbs and distilled water ( 4 5 min ) . finally , sections were embedded in 2% uranyl acetate and 1.8% methyl cellulose mixture ( 1:9 parts ) for 5 min and dried before viewing under the cm120 biotwin transmission electron microscope ( philips , the netherlands ) . afm : atomic force microscope tem : transmission electron microscope sem : scanning electron microscope pbs : phosphate buffered saline bsa : bovine serum albumin h p.i . : hours post infection nml conceived of the study , and participated in its design and coordination . both authors read and approved the final manuscript .

backgroundmicroscopic imaging of viruses and their interactions with and effects on host cells are frequently held back by limitations of the microscope 's resolution or the invasive nature of the sample preparation procedures . it is also difficult to have a technique that would allow simultaneous imaging of both surface and sub - surface on the same cell . this has hampered endeavours to elucidate virus - host interactions . atomic force microscopy ( afm ) , which is commonly used in the physical sciences , is now becoming a good correlative form of microscopy used to complement existing optical , confocal and electron microscopy for biological applicationsresultsin this study , the west nile ( sarafend ) virus - infected vero cell model was used . the atomic force microscope was found to be useful in producing high resolution images of virus - host events with minimal sample processing requirements . the afm was able to image the budding of the west nile ( sarafend ) virus at the infected cell surface . proliferation of the filopodia and thickening of clusters of actin filaments accompanied west nile virus replication.conclusionsthe study shows that the afm is useful for virus - host interaction studies . the technique provides morphological information on both the virus and the host cell during the infection stages .

Background Results and Discussion West Nile (Sarafend) virus-induced changes in infected cells at late stage infection Conclusions Methods Cells Virus Flavivirus infection of cells Sample preparation AFM Cryo-Immunolabelling Electron Microscopy List of Abbreviations Used Authors Contributions Acknowledgement

viral infections represent a particularly difficult type of challenge to overcome . this is due in large part to the lack of understanding of virus interactions with their host cells . the small scale of viruses results in difficulties in the efficient imaging of virus - infected samples . to date , the microscopy forms that are able to achieve the level of magnification and resolution for imaging viruses are the scanning electron microscope ( sem ) and transmission electron microscope ( tem ) . the invention of the atomic force microscope ( afm ) by binnig and colleagues has allowed for high - resolution imaging of nanostructures in living samples [ 2 - 4 ] . a number of these studies involved the observation of purified samples of biomolecules including large viruses , for example , the tobacco mosaic virus , pox virus and human immunodeficiency virus [ 5 - 7 ] . the homogeneity in the sample appears to lend itself to better afm imaging . the afm is able to produce images in several different formats , of which three were consistently analysed : height , phase and amplitude . the height data image is obtained via recording the changes in afm scanner height as it shifts to keep the vibrational amplitude of the cantilever of the probe constant . the phase data image produces an image that provides information of the differing materials / texture in the sample by analyzing the different responses of the probe on such materials . the amplitude data image produces an emphasized view of the height data image by describing the change in the amplitude of the probe directly . while structures are not distorted in the amplitude data image , accurate quantitative measurements are more likely garnered from the height data image . the afm does not require much in sample processing : in fact , its main requirement is that the sample is well - adhered to a substrate such that it does not move around when the afm probe engages it . beyond that , additional sample preparation such as fixation or labelling is decided on a sample - specific basis . while a live sample would be an ideal specimen to study , biosafety concerns stipulated the use of mild fixation in our virus infected samples . fixation was preformed on mock - infected samples for the sake of consistency . as a result the objectives of this study are to investigate the suitability of atomic force microscopy for virus - host interaction studies using the flavivirus model . at the late stages of west nile ( sarafend ) virus infection in vero cells , several interesting aspects warranted investigation . active budding was also present at the proliferated filopodia . in conjunction with the budding of the maturing virus particles , there was a progressive lengthening and thickening of the actin filaments at the cell peripheral . the postulation was that the vectorial force of the growing length of the actin filament provided the bending force to expel the virus particles . 1b shows a higher resolution scan of the extruding virion , with the envelope clearly surrounding the virus nucleocapsid ( arrowhead ) . a tem image of a virus particle ( fig . ( a ) afm height scan of west nile virus - infected vero cells at 24 h p.i . a progeny virus ( arrow ) budding from the plasma membrane of the host cell . ( b ) afm height scan of west nile virus - infected vero cells at 24 h p.i . ( c ) tem image of west nile virus - infected vero cells ( arrowheads ) budding from host cell . the electron dense dots are the immunogold particles targeted against the envelope protein of the wnv . the hard tapping afm technique used provided a certain degree of " translucency " to the plasma membrane , which enabled the imaging of sub - surface structures in addition to producing surface morphological data . by adjusting the probe to engage the sample with greater force , the probe tip physically pushes against the soft sample surface to image sub - surface structures . it was through this ability that the afm was able to observe the up - regulation of actin filaments in the late infection stages . 2 the formation of actin filaments near the cell periphery in infected cells was seen over time . 2a shows some degree of enhancement of the cytoskeleton network around the cell periphery ( arrow ) in the west nile ( sarafend ) virus - infected vero cells at 12 h p.i . it is noted that the formation of cytoskeleton clustering in the infected cells appears to be proximal to the formation of filopodia . a lower magnification scan of a mock - infected vero control cell is provided in fig . while the endoplasmic reticulum region is clearly observed at the perinuclear region ( arrow ) , there is the lack of the filopodia and cytoskeleton clustering that is observed in the west nile virus - infected cells at the periphery of the plasma membrane . ( a ) afm height scan of west nile virus infected vero cells at 12 h p.i . ( b ) afm height scan of west nile virus infected vero cells at 24 h p.i . arrows show greater degree of thickening and clumping of actin filaments in close proximity to the cell periphery of the infected cell at 16 h p.i . arrows show the actin filaments forming near the cell edge and the nascent filopodia ( arrowheads ) . ( d ) afm height scan of mock - infected vero cells at 24 h p.i . this control cell shows a lack of cytoskeleton thickening and clumping at the periphery of the cell plasma membrane . these structures were not seen in mock - infected vero cells at 36 h p.i . these control cells also showed a lack of cytoskeletal formation at the periphery of the plasma membrane when compared to infected cells . 3b shows the filopodia ( arrows ) formation between four infected host cells at 36 h p.i . 3c shows bags of virus particles ( thin arrows ) liberated from the cells surface as well as the filopodia at 36 h p.i . during this stage of infection when the cytopathic effects were advanced , arrowhead shows the filopodia , but these were not as pronounced as in the afm imaging due to required ultrathin sectioning of the sample . ( a ) afm height scan of mock infected vero cells at 36 h p.i . in the mock - infected control sample , ( b ) afm amplitude scan of west nile virus infected vero cells at 36 h p.i . ( c ) afm height scan of west nile virus infected vero cells at 36 h p.i . thin arrows show enveloped bags containing progeny west nile viruses as they egress from host vero cells . ( d ) the transmission electron micrograph shows similar image as in ( c ) . the bags of virus ( arrows ) are seen exiting from the cell filopodia ( filopodia ) . at the late stages of west nile ( sarafend ) virus infection in vero cells , several interesting aspects warranted investigation . active budding was also present at the proliferated filopodia . in conjunction with the budding of the maturing virus particles , there was a progressive lengthening and thickening of the actin filaments at the cell peripheral . the postulation was that the vectorial force of the growing length of the actin filament provided the bending force to expel the virus particles . 1b shows a higher resolution scan of the extruding virion , with the envelope clearly surrounding the virus nucleocapsid ( arrowhead ) . a tem image of a virus particle ( fig . ( a ) afm height scan of west nile virus - infected vero cells at 24 h p.i . a progeny virus ( arrow ) budding from the plasma membrane of the host cell . ( b ) afm height scan of west nile virus - infected vero cells at 24 h p.i . ( c ) tem image of west nile virus - infected vero cells ( arrowheads ) budding from host cell . the electron dense dots are the immunogold particles targeted against the envelope protein of the wnv . the hard tapping afm technique used provided a certain degree of " translucency " to the plasma membrane , which enabled the imaging of sub - surface structures in addition to producing surface morphological data . by adjusting the probe to engage the sample with greater force , the probe tip physically pushes against the soft sample surface to image sub - surface structures . it was through this ability that the afm was able to observe the up - regulation of actin filaments in the late infection stages . 2 the formation of actin filaments near the cell periphery in infected cells was seen over time . 2a shows some degree of enhancement of the cytoskeleton network around the cell periphery ( arrow ) in the west nile ( sarafend ) virus - infected vero cells at 12 h p.i . it is noted that the formation of cytoskeleton clustering in the infected cells appears to be proximal to the formation of filopodia . this detailed progression of cytoskeleton enhancement can not be visualized by the sem ( as it can only provide surface morphology data ) or tem ( as it can only provide 2-dimensional data ) . a lower magnification scan of a mock - infected vero control cell is provided in fig . while the endoplasmic reticulum region is clearly observed at the perinuclear region ( arrow ) , there is the lack of the filopodia and cytoskeleton clustering that is observed in the west nile virus - infected cells at the periphery of the plasma membrane . ( a ) afm height scan of west nile virus infected vero cells at 12 h p.i . ( b ) afm height scan of west nile virus infected vero cells at 24 h p.i . arrows show greater degree of thickening and clumping of actin filaments in close proximity to the cell periphery of the infected cell at 16 h p.i . arrows show the actin filaments forming near the cell edge and the nascent filopodia ( arrowheads ) . ( d ) afm height scan of mock - infected vero cells at 24 h p.i . this control cell shows a lack of cytoskeleton thickening and clumping at the periphery of the cell plasma membrane . these structures were not seen in mock - infected vero cells at 36 h p.i . these control cells also showed a lack of cytoskeletal formation at the periphery of the plasma membrane when compared to infected cells . 3b shows the filopodia ( arrows ) formation between four infected host cells at 36 h p.i . 3c shows bags of virus particles ( thin arrows ) liberated from the cells surface as well as the filopodia at 36 h p.i . during this stage of infection when the cytopathic effects were advanced , arrowhead shows the filopodia , but these were not as pronounced as in the afm imaging due to required ultrathin sectioning of the sample . ( a ) afm height scan of mock infected vero cells at 36 h p.i . in the mock - infected control sample , ( b ) afm amplitude scan of west nile virus infected vero cells at 36 h p.i . ( c ) afm height scan of west nile virus infected vero cells at 36 h p.i . thin arrows show enveloped bags containing progeny west nile viruses as they egress from host vero cells . the bags of virus ( arrows ) are seen exiting from the cell filopodia ( filopodia ) . the afm successfully imaged the west nile virus - induced changes in infected vero cells . this is the first study using atomic force microscopy for flaviviruses , a more cautious approach was taken . mildly - fixed infected cells were used to optimize the afm technique for virus - host interaction studies . to gain further confidence with this technique , as these samples were fixed and dried prior to imaging , certain considerations had to be addressed . the effects of this drying procedure may cause a certain degree of flattening to the nanostructures on the sample , and this was taken into account during interpretation of the results . however , the effect of fixation on these samples brings the samples morphologically more similar to the tem images that we used for comparison , and made such comparisons more meaningful . at such an early stage of research , confirmations and corroboration from other more established microscopy forms are essential to successfully interpret the results from the afm . the alternative contact mode involves the constant contact between the probe and the sample , thus causing damage to the soft biological samples . hard tapping was applied when there was a need to image sub - surface structures . these modes proved to be very compatible for virus and cell imaging . in the case of west nile ( sarafend ) virus infections , the new information provided by the afm gave further insight into virus - induced changes during the infection . nanostructures such as the budding of individual progeny viruses ( fig.1 ) or of bags containing progeny virus were observed as enveloped sacs associated with the filopodia extending from the plasma membrane in the west nile virus - infected vero cells ( fig . the afm can image the virus - induced changes in the host cell as well as minute structures ( 50 nm ) such as west nile virus particles illustrates that the afm is able to provide both the macro and nano data germane to virus - host interaction studies . the usefulness of hard tapping techniques was most notable in the observation of actin filament formation near the periphery of the infected cell membrane . the time course of west nile virus infection through the vero cell has been documented in previous studies [ 8 - 10 ] using other microscopy forms , and they corroborate the observations made in this study using the afm . the virus studied here is of great medical and economical impact on the world . flaviviruses have long been a medical problem in many parts of the world , especially with the resurgence of west nile virus infections in north america . with emerging and re - emerging viruses such as this , new and pertinent data of how these viruses replicate in their host cells is becoming increasingly and the nano - biotechnological field of afm represents a new set of " eyes " in which virologists may use to meet these new challenges . west nile ( sarafend ) virus , a kind gift from professor e. g. westaway , sir albert sakzewski virus research laboratory , queensland , australia ) , was propagated in vero cells throughout this study . the vero cells were infected with west nile ( sarafend ) virus at a multiplicity of infection ( m.o.i . ) the cells grown on the coverslips were washed once with pbs , before infection with 100 l of viruses for 1 h. the coverslips were rocked gently every 15 min to ensure the uniform spreading of the virus solution over the cell monolayer on the coverslip . after 1 h of infection , 1.5 ml of the appropriate growth media supplemented with 2% fcs was added to the coverslips , and these were left to incubate in a 5% co2 incubator at 37c . as this is a pioneering study on the flavivirus , mildly fixed samples were used . a final wash with deionised water was followed by gentle drying with a nitrogen gas gun . the samples were then imaged in the afm using the force modulation etched silicon probe ( fesp ) , ( veeco , usa ) which has a length of 219 m and a vibrational frequency of 69 - 88 khz . the cell pellet was resuspended in 10% gelatin at room temperature and spin , and the gelatin with the pelleted cells was solidified at 4c for 10 min . ultrathin sections of the infected cells were picked up onto formvar - coated grids and immunolabelling was carried out at room temperature . finally , sections were embedded in 2% uranyl acetate and 1.8% methyl cellulose mixture ( 1:9 parts ) for 5 min and dried before viewing under the cm120 biotwin transmission electron microscope ( philips , the netherlands ) . west nile ( sarafend ) virus , a kind gift from professor e. g. westaway , sir albert sakzewski virus research laboratory , queensland , australia ) , was propagated in vero cells throughout this study . the vero cells were infected with west nile ( sarafend ) virus at a multiplicity of infection ( m.o.i . ) the cells grown on the coverslips were washed once with pbs , before infection with 100 l of viruses for 1 h. the coverslips were rocked gently every 15 min to ensure the uniform spreading of the virus solution over the cell monolayer on the coverslip . after 1 h of infection , 1.5 ml of the appropriate growth media supplemented with 2% fcs was added to the coverslips , and these were left to incubate in a 5% co2 incubator at 37c . as this is a pioneering study on the flavivirus , mildly fixed samples were used . the samples were washed thrice with pbs for the duration of 5 min each time , after which they were fixed with a 2% paraformaldehyde/0.2% glutaraldehyde solution at ph 7.4 for 30 min . a final wash with deionised water was followed by gentle drying with a nitrogen gas gun . the samples were then imaged in the afm using the force modulation etched silicon probe ( fesp ) , ( veeco , usa ) which has a length of 219 m and a vibrational frequency of 69 - 88 khz . , vero cells infected with wnv were washed twice with cold pbs and fixed with 0.2% glutaraldehyde/ 4 % paraformaldehyde for 30 min . the cell pellet was resuspended in 10% gelatin at room temperature and spin , and the gelatin with the pelleted cells was solidified at 4c for 10 min . ultrathin sections of the infected cells were picked up onto formvar - coated grids and immunolabelling was carried out at room temperature . this was followed by three washes ( at 5 min each ) with 0.05 m glycine in pbs ( to block the aldehyde groups ) before incubating for 30 min in pbs with 5% bsa ( protein block ) . sections were then washed in 0.1% bsa in pbs ( 3 5 min ) and exposed to the primary antibody ( against the wnv envelope protein ) at 1:100 dilution ( 0.1% bsa in pbs ) for 1 h. six washes ( at 5 min each ) in 1% bsa in pbs was done before exposing the cells to the protein a colloidal gold ( 10 nm at 1:20 dilution to label envelope protein ) for 1 h. samples were then postfixed in 2% glutaraldehyde in pbs for 5 min and washed ( 2 5 min ) in pbs and distilled water ( 4 5 min ) . finally , sections were embedded in 2% uranyl acetate and 1.8% methyl cellulose mixture ( 1:9 parts ) for 5 min and dried before viewing under the cm120 biotwin transmission electron microscope ( philips , the netherlands ) . afm : atomic force microscope tem : transmission electron microscope sem : scanning electron microscope pbs : phosphate buffered saline bsa : bovine serum albumin h p.i . : hours post infection nml conceived of the study , and participated in its design and coordination . both authors read and approved the final manuscript .

the chemical and physical principles underlying protein function can only be unraveled by gaining insight into both structural and dynamic features . nuclear magnetic resonance spectroscopy is unmatched in its ability to provide such insight at atomic resolution . nuclear spin relaxation , i.e. , the return of the perturbed magnetization to its equilibrium , allows one to characterize internal protein dynamics in two distinct ranges : fast pico- to nanosecond and slow micro- to millisecond time scales . overall rotational diffusion of proteins , which occurs typically on time scales c on the order of a few nanoseconds to several tens of nanoseconds , and internal motions on time scales int c lead to stochastic fluctuations of orientation - dependent spin interactions such as dipole dipole couplings and anisotropic chemical shifts . the resulting relaxation rates depend on spectral density functions j( ) , which are defined as fourier transforms of the correlation functions c(t ) of the orientation - dependent interactions . the measurement of a series of relaxation rates allows one to map the spectral density functions j( ) , thus providing quantitative parameters for models of overall and internal motions . most models employed to date rely on the hypothesis that internal motions are statistically independent of the rotational diffusion of a macromolecule , which is usually justified by the separation of the time scales of overall and internal motions , the latter being hitherto considered to be at least an order of magnitude faster than the former . internal motions are usually described by one or two order parameters and discrete internal correlation times or by a distribution of such internal correlation times . the hypothesis that the time scales of overall and local motions can be separated and that they are statistically independent has however been challenged . the discrimination between different models of motions has proven to be difficult because the spectral density functions could only be properly determined over few narrow ranges of fairly high frequencies i.e. , by measuring relaxation rates over a wide range of magnetic fields , typically from 1 t to 3 t. so far , this could only be achieved at the expense of sensitivity and resolution . relaxometry can be reconciled with high - resolution high - field nmr by rapidly shuttling slower shuttling has allowed fruitful studies of slowly relaxing phosphorus-31 and carbon-13 nuclei in lipids , but relaxation of nitrogen-15 in proteins is so fast at low fields that the shuttling must be carried out very rapidly . to the best of our knowledge , there is only one pioneering study , albeit shuttling was limited to a fairly narrow range of magnetic fields ( down to 4 t ) . here , we use high - resolution relaxometry combined with traditional high - field measurements to measure relaxation rates over nearly 2 orders of magnitude ( 0.522.3 t. ) we illustrate the power of this method by revealing internal nanosecond - time scale dynamics in the protein ubiquitin . ( poly)ubiquitination is a mechanism involved in many biological cell - signaling processes , from protein degradation to dna damage response . signaling is mediated by interactions of ubiquitin and polyubiquitin with a broad range of protein partners . such a diversity is made possible through the conformational flexibility of its 76 amino acid chain . this observation was highlighted in a recent study where the internal dynamics of ubiquitin were modified to bind selectively to a single partner , primarily by reducing the flexibility of its 12 turn ( residues 713 ) . it has been shown that the intrinsic flexibility of ubiquitin in its free apo form leads to a rich conformational landscape , which is similar to the conformational diversity in ubiquitin complexes . binding to a given partner can be described by an induced fit , a conformational selection , or an intermediate mechanism , depending on the time scales of conformational transitions and the lifetimes of encounter complexes . unraveling the time scales of internal motions in ubiquitin is obviously a prerequisite to understanding the kinetic pathways of binding reactions . many nmr studies have sought to identify signatures of chemical exchange in ubiquitin in solution , demonstrating the presence of internal motions on slow time scales ( 10 int 100 s ) . residual dipolar couplings ( rdcs ) also point to the presence of extensive slow supra-c time scales of 5 ns int 10 ms ( with c 5 ns ) , which could not be detected by high - field relaxation or by chemical exchange phenomena . several studies of faster sub-c motions with int c = 5 ns have been carried out using n or c relaxation . most of these studies rely on data acquired at a single high magnetic field and use simple spectral density functions . the results are often compared to those derived from rdc measurements , and discrepancies are attributed to contributions of supra-c motions . we show that high - resolution relaxometry can reveal a surprising complexity of internal protein motions with time scales comparable to overall tumbling that were overlooked in high - field relaxation studies . we show that slower supra-c motions have likely been overestimated so far , in particular in the essential extended 12 turn . the sensitivity of relaxometry to motions on nanosecond time scales is illustrated in figure 1 . we have simulated the longitudinal relaxation rates of nitrogen-15 at several magnetic fields , assuming motions over a range of time scales and amplitudes . an extended model - free spectral density function was assumed with variable order parameters and time scales for slow motions on nanosecond time scales . the longitudinal relaxation rates vary strongly with the magnetic field , depending on the parameters of local motions . both high- and low - field relaxation rates are remarkably sensitive to slow sub-c motions if the order parameters are low . the rates at low fields are more sensitive to internal motions when their correlation times are comparable to the overall tumbling time . relaxation rates at 0.5 t are more sensitive to internal motions than those recorded at 3 t , which underscores the advantages of studying relaxation at low magnetic fields . longitudinal relaxation rates are largely insensitive to internal motions with slow time scales int c/2 at 14.1 t , i.e. , at high magnetic fields where many studies of internal dynamics in ubiquitin have been carried out so far . the different patterns for the dependence of longitudinal relaxation rates at high and low fields underline the enhanced sensitivity to nanosecond motions of relaxation measurements over a broad range of magnetic fields . simulated dependence of longitudinal nitrogen-15 relaxation rates on internal motions with nanosecond time scales , i.e. , below and above the correlation time for overall tumbling c = 5 ns . an extended model - free spectral density function was used with the following parameters : c = 5 ns ; correlation time for fast internal motions fast = 10 ps ; order parameters for fast internal motions sfast = 0.8 ; order parameters for slow internal motions 0.1 < sslow < 1.0 ( x axis ) ; 1 ns < int < 15 ns ( y axis ) . we developed a pneumatic system for fast shuttling , based on a system that was originally developed for liquid - state dynamic nuclear polarization ( dnp ) studies where the proton polarization observed at 14.1 t can be enhanced by saturating epr transitions at 0.34 t. our shuttle consists of a custom - designed probe ( figure 2a , b ) , a transfer system , and a control unit , as described in more detail in supporting information . the probe uses two saddle coils , like in standard high - resolution probes . the inner coil is doubly tuned for c and n , while the orthogonal outer coil is doubly tuned for h and h. this reduces interactions between the sample and the electric component of the rf field , albeit at the expense of a slight loss of sensitivity . the design affords a spectral resolution and line shapes comparable to those obtained using state - of - the - art high - resolution probes at 600 mhz . special care was taken ( figure 2a ) to reduce vibrations arising from the abrupt landing of the shuttle at the lower end ( see supporting information ) . the upper position of the shuttle is controlled by an adjustable inner tube . the inner tube is connected to the guiding tube by another damping system to reduce vibrations . synthetic amorphous quartz glass with a low magnetic susceptibility can resist a large number of shocks . ( a single container was used for more than 500 000 shuttling events in the course of this study . ) in the shuttle container , a 100 l sample compartment is separated from a 10 l bubble catcher compartment by a narrow capillary ( 150 m inner diameter ) . bubbles appearing in the course of extensive experimental series can be centrifuged into the bubble catcher before resuming the experiments . the active volume that lies within the rf coils is 60 l ( figure 2d ) . the resulting sensitivity of this system is about an order of magnitude lower than the sensitivity of a room - temperature txi probe used with a large - volume sample . this currently limits applications to the study of ( bio)molecules that have a good solubility or favorable relaxation properties . schematic views of the fast shuttling system : ( a ) expanded view of the upper part of the probe ; ( b ) coil assembly with shuttle container ; ( c ) shuttle container ; ( d ) schematic view ( not to scale ) of the full assembly in the bore of a high - field magnet . we used this shuttling system to measure the longitudinal relaxation of backbone nitrogen-15 nuclei in uniformly nitrogen-15 labeled human ubiquitin in acetate buffer at ph 4.5 and t = 296.6 0.6 k. the pulse sequence used for these measurements is shown in figure 3 . after a recovery delay in high field ( b0 = 14.1 t ) to allow the boltzmann polarization to build up , the temperature to be regulated , and the field - frequency lock to stabilize the field , the longitudinal polarization nz of nitrogen-15 is enhanced using the refocused inept method . the sample is then transferred in 41 < up < 54 ms to a predetermined position in the stray field 27 < z < 46 cm above the magnetic center . the polarization is then allowed to relax in a low field b0 for a duration trel and transferred back to high field b0 in 40 < down < 70 ms . during a stabilization delay st = 100 ms , convection and vibrations finally , the longitudinal nitrogen-15 polarization nz is converted back into transverse proton magnetization for detection . the average signal - to - noise ratios in two - dimensional ( 2d ) spectra obtained for the shortest relaxation delays trel = 39 ms were s / n = 66 at b0 = 5 t ( at z = 27 cm above the magnetic center ) and s / n = 24 for trel = 51 ms at b0 = 0.5 t ( z = 46 cm ) when 16 transients were recorded for each of 64 complex points in the indirect t1 dimension ( the experimental time was 85 min for each interval trel ) . in addition , a full set of conventional nitrogen-15 relaxation experiments was recorded without shuttling at 14.1 , 18.8 , and 22.3 t ( 600 , 800 , and 950 mhz for h ) using state - of - the - art methods to cancel cross - correlation effects . the parameters of rotational diffusion were derived from relaxation rates using the program rotdif . diffusion tensors obtained at all three high fields are virtually identical ( see supporting information ) . on the basis of a solution - state structure of ubiquitin ( pdb code 1d3z ) , we obtained an axially symmetric diffusion tensor with d/d = 1.18 0.08 and an overall correlation time c = ( 6tr(d ) ) = 4.84 0.2 ns , where tr(d ) is the trace of the diffusion tensor from our measurements at 18.8 t. at a lower concentration ( 200 m ) at 14.1 t we found a slightly shorter overall correlation time c = 4.22 0.15 ns . the small variation of c and the fact that the anisotropy and orientation of the diffusion tensor were found to be independent of concentration ( see supporting information ) suggest that the protein is in monomeric form at ph 4.5 and not in a monomer / dimer equilibrium , as found at neutral ph . in our case , the low ph leads to the protonation of his68 and is likely to reduce the binding affinity of symmetric dimers that associate at the hydrophobic patch at neutral ph , as shown in diubiquitin . therefore , we believe that the slight increase of the overall correlation time c was due to nonspecific intermolecular interactions at high concentration so that we could describe the overall rotational diffusion by a single time - independent tensor . pulse sequence for the measurement of longitudinal nitrogen-15 relaxation of amide nitrogen nuclei in proteins at various low fields b0 and recovery and detection at high field b0 . narrow ( filled ) and wide ( open ) rectangles represent 90 and 180 pulses , respectively . pulse phases are along the x - axis of the rotating frame unless otherwise specified . all delays a are set to 1/|4jnh| , with jnh = 92 hz . the stabilization delay st = 100 ms allows for convection currents and vibrations to settle . . their peak amplitudes are g1 = 25 , g2 = 40 , g3 = 11.5 , g4 = 20.5 , g5 = 40 , g6 = 15 g cm . the phase cycles were 1 = { y , y , y , y , y , y , y , y } ; 2 = { x , x } ; 3 = { y , y , y , y , y , y , y , y , y , y , y , y , y , y , y , y } ; 4 = { x , x , x , x } ; acq = { x , x , x , x , x , x , x , x , x , x , x , x , x , x , x , x}. particular care has been taken to control the temperature in these experiments , since temperature regulation at low field b0 is not yet feasible in our prototype . we have measured differences of chemical shifts between subsets of signals and calibrated them as a function of temperature . the actual sample temperature was then derived from these chemical shift differences in each experiment . only experiments with 296 t 297.2 k were retained in the analysis so that systematic errors can be safely neglected ( see supporting information ) . conventional high field - relaxation measurements at 14.1 and 18.8 t were carried out at 296.5 k. rates measured at 22.3 t and 298.5 k were corrected to account for the change in c . the longitudinal relaxation rates r1(b0 ) = 1/t1(b0 ) of n nuclei in the backbone of ubiquitin determined at 10 different fields are shown in figure 4 . cross - relaxation pathways lead to multiexponential decays , which in high fields can usually be transformed into monoexponential decays by suitable pulse sequences . since in our prototype it is not possible to apply any rf pulses in low fields , systematic deviations from monoexponential decays must be taken into account in the analysis . we have developed a protocol dubbed iterative correction for the analysis of relaxation under shuttling first , the analysis of relaxation rates was carried out ( in terms of overall tumbling and microdynamics ) at all three high fields 14.1 , 18.8 , and 22.3 t using the programs rotdif and dynamics . the parameters resulting from this initial step were then used to predict the deviations from simple exponential decays in a spin system that comprises one n h pair and two remote protons ( see supporting information ) . for each field b0 , we simulated the relaxation of each n h pair in ubiquitin during shuttling , with constant velocity ( see supporting information ) . the deviations between the calculated apparent nitrogen-15 relaxation rates and the true low - field relaxation rates were then used to correct for systematic errors in the experimental rates . in a second iteration , longitudinal relaxation rates at all 10 fields and n{h } noes at the three high fields 14.1 , 18.8 , and 22.3 t were used as input to the program dynamics . this cycle was reiterated four times to achieve a satisfactory convergence for all residues . cross - correlations of the fluctuations of nitrogen-15 chemical shift anisotropies and n h dipolar couplings are dominant above 3 t , with average corrections ranging from 5.1% at 3 t to 9.2% at 5 t , while n h dipolar cross - relaxation dominates below 2 t , with corrections on the order of 11% at fields below 1 t. experimental longitudinal relaxation rates r1 = 1/t1 of n in backbone amide groups of ubiquitin as a function of magnetic fields 0.5 b0 5 t and 14.1 b0 22.3 t. from bottom to top : b0 = 22.3 , 18.8 , 14.1 ( red squares , magenta crosses , blue triangles ) ; b0 = 22.3 , 18.8 , 14.1 5.0 , 3.0 , 2.0 , 1.4 , 1.0 , 0.74 , and 0.5 t ( cyan diamonds , green circles , orange squares , red crosses , magenta triangles , blue diamonds , cyan circles ) . the lower b0 is , the greater are the variations of the rates along the backbone . the results of this analysis are shown in figure 5 . as expected from studies of n and c relaxation , residual dipolar couplings , and molecular dynamics , we find that ubiquitin is fairly rigid . however , we determined the order parameters to be significantly lower than in earlier relaxation - based studies . we may compare ( figure 4 ) ( i ) the order parameters resulting from all 10 fields , ( ii ) those obtained from relaxation rates at 14.1 t only , and ( iii ) those obtained at three high fields ( 14.1 , 18.8 , and 22.3 t ) . with few exceptions , the order parameters resulting from our analysis of relaxation at 10 magnetic fields are the lowest . in particular , the dynamics of the crucial 12 turn ( residues 712 ) can be best described by an extended model - free spectral density function with similar time scales for all six residues ( see supporting information ) . a global fit of these six residues gives a common effective time scale 712 = 2 ns . this is in good agreement with the well - documented hypothesis of a collective motion . this motion was so far believed to occur on a much slower , so - called supra-c time scale 712 > c , since rdc studies indicated large - amplitude motions while relaxation at a single high field failed to detect such motions . interestingly , lower order parameters and motions on similar time scales ( see supporting information ) are also found at the c - terminus of helix 1 and loop 14 ( residues 33 and 36 ) , which participate , along with the 12 turn , in the principal mode of ubiquitin dynamics . the relaxation of this principal mode could be described in silico with two time scales , 0.4 and 13 ns . our analysis assumes only one single time scale for the semilocal motions , and the fit leads to an intermediate value , 712 = 2 ns , which could well result from effective averaging between these two time scales . more complex models of spectral density functions should open the way to a better agreement between experimental rates and theory . ( a ) order parameters s in ubiquitin obtained from the analysis of nitrogen-15 relaxation rates , taking into account ( red ) relaxation rates at 14.1 t only ; ( black ) relaxation data at three fields 14.1 , 18.8 , and 22.3 t ; ( green ) relaxation rates at all 10 fields from 0.5 to 22.3 t. ( b ) comparison of order parameters obtained from relaxation rates at all fields ( green ) and from analysis of residual dipolar couplings ( rdcs ) in large sets of orienting media , either by gaf ( blue ) or scrm ( purple ) . the inability of high - field relaxation studies to identify these motions is hardly surprising , since most studies have only been carried out at a single field . therefore , the sampling of the spectral density function was insufficient and did not characterize sufficiently well motions on nanosecond time scales . the consequences of undersampling of the spectral density function are exacerbated if one uses simple models that do not properly reproduce the actual spectral density functions . strikingly , the order parameters for fast motions obtained by extended - model free analysis of all relaxation rates match very well with order parameters obtained from the analysis of relaxation data at 14.1 t only ( see supporting information ) . this point is also nicely illustrated by the analysis of a 1.2 s molecular dynamics trajectory of ubiquitin . in this study , order parameters s derived from the average orientations of nh vectors were low for the 12 turn . however , when the nonexponential correlation functions were forced to fit with a simple extended model - free correlation function , the order parameters became significantly higher , similar to those found in relaxation studies at 14.1 t. figure 5b presents the comparison of orders parameters obtained ( i ) from our relaxometry analysis of relaxation at 10 magnetic fields and ( ii ) from two independent analyses of residual dipolar couplings ( rdcs ) in large sets of oriented media using the gaussian axial fluctuations ( gaf ) or the self - consistent rdc - based model - free analysis ( scrm ) approaches . the three profiles are similar . in particular , the order parameters in the 12 turn ( residues 712 ) are almost identical so that one expects the amplitudes of motions in this turn that can not be detected by relaxation to be very small . significant correlations between slow supra-c motions of the 12 turn and those of the sheet are therefore unlikely , although correlated motions in the core of the -sheet can not be excluded . our relaxometry data show that the whole 2 strand , which lies at the edge of the -sheet , is significantly dynamic . similarly , studies of the third immunoglobin binding domain of streptococcal protein g ( gb3 ) have also shown the presence of enhanced motions in the last strand of an otherwise fairly rigid -sheet . these results differ from those of a gaf analysis of dynamics in ubiquitin , where the 2-strand is found to be rigid . mobile residues with intermediate and small order parameters are shown in yellow ( 0.70 < s < 0.75 ) , orange ( 0.60 < s < 0.70 ) , and red ( s < 0.6 . ) . the main interface with binding partners comprises the side chains of residues leu8 , ile44 , his68 , and val70 represented by space - filling models . in addition to this region , our relaxometry method allowed us to detect enhanced dynamics in several loops : 21 , 42 , and 25 as well as in the 34 turn and 4 strand , which lies at the opposite edge of the sheet . this is , again , in good qualitative agreement with both rdc studies ( see figure 5b ) . the agreement with accelerated molecular dynamics simulations is excellent , with a good correlation coefficient ( r = 0.91 ) between the two data sets ( see supporting information ) . note , however , that the order parameters found by relaxometry are systematically ( albeit only slightly ) lower than those obtained by molecular dynamics . the edges of the -sheet are occupied by the 12 turn and the flexible c - terminal tail at one end and by the 25 loop and 34 turn at the other end , with the 2 and 4 strands on each side . all of these regions are found to be dynamic , albeit to different extent . the picture that emerges is a hierarchy of time scales near the main binding interface of ubiquitin that consists of a -sheet with a core that is flexible on a slow time scale of about 50 s , while its edges are mobile on faster nanosecond time scales . between these two time scales , small correlated fluctuations of the -sheet . the ability of the edges of the interface to undergo conformational rearrangements on nanosecond time scales would be compatible with an induced fit mechanism in the early stage of binding . longitudinal relaxation rates r1(b0 ) as a function of the static field ( so - called relaxometry dispersion profiles ) for eight selected residues in ubiquitin . note that the vertical scale is expanded by a factor of 5 for the two c - terminal glycines ( bottom right ) . the blue and red dots show corrected longitudinal relaxation rates , adjusted to compensate for relaxation during shuttling , while the lines show dispersion profiles calculated from the microdynamic parameters obtained in our analysis . some dispersion profiles feature systematic discrepancies , which highlight the limitations of current models of spectral density functions . our experimental data call for the development of new , more sophisticated models . some relaxation profiles , e.g. , for the highly mobile c - terminal glycine residues g75 and g76 , present deviations from the theoretical profiles at both high and low magnetic fields , even when postulating a spectral density function comprising a sum of three lorentzian functions with five adjustable parameters , thus suggesting the presence of a distribution of time scales . in particular , with only two time scales for internal motions , the fitted spectral density function is rather flat at high frequencies . hence , contributions of the chemical shift anisotropy to relaxation lead to an increase in the r1(b0 ) curve between 14 and 23 t , in contradiction with experimental results . similarly , a small but systematic underestimation of the spectral density j(=0 ) in some of the most mobile regions , and hence of the back - predicted transverse relaxation rates ( see supporting information ) , can be understood by postulating a rapid initial decay of the spectral density function at low frequencies . interestingly , no significant contribution of chemical exchange to transverse relaxation rex could be detected ( see supporting information ) . the analysis of relaxation at three high fields also leads to underestimate j(=0 ) , as illustrated by the need for urealistic rex contributions to fit all relaxation data . nanosecond fluctuations of the overall diffusion tensor associated with motions of the c - terminal tail , transient oligomerization , and mode coupling of local and global motions may be responsible for these unexpected features . we have measured and analyzed residue - specific relaxation rates in a protein over a range of nearly 2 orders of magnitude of magnetic fields . in particular , the motion of the 12 turn appears to have larger amplitudes than could be previously identified by relaxation at high fields , in agreement with rdcs and md . until now , discrepancies between high - field relaxation and rdc - based methods were attributed to the cutoff of internal motions by overall rotation . high - field relaxation studies have led to underestimate sub-c and near-c motions because relaxation rates in high fields are not sufficiently sensitive to motions in the nanosecond frequency range . although many proteins , and ubiquitin in particular , are mobile on slow supra-c time scales ( slower than overall rotational diffusion ) , a mere comparison of order parameters obtained from high - field relaxation and rdcs is likely to overestimate such slow motions . this study shows that high - resolution relaxometry with fast sample shuttling allows one to map the spectral density functions in exquisite detail and offers unprecedented information about local motions in proteins on time scales that are faster than or comparable to their overall tumbling . the magnetic field was measured as a function of the height above the magnetic center in steps of 1 mm using a homemade mapping device with two calibrated triple - axes hall probes ( senis ) with a precision of 0.1% . a ch3a10me3d transducer was used for measurements from 0.05 to 2 t , while a 03a05f - a20t0k5q transducer was used between 1 and 13 t. the experiments were carried out on samples of 0.2 and 3 mm uniformly n labeled human ubiquitin ( giotto ) in 50 mm acetate buffer ( ph 4.5 ) in h2o / d2o ( 90/10 v / v ) at 296 t 297.2 k using a 600 mhz bruker avance iii spectrometer equipped with our pneumatic sample shuttle for measurements at low field . the pulse sequence shown in figure 3 was used for 0.5 < b0 < 5 t , with a recovery delay of 2.2 s. all experiments were acquired with 16 transients and 64 complex points in the indirect t1 dimension . water - flip back pulses were applied to minimize the saturation of the water resonance ; the watergate scheme was used prior to detection . frequency sign discrimination in the 1 domain was achieved with the states tppi method . a full relaxation decay comprising 78 interleaved spectra could be recorded in 1012 h. all signals were recorded at 14.1 t with a prototype probe equipped with z axis gradients and processed and analyzed with nmrpipe . the analysis of relaxation rates was performed with our icarus process , which uses rotdif and dynamics at each iteration . a full description is given in supporting information . in order to account for systematic errors , a jack - knife procedure was used : the analysis was repeated seven times while excluding one of the seven low - field relaxation rates . the order parameters shown in figure 5 result from the average over the seven analyses , and the errors correspond to the standard deviation of all seven values weighted by 6 . the analysis was carried out with similar parameters as in many other nmr studies of protein dynamics . an internuclear nitrogen hydrogen distance dnh = 1.02 and a n chemical shift anisotropy of 160 ppm were assumed to be common to all peptide bonds . the effective distances between the h amide proton and the two additional protons are critical for scaling the corrections in the icarus procedure . in order to determine these distances , we measured longitudinal relaxation rates at 14.1 t with experiments similar to the shuttling method but where the longitudinal nitrogen-15 polarization is allowed to evolve during fixed intervals before and after the relaxation delay , during which no rf pulses are applied . the optimal distances dhh were found to vary between 1.6 and 2.7 with an average of 2.1 . this result is confirmed by a computation of the sum of dipolar interactions with all protons ( see supporting information ) where the median value corresponds to an effective distance dhh = 2.07 . unfortunately , site - specific variations of dhh obtained in the two approaches were only weakly correlated so that we decided to use an average dhh = 2.1 for all residues . in order to evaluate the potential systematic errors of the resulting order parameters , we carried out a complete icarus analysis for a series of distances 1.7 < dhh < 2.6 . order parameters of some sites tend to be sensitive to the distance dhh , but the main features of our analysis , in particular the low order parameters found in the 12 turn , remain stable regardless of the distance dhh .

understanding the molecular determinants underlying protein function requires the characterization of both structure and dynamics at atomic resolution . nuclear relaxation rates allow a precise characterization of protein dynamics at the larmor frequencies of spins . this usually limits the sampling of motions to a narrow range of frequencies corresponding to high magnetic fields . at lower fields one can not achieve sufficient sensitivity and resolution in nmr . here , we use a fast shuttle device where the polarization builds up and the signals are detected at high field , while longitudinal relaxation takes place at low fields 0.5 < b0 < 14.1 t. the sample is propelled over a distance up to 50 cm by a blowgun - like system in about 50 ms . the analysis of nitrogen-15 relaxation in the protein ubiquitin over such a wide range of magnetic fields offers unprecedented insights into molecular dynamics . some key regions of the protein feature structural fluctuations on nanosecond time scales , which have so far been overlooked in high - field relaxation studies . nanosecond motions in proteins may have been underestimated by traditional high - field approaches , and slower supra-c motions that have no effect on relaxation may have been overestimated . high - resolution relaxometry thus opens the way to a quantitative characterization of nanosecond motions in proteins .

Introduction Results and Discussion Methods

, the return of the perturbed magnetization to its equilibrium , allows one to characterize internal protein dynamics in two distinct ranges : fast pico- to nanosecond and slow micro- to millisecond time scales . overall rotational diffusion of proteins , which occurs typically on time scales c on the order of a few nanoseconds to several tens of nanoseconds , and internal motions on time scales int c lead to stochastic fluctuations of orientation - dependent spin interactions such as dipole dipole couplings and anisotropic chemical shifts . the resulting relaxation rates depend on spectral density functions j( ) , which are defined as fourier transforms of the correlation functions c(t ) of the orientation - dependent interactions . most models employed to date rely on the hypothesis that internal motions are statistically independent of the rotational diffusion of a macromolecule , which is usually justified by the separation of the time scales of overall and internal motions , the latter being hitherto considered to be at least an order of magnitude faster than the former . , by measuring relaxation rates over a wide range of magnetic fields , typically from 1 t to 3 t. so far , this could only be achieved at the expense of sensitivity and resolution . relaxometry can be reconciled with high - resolution high - field nmr by rapidly shuttling slower shuttling has allowed fruitful studies of slowly relaxing phosphorus-31 and carbon-13 nuclei in lipids , but relaxation of nitrogen-15 in proteins is so fast at low fields that the shuttling must be carried out very rapidly . to the best of our knowledge , there is only one pioneering study , albeit shuttling was limited to a fairly narrow range of magnetic fields ( down to 4 t ) . here , we use high - resolution relaxometry combined with traditional high - field measurements to measure relaxation rates over nearly 2 orders of magnitude ( 0.522.3 t. ) we illustrate the power of this method by revealing internal nanosecond - time scale dynamics in the protein ubiquitin . binding to a given partner can be described by an induced fit , a conformational selection , or an intermediate mechanism , depending on the time scales of conformational transitions and the lifetimes of encounter complexes . unraveling the time scales of internal motions in ubiquitin is obviously a prerequisite to understanding the kinetic pathways of binding reactions . residual dipolar couplings ( rdcs ) also point to the presence of extensive slow supra-c time scales of 5 ns int 10 ms ( with c 5 ns ) , which could not be detected by high - field relaxation or by chemical exchange phenomena . we show that high - resolution relaxometry can reveal a surprising complexity of internal protein motions with time scales comparable to overall tumbling that were overlooked in high - field relaxation studies . we show that slower supra-c motions have likely been overestimated so far , in particular in the essential extended 12 turn . the sensitivity of relaxometry to motions on nanosecond time scales is illustrated in figure 1 . we have simulated the longitudinal relaxation rates of nitrogen-15 at several magnetic fields , assuming motions over a range of time scales and amplitudes . an extended model - free spectral density function was assumed with variable order parameters and time scales for slow motions on nanosecond time scales . the longitudinal relaxation rates vary strongly with the magnetic field , depending on the parameters of local motions . both high- and low - field relaxation rates are remarkably sensitive to slow sub-c motions if the order parameters are low . relaxation rates at 0.5 t are more sensitive to internal motions than those recorded at 3 t , which underscores the advantages of studying relaxation at low magnetic fields . longitudinal relaxation rates are largely insensitive to internal motions with slow time scales int c/2 at 14.1 t , i.e. , at high magnetic fields where many studies of internal dynamics in ubiquitin have been carried out so far . the different patterns for the dependence of longitudinal relaxation rates at high and low fields underline the enhanced sensitivity to nanosecond motions of relaxation measurements over a broad range of magnetic fields . simulated dependence of longitudinal nitrogen-15 relaxation rates on internal motions with nanosecond time scales , i.e. we developed a pneumatic system for fast shuttling , based on a system that was originally developed for liquid - state dynamic nuclear polarization ( dnp ) studies where the proton polarization observed at 14.1 t can be enhanced by saturating epr transitions at 0.34 t. our shuttle consists of a custom - designed probe ( figure 2a , b ) , a transfer system , and a control unit , as described in more detail in supporting information . the inner coil is doubly tuned for c and n , while the orthogonal outer coil is doubly tuned for h and h. this reduces interactions between the sample and the electric component of the rf field , albeit at the expense of a slight loss of sensitivity . in the shuttle container , a 100 l sample compartment is separated from a 10 l bubble catcher compartment by a narrow capillary ( 150 m inner diameter ) . schematic views of the fast shuttling system : ( a ) expanded view of the upper part of the probe ; ( b ) coil assembly with shuttle container ; ( c ) shuttle container ; ( d ) schematic view ( not to scale ) of the full assembly in the bore of a high - field magnet . after a recovery delay in high field ( b0 = 14.1 t ) to allow the boltzmann polarization to build up , the temperature to be regulated , and the field - frequency lock to stabilize the field , the longitudinal polarization nz of nitrogen-15 is enhanced using the refocused inept method . the sample is then transferred in 41 < up < 54 ms to a predetermined position in the stray field 27 < z < 46 cm above the magnetic center . the polarization is then allowed to relax in a low field b0 for a duration trel and transferred back to high field b0 in 40 < down < 70 ms . in addition , a full set of conventional nitrogen-15 relaxation experiments was recorded without shuttling at 14.1 , 18.8 , and 22.3 t ( 600 , 800 , and 950 mhz for h ) using state - of - the - art methods to cancel cross - correlation effects . on the basis of a solution - state structure of ubiquitin ( pdb code 1d3z ) , we obtained an axially symmetric diffusion tensor with d/d = 1.18 0.08 and an overall correlation time c = ( 6tr(d ) ) = 4.84 0.2 ns , where tr(d ) is the trace of the diffusion tensor from our measurements at 18.8 t. at a lower concentration ( 200 m ) at 14.1 t we found a slightly shorter overall correlation time c = 4.22 0.15 ns . the small variation of c and the fact that the anisotropy and orientation of the diffusion tensor were found to be independent of concentration ( see supporting information ) suggest that the protein is in monomeric form at ph 4.5 and not in a monomer / dimer equilibrium , as found at neutral ph . therefore , we believe that the slight increase of the overall correlation time c was due to nonspecific intermolecular interactions at high concentration so that we could describe the overall rotational diffusion by a single time - independent tensor . pulse sequence for the measurement of longitudinal nitrogen-15 relaxation of amide nitrogen nuclei in proteins at various low fields b0 and recovery and detection at high field b0 . the longitudinal relaxation rates r1(b0 ) = 1/t1(b0 ) of n nuclei in the backbone of ubiquitin determined at 10 different fields are shown in figure 4 . since in our prototype it is not possible to apply any rf pulses in low fields , systematic deviations from monoexponential decays must be taken into account in the analysis . we have developed a protocol dubbed iterative correction for the analysis of relaxation under shuttling first , the analysis of relaxation rates was carried out ( in terms of overall tumbling and microdynamics ) at all three high fields 14.1 , 18.8 , and 22.3 t using the programs rotdif and dynamics . the deviations between the calculated apparent nitrogen-15 relaxation rates and the true low - field relaxation rates were then used to correct for systematic errors in the experimental rates . in a second iteration , longitudinal relaxation rates at all 10 fields and n{h } noes at the three high fields 14.1 , 18.8 , and 22.3 t were used as input to the program dynamics . cross - correlations of the fluctuations of nitrogen-15 chemical shift anisotropies and n h dipolar couplings are dominant above 3 t , with average corrections ranging from 5.1% at 3 t to 9.2% at 5 t , while n h dipolar cross - relaxation dominates below 2 t , with corrections on the order of 11% at fields below 1 t. experimental longitudinal relaxation rates r1 = 1/t1 of n in backbone amide groups of ubiquitin as a function of magnetic fields 0.5 b0 5 t and 14.1 b0 22.3 t. from bottom to top : b0 = 22.3 , 18.8 , 14.1 ( red squares , magenta crosses , blue triangles ) ; b0 = 22.3 , 18.8 , 14.1 5.0 , 3.0 , 2.0 , 1.4 , 1.0 , 0.74 , and 0.5 t ( cyan diamonds , green circles , orange squares , red crosses , magenta triangles , blue diamonds , cyan circles ) . as expected from studies of n and c relaxation , residual dipolar couplings , and molecular dynamics , we find that ubiquitin is fairly rigid . we may compare ( figure 4 ) ( i ) the order parameters resulting from all 10 fields , ( ii ) those obtained from relaxation rates at 14.1 t only , and ( iii ) those obtained at three high fields ( 14.1 , 18.8 , and 22.3 t ) . interestingly , lower order parameters and motions on similar time scales ( see supporting information ) are also found at the c - terminus of helix 1 and loop 14 ( residues 33 and 36 ) , which participate , along with the 12 turn , in the principal mode of ubiquitin dynamics . our analysis assumes only one single time scale for the semilocal motions , and the fit leads to an intermediate value , 712 = 2 ns , which could well result from effective averaging between these two time scales . more complex models of spectral density functions should open the way to a better agreement between experimental rates and theory . ( a ) order parameters s in ubiquitin obtained from the analysis of nitrogen-15 relaxation rates , taking into account ( red ) relaxation rates at 14.1 t only ; ( black ) relaxation data at three fields 14.1 , 18.8 , and 22.3 t ; ( green ) relaxation rates at all 10 fields from 0.5 to 22.3 t. ( b ) comparison of order parameters obtained from relaxation rates at all fields ( green ) and from analysis of residual dipolar couplings ( rdcs ) in large sets of orienting media , either by gaf ( blue ) or scrm ( purple ) . the inability of high - field relaxation studies to identify these motions is hardly surprising , since most studies have only been carried out at a single field . therefore , the sampling of the spectral density function was insufficient and did not characterize sufficiently well motions on nanosecond time scales . strikingly , the order parameters for fast motions obtained by extended - model free analysis of all relaxation rates match very well with order parameters obtained from the analysis of relaxation data at 14.1 t only ( see supporting information ) . this point is also nicely illustrated by the analysis of a 1.2 s molecular dynamics trajectory of ubiquitin . however , when the nonexponential correlation functions were forced to fit with a simple extended model - free correlation function , the order parameters became significantly higher , similar to those found in relaxation studies at 14.1 t. figure 5b presents the comparison of orders parameters obtained ( i ) from our relaxometry analysis of relaxation at 10 magnetic fields and ( ii ) from two independent analyses of residual dipolar couplings ( rdcs ) in large sets of oriented media using the gaussian axial fluctuations ( gaf ) or the self - consistent rdc - based model - free analysis ( scrm ) approaches . in particular , the order parameters in the 12 turn ( residues 712 ) are almost identical so that one expects the amplitudes of motions in this turn that can not be detected by relaxation to be very small . significant correlations between slow supra-c motions of the 12 turn and those of the sheet are therefore unlikely , although correlated motions in the core of the -sheet can not be excluded . our relaxometry data show that the whole 2 strand , which lies at the edge of the -sheet , is significantly dynamic . similarly , studies of the third immunoglobin binding domain of streptococcal protein g ( gb3 ) have also shown the presence of enhanced motions in the last strand of an otherwise fairly rigid -sheet . in addition to this region , our relaxometry method allowed us to detect enhanced dynamics in several loops : 21 , 42 , and 25 as well as in the 34 turn and 4 strand , which lies at the opposite edge of the sheet . the edges of the -sheet are occupied by the 12 turn and the flexible c - terminal tail at one end and by the 25 loop and 34 turn at the other end , with the 2 and 4 strands on each side . the picture that emerges is a hierarchy of time scales near the main binding interface of ubiquitin that consists of a -sheet with a core that is flexible on a slow time scale of about 50 s , while its edges are mobile on faster nanosecond time scales . between these two time scales , small correlated fluctuations of the -sheet . the ability of the edges of the interface to undergo conformational rearrangements on nanosecond time scales would be compatible with an induced fit mechanism in the early stage of binding . longitudinal relaxation rates r1(b0 ) as a function of the static field ( so - called relaxometry dispersion profiles ) for eight selected residues in ubiquitin . the blue and red dots show corrected longitudinal relaxation rates , adjusted to compensate for relaxation during shuttling , while the lines show dispersion profiles calculated from the microdynamic parameters obtained in our analysis . similarly , a small but systematic underestimation of the spectral density j(=0 ) in some of the most mobile regions , and hence of the back - predicted transverse relaxation rates ( see supporting information ) , can be understood by postulating a rapid initial decay of the spectral density function at low frequencies . we have measured and analyzed residue - specific relaxation rates in a protein over a range of nearly 2 orders of magnitude of magnetic fields . until now , discrepancies between high - field relaxation and rdc - based methods were attributed to the cutoff of internal motions by overall rotation . high - field relaxation studies have led to underestimate sub-c and near-c motions because relaxation rates in high fields are not sufficiently sensitive to motions in the nanosecond frequency range . although many proteins , and ubiquitin in particular , are mobile on slow supra-c time scales ( slower than overall rotational diffusion ) , a mere comparison of order parameters obtained from high - field relaxation and rdcs is likely to overestimate such slow motions . this study shows that high - resolution relaxometry with fast sample shuttling allows one to map the spectral density functions in exquisite detail and offers unprecedented information about local motions in proteins on time scales that are faster than or comparable to their overall tumbling . a ch3a10me3d transducer was used for measurements from 0.05 to 2 t , while a 03a05f - a20t0k5q transducer was used between 1 and 13 t. the experiments were carried out on samples of 0.2 and 3 mm uniformly n labeled human ubiquitin ( giotto ) in 50 mm acetate buffer ( ph 4.5 ) in h2o / d2o ( 90/10 v / v ) at 296 t 297.2 k using a 600 mhz bruker avance iii spectrometer equipped with our pneumatic sample shuttle for measurements at low field . the pulse sequence shown in figure 3 was used for 0.5 < b0 < 5 t , with a recovery delay of 2.2 s. all experiments were acquired with 16 transients and 64 complex points in the indirect t1 dimension . the analysis of relaxation rates was performed with our icarus process , which uses rotdif and dynamics at each iteration . in order to account for systematic errors , a jack - knife procedure was used : the analysis was repeated seven times while excluding one of the seven low - field relaxation rates . the analysis was carried out with similar parameters as in many other nmr studies of protein dynamics . in order to determine these distances , we measured longitudinal relaxation rates at 14.1 t with experiments similar to the shuttling method but where the longitudinal nitrogen-15 polarization is allowed to evolve during fixed intervals before and after the relaxation delay , during which no rf pulses are applied . this result is confirmed by a computation of the sum of dipolar interactions with all protons ( see supporting information ) where the median value corresponds to an effective distance dhh = 2.07 .

we performed a prospective observational study at cipto mangunkusumo and persahabatan hospitals , jakarta , indonesia . patients with dengue infection who were aged > 14 years who were admitted to the hospital on the third day of fever from november 2013 through august 2015 were included in the study . patients with dengue infection were diagnosed based on clinical manifestation of acute fever and a positive result for dengue nonstructural protein 1 ( ns1 ) antigen , as confirmed by both conventional reverse - transcriptase polymerase chain reaction ( rt - pcr ) and simplexa dengue real - time rt - pcr assay [ 22 , 23 ] . the clinical characteristics and laboratory parameters of each patient were recorded at the ward before enrollment . the subjects were classified as having plasma leakage based on world health organization criteria ( ie , an increase in hematocrit > 20% , hypoalbuminemia , or the presence of pleural effusions / ascites detected by ultrasonography ) . subjects with plasma leakage and a serum albumin level > 3 g / dl were categorized as having dhf ( group 2 ) . subjects with plasma leakage and a serum albumin level 3 g / dl were categorized as having severe plasma leakage ( group 3 ) . albumin measurement and abdominal ultrasonography were performed 24 hours after defervescence [ 2 , 4 ] . biomarkers of the ve components were measured in duplicate using the following commercially available enzyme - linked immunosorbent assays ( elisas ) , performed according to the manufacturers recommendations : human scd138 ( syndecan-1 ) elisa ( diaclone ) , human heparan sulfate elisa ( mybiosource ) , human chondroitin sulfate elisa ( mybiosource ) , human hyaluronan quantikine elisa ( r&d systems ) , human claudin 5 elisa ( mybiosource ) , and human ve - cadherin quantikine elisa ( r&d systems ) . serum samples were diluted according to the manufacturers recommendation to obtain the best dilution factors to generate an optical density within the range of detection of the elisa reader . the measurements were performed using a multiskan microplate reader with a wavelength set to 450 nm . biomarkers of ve components in all 3 groups were measured twice : on the third day of fever ( febrile phase ) and 24 hours after defervescence ( critical phase ) . the study was approved by the faculty of medicine universitas indonesia ethics committee , and written informed consent was obtained from all patients . the sample size calculation was performed by estimation of the heparan sulfate population variance as 3.24 , which was the highest population variance among all of the ve biomarkers studied . assuming a difference in the population mean of 1.25 , with a confidence level of 95% and a power of 80% , the minimal total sample size required for the 3 groups was 99 patients . a bivariate analysis with a mann whitney test for nonparametric data regarding the ve biomarkers and the cytokine and chemokine studied was performed between pairs among the 3 groups . the ve biomarker variables that significantly differed based on the severity of plasma leakage in the bivariate analysis were then entered into a multivariate analysis to determine the adjusted association . the statistical analyses were performed using spss version 20.0 and graphpad prism version 7.00 for windows . we performed a prospective observational study at cipto mangunkusumo and persahabatan hospitals , jakarta , indonesia . patients with dengue infection who were aged > 14 years who were admitted to the hospital on the third day of fever from november 2013 through august 2015 were included in the study . patients with dengue infection were diagnosed based on clinical manifestation of acute fever and a positive result for dengue nonstructural protein 1 ( ns1 ) antigen , as confirmed by both conventional reverse - transcriptase polymerase chain reaction ( rt - pcr ) and simplexa dengue real - time rt - pcr assay [ 22 , 23 ] . the clinical characteristics and laboratory parameters of each patient were recorded at the ward before enrollment . the subjects were classified as having plasma leakage based on world health organization criteria ( ie , an increase in hematocrit > 20% , hypoalbuminemia , or the presence of pleural effusions / ascites detected by ultrasonography ) . subjects with plasma leakage and a serum albumin level > 3 g / dl were categorized as having dhf ( group 2 ) . subjects with plasma leakage and a serum albumin level 3 g / dl were categorized as having severe plasma leakage ( group 3 ) . albumin measurement and abdominal ultrasonography were performed 24 hours after defervescence [ 2 , 4 ] . biomarkers of the ve components were measured in duplicate using the following commercially available enzyme - linked immunosorbent assays ( elisas ) , performed according to the manufacturers recommendations : human scd138 ( syndecan-1 ) elisa ( diaclone ) , human heparan sulfate elisa ( mybiosource ) , human chondroitin sulfate elisa ( mybiosource ) , human hyaluronan quantikine elisa ( r&d systems ) , human claudin 5 elisa ( mybiosource ) , and human ve - cadherin quantikine elisa ( r&d systems ) . serum samples were diluted according to the manufacturers recommendation to obtain the best dilution factors to generate an optical density within the range of detection of the elisa reader . the measurements were performed using a multiskan microplate reader with a wavelength set to 450 nm . biomarkers of ve components in all 3 groups were measured twice : on the third day of fever ( febrile phase ) and 24 hours after defervescence ( critical phase ) . the study was approved by the faculty of medicine universitas indonesia ethics committee , and written informed consent was obtained from all patients . the sample size calculation was performed by estimation of the heparan sulfate population variance as 3.24 , which was the highest population variance among all of the ve biomarkers studied . assuming a difference in the population mean of 1.25 , with a confidence level of 95% and a power of 80% , the minimal total sample size required for the 3 groups was 99 patients . a bivariate analysis with a mann whitney test for nonparametric data regarding the ve biomarkers and the cytokine and chemokine studied was performed between pairs among the 3 groups . the ve biomarker variables that significantly differed based on the severity of plasma leakage in the bivariate analysis were then entered into a multivariate analysis to determine the adjusted association . the statistical analyses were performed using spss version 20.0 and graphpad prism version 7.00 for windows . a total of 30 patients ( 29.1% ) were classified as having df , 50 patients ( 48.5% ) were classified as having dhf , and 23 patients ( 22.3% ) were classified as having dengue with severe plasma leakage . dengue - infected patients : comparison of patients with dengue fever , dengue hemorrhagic fever , and severe plasma leakage abbreviations : denv , dengue virus ; df , dengue fever ; dhf , dengue hemorrhagic fever ; iqr , interquartile range . in the bivariate analysis in the febrile phase , there were significantly increased levels of syndecan-1 ( p = .002 ) , chondroitin sulfate ( p = .007 ) , and hyaluronan ( p = .03 ) in the dhf group compared with the df group . levels of syndecan-1 ( p < .001 ) , chondroitin sulfate ( p = .02 ) , and hyaluronan ( p = .002 ) were significantly higher in the group with severe plasma leakage compared with the df group as well . we also found a significantly increased level of claudin-5 ( p = .002 ) in the severe plasma leakage group . between the dhf and severe plasma leakage groups , there were significant differences in levels of syndecan-1 ( p = .04 ) and claudin-5 ( p = .04 ) ( table 2 ) . in the multivariate analysis of the biomarkers , there was an adjusted association for syndecan-1 between the df and dhf groups as well as between the df and severe plasma leakage groups . there was also an adjusted association for claudin-5 between the dhf and severe plasma leakage groups ( table 3 ) . plasma concentration of the endothelial glycocalyx layer and tight and adherens junction markers measured in the febrile and critical phases data presented as median ( interquartile range ) for each group . the mann multivariate analysis of the plasma concentrations of the endothelial glycocalyx layer and tight and adherens junction markers measured in the febrile phase between groups abbreviations : ci , confidence interval ; df , dengue fever ; dhf , dengue hemorrhagic fever . in the critical phase , we found significantly different levels of syndecan-1 , chondroitin sulfate , and hyaluronan ( p < .001 for each comparison ) between the df and dhf groups . between the df and severe plasma leakage groups , there were significantly different levels of syndecan-1 , chondroitin sulfate , hyaluronan , and claudin-5 ( p < .001 for each comparison ) . furthermore , between the dhf and severe plasma leakage groups , we found significantly different levels of syndecan-1 ( p = .03 ) and claudin-5 ( p < .001 ) ( table 2 ) . in the multivariate analysis between the df and dhf groups , we found an adjusted association for syndecan-1 and chondroitin sulfate . between the df and severe plasma leakage groups finally , between the dhf and severe plasma leakage groups , we found an adjusted association for claudin-5 ( table 4 ) . multivariate analysis of the plasma concentrations of the endothelial glycocalyx layer and tight and adherens junction markers measured in the critical phase between groups abbreviations : ci , confidence interval ; df , dengue fever ; dhf , dengue hemorrhagic fever . we found a significantly increased ( p = .03 ) il-10 in the dhf group ( median = 9.47 pg / ml ; interquartile range [ iqr ] = 7.65 - 13.83 ) compared with the df group ( median = 7.29 pg / ml ; iqr = 6.29 - 10.36 ) and also in the severe plasma leakage group ( median = 11.23 pg / ml ; iqr = 8.06 - 17.06 ) compared with the df group ( p = .02 ) ( figure 1 ) . moreover , there was a significantly increased ( p = .01 ) level of cxcl10/ip10 in the dhf group ( median = 46.07 pg / ml ; iqr = 26.02 - 56.84 ) compared with the df group ( median = 23.69 pg / ml ; iqr = 17.78 - 41.51 ) . finally , there was a significantly increased ( p = .006 ) level of cxcl10/ip10 in the severe plasma leakage group ( median = 61.39 pg / ml ; iqr = 22.24 - 108.13 ) compared with the df group ( figure 2 ) . scatter plot of the serum interleuking 10 ( il-10 ) levels in the dengue fever ( df ) , dengue hemorrhagic fever ( dhf ) , and severe plasma leakage groups . whitney test ( p = .03 for the difference between the df and the dhf groups ; p = .02 for the difference between the df and the severe plasma leakage groups ) . scatter plot of the serum chemokine ( c - x - c motif ) ligand 10/interferon -inducible protein 10 ( cxcl10/ip10 ) levels in the dengue fever ( df ) , dengue hemorrhagic fever ( dhf ) , and severe plasma leakage groups . whitney test ( p = .01 for the difference between the df and the dhf groups ; p = .006 for the difference between the df and the severe plasma leakage groups ) . a total of 30 patients ( 29.1% ) were classified as having df , 50 patients ( 48.5% ) were classified as having dhf , and 23 patients ( 22.3% ) were classified as having dengue with severe plasma leakage . dengue - infected patients : comparison of patients with dengue fever , dengue hemorrhagic fever , and severe plasma leakage abbreviations : denv , dengue virus ; df , dengue fever ; dhf , dengue hemorrhagic fever ; iqr , interquartile range . in the bivariate analysis in the febrile phase , there were significantly increased levels of syndecan-1 ( p = .002 ) , chondroitin sulfate ( p = .007 ) , and hyaluronan ( p = .03 ) in the dhf group compared with the df group . levels of syndecan-1 ( p < .001 ) , chondroitin sulfate ( p = .02 ) , and hyaluronan ( p = .002 ) were significantly higher in the group with severe plasma leakage compared with the df group as well . we also found a significantly increased level of claudin-5 ( p = .002 ) in the severe plasma leakage group . between the dhf and severe plasma leakage groups , there were significant differences in levels of syndecan-1 ( p = .04 ) and claudin-5 ( p = .04 ) ( table 2 ) . in the multivariate analysis of the biomarkers , there was an adjusted association for syndecan-1 between the df and dhf groups as well as between the df and severe plasma leakage groups . there was also an adjusted association for claudin-5 between the dhf and severe plasma leakage groups ( table 3 ) . plasma concentration of the endothelial glycocalyx layer and tight and adherens junction markers measured in the febrile and critical phases data presented as median ( interquartile range ) for each group . the mann multivariate analysis of the plasma concentrations of the endothelial glycocalyx layer and tight and adherens junction markers measured in the febrile phase between groups abbreviations : ci , confidence interval ; df , dengue fever ; dhf , dengue hemorrhagic fever . in the critical phase , we found significantly different levels of syndecan-1 , chondroitin sulfate , and hyaluronan ( p < .001 for each comparison ) between the df and dhf groups . between the df and severe plasma leakage groups , there were significantly different levels of syndecan-1 , chondroitin sulfate , hyaluronan , and claudin-5 ( p < .001 for each comparison ) . furthermore , between the dhf and severe plasma leakage groups , we found significantly different levels of syndecan-1 ( p = .03 ) and claudin-5 ( p < .001 ) ( table 2 ) . in the multivariate analysis between the df and dhf groups , we found an adjusted association for syndecan-1 and chondroitin sulfate . between the df and severe plasma leakage groups finally , between the dhf and severe plasma leakage groups , we found an adjusted association for claudin-5 ( table 4 ) . multivariate analysis of the plasma concentrations of the endothelial glycocalyx layer and tight and adherens junction markers measured in the critical phase between groups abbreviations : ci , confidence interval ; df , dengue fever ; dhf , dengue hemorrhagic fever . we found a significantly increased ( p = .03 ) il-10 in the dhf group ( median = 9.47 pg / ml ; interquartile range [ iqr ] = 7.65 - 13.83 ) compared with the df group ( median = 7.29 pg / ml ; iqr = 6.29 - 10.36 ) and also in the severe plasma leakage group ( median = 11.23 pg / ml ; iqr = 8.06 - 17.06 ) compared with the df group ( p = .02 ) ( figure 1 ) . moreover , there was a significantly increased ( p = .01 ) level of cxcl10/ip10 in the dhf group ( median = 46.07 pg / ml ; iqr = 26.02 - 56.84 ) compared with the df group ( median = 23.69 pg / ml ; iqr = 17.78 - 41.51 ) . finally , there was a significantly increased ( p = .006 ) level of cxcl10/ip10 in the severe plasma leakage group ( median = 61.39 pg / ml ; iqr = 22.24 - 108.13 ) compared with the df group ( figure 2 ) . scatter plot of the serum interleuking 10 ( il-10 ) levels in the dengue fever ( df ) , dengue hemorrhagic fever ( dhf ) , and severe plasma leakage groups . whitney test ( p = .03 for the difference between the df and the dhf groups ; p = .02 for the difference between the df and the severe plasma leakage groups ) . scatter plot of the serum chemokine ( c - x - c motif ) ligand 10/interferon -inducible protein 10 ( cxcl10/ip10 ) levels in the dengue fever ( df ) , dengue hemorrhagic fever ( dhf ) , and severe plasma leakage groups . whitney test ( p = .01 for the difference between the df and the dhf groups ; p = .006 for the difference between the df and the severe plasma leakage groups ) . this is the first indonesian study to assess the role of egl and endothelial cell junction components in determining the severity of plasma leakage , which is responsible for the life - threatening nature of dhf . previous studies reported that the level of serum albumin was associated with the severity of dengue infection and could be used as a surrogate indicator of severe plasma leakage [ 8 , 9 ] . bivariate analysis of egl components in the febrile phase showed significantly increased levels of syndecan-1 , chondroitin sulfate , and hyaluronan in the dhf and severe plasma leakage groups compared with the df group . an explanation for these findings is that there are several possible mechanisms of vascular permeability alteration due to egl degradation in dengue infection based on previous in vitro studies . the first mechanism is release of cytokines and chemokines by peripheral blood mononuclear cells through direct activation of toll - like receptor 4 by ns1 . the second mechanism is complement activation induced by either attachment of ns1 to the glycosaminoglycans heparan sulfate and chondroitin sulfate e or binding of denv to a specific syndecan-2 proteoglycan receptor [ 2729 ] . the third mechanism is activation of endothelial cell - intrinsic pathways caused by binding of denv ns1 to the glycocalyx . these 3 possible mechanisms may explain the degradation and shedding of the egl components in the circulation [ 26 , 29 ] . however , we found no significant increase in the level of heparan sulfate within the 3 groups . furthermore , in the multivariate analysis , the only egl component that had a strong association with the severity of plasma leakage was syndecan-1 . our finding may be explained based on the mechanism that regulates synthesis of the syndecan-1 core protein by heparan sulfate . in endothelial cells , heparanase then cleaves heparan sulfate chains , resulting in degradation of the heparan sulfate attached to syndecan-1 . diminished levels of heparan sulfate chains on the syndecan-1 core protein cause a significant increase in shedding of syndecan-1 . this effect is specifically induced by ns1 from denv-14 , and not by proteins from other flaviviruses . in this study , both bivariate and multivariate analyses of tight junction markers showed an adjusted association for claudin-5 between the dhf and severe plasma leakage groups . this finding is consistent with an in vitro study that showed disrupted tight junctions among human ve cells after exposure to monocyte chemoattractant protein-1 ( mcp-1 ) from the sera of dss patients . bivariate analysis of adherens junction markers in the febrile phase showed there was no difference in ve - cadherin levels within the subject groups . in fact , in patients with hemolytic uremic syndrome ( hus ) caused by shiga toxin producing escherichia coli ( stec ) infection , soluble ve - cadherin levels are significantly higher than in non - hus patients ; this finding indicates that ve - cadherin is released when membrane damage occurs . in contrast with hus , plasma leakage in dengue infection occurs without morphological damage to the vascular endothelium . this feature may explain the lack of a difference in ve - cadherin levels within the groups of dengue patients with different severities of plasma leakage in this study . however , further studies are needed to determine the role of ve - cadherin in the most severe dengue infections , such as in dss . our findings regarding biomarker levels in the febrile phase suggest that syndecan-1 plays a dual role as a risk factor for plasma leakage occurring at 24 days after defervescence : first , as a risk factor for plasma leakage in general , and second , as a risk factor for severe plasma leakage together with claudin-5 . as has been discussed previously , syndecan-1 plays a vital role in the early phase , serving as a risk factor for plasma leakage , and this role has also been confirmed in the critical phase . we also found significantly elevated chondroitin sulfate in the dhf group compared with the df group . however , based on the multivariate analysis , the adjusted association of elevated chondroitin sulfate only persisted in the critical phase . this finding can be explained by the fact that plasma leakage is most commonly detected in the critical phase [ 2 , 3 ] . in addition , the median level of chondroitin sulfate was lower in the critical phase compared with the febrile phase . a previous study characterizing circulating glycosaminoglycans during critical illness reported a decline in circulating chondroitin sulfate 72 hours after study enrollment ; this finding may reflect glycosaminoglycan clearance by the liver or kidney . physiologically , syndecan-1 contains 3 attachment sites for heparan sulfate and 2 additional attachment sites for chondroitin sulfate . we postulate that the peak shedding of syndecan-1 is followed by a significant increase in the level of chondroitin sulfate . in contrast , due to heparanase activity , there is no increase in the level of heparan sulfate . although hyaluronan showed a significant association in the bivariate analysis in both the febrile and the critical phases , the association did not persist in the multivariate analysis . this finding may indicate a less significant role for hyaluronan , which is located on the apical surface of endothelial cells , compared with syndecan-1 and chondroitin sulfate in terms of the process of plasma leakage in dengue infection . in vitro studies have reported that hyaluronidase treatment to remove hyaluronan results only in minimal changes in permeability . it appears that the components of the egl other than hyaluronan can stabilize the properties of the glycocalyx . we found significantly increased levels of the cytokine il-10 and the chemokine cxcl10/ip10 in the dhf group compared with the df group in the critical phase . a previous study in pediatric patients reported that circulating levels of il-10 and cxcl10/ip10 significantly increased in dengue patients with vascular leakage . a study in hemorrhagic shock patients reported a significantly increased level of syndecan-1 after injury and a positive correlation between the il-10 level and syndecan-1 shedding . furthermore , an in vitro model of endothelial injury showed that syndecan-1 shedding correlated with changes in endothelial permeability , and another in vitro study revealed that chemokines stimulate cell metalloproteinases , which are involved in syndecan shedding . we found no significant association between df and dhf and either tight or adherens junction components in the critical phase . the glycocalyx plays an important role as an endothelial barrier because of its net negative charge . this role is supported by experiments that characterized the effect of disrupting the glycocalyx or neutralizing its negative charge . in particular , treatment with neuraminidase , causing removal of the majority of the glycocalyx , increased albumin flux across the endothelium . moreover , neutralization of the apical endothelial negative charge with cationic agents ( eg , ferritin or protamine ) increased the transendothelial permeability of albumin . the transcellular pathway is initiated by binding of albumin to the protein gp60 , which induces the phosphorylation of the src protein tyrosine kinases ( ptks ) , leading to phosphorylation of caveolin-1 and dynamin-2 . the activation of these proteins is required for caveolar fission and the internalization of albumin within caveolae , and inflammatory cytokines such as tnf - may directly increase the phosphorylation of the src ptks . moreover , an in vitro study using human dendritic cells showed that chemokines could stimulate the activation of src ptks in both immature and mature dendritic cells . another in vitro study found that denv infection enhances caveolae - mediated albumin transcytosis , which consequently induces vascular leakage . these mechanisms may explain the increase in albumin leakage through transcytosis in dhf , independent of the paracellular pathway . consistent with our findings in the febrile phase , in the critical phase , there was an adjusted association for syndecan-1 and claudin-5 between the df and severe plasma leakage groups . we also found that claudin-5 had an adjusted association between the dhf and the severe plasma leakage groups . in addition , there were increased levels of il-10 and cxcl10/ip10 in the severe plasma leakage group compared with the df group . the disruption of tight junctions has been elucidated by in vitro studies that have determined that the mechanism of severe dengue is based on excessive release of various cytokines and chemokines , known as cytokine storm , as observed in u937-derived macrophages infected with a humanized monoclonal antibody recognizing protein e . this degradation of tight junctions allows the passage of albumin across the endothelial barrier , causing hypoalbuminemia , even without degradation of adherens junctions . the potential explanation for this paracellular pathway is that endothelial - cell retraction may lead to opening of intercellular gaps . this reversible effect may be caused by histamine , thrombin , and ve growth factors , without affecting vascular damage [ 11 , 43 ] . in sum , the results reflect major roles for syndecan-1 and claudin-5 in the mechanism of albumin leakage through transcellular and paracellular pathways during severe plasma leakage . a proposed mechanism of microvascular leakage in dengue - infected patients based on the overall results of our study is shown in figure 3 . plasma leakage : dengue virus ( denv ) nonstructural protein 1 ( ns1)induced degradation of syndecan-1 and chondroitin sulfate results in gp60 activation by albumin . gp60 activation leads to phosphorylation of the src protein tyrosine kinases ( ptks ) and caveolin-1 , which is responsible for regulation of caveolar fission . severe plasma leakage : claudin-5 degradation causes retraction of endothelial cells and opening of intercellular gaps , allowing albumin movement into the extravascular space through paracellular pathways . albumin leakage that occurs through both paracellular and transcellular pathways results in severe plasma leakage . a limitation of our study is the measurement of egl components and tight and adherens junction markers in the plasma . in particular , the predominant source of these components is endothelial cells , but these components are also present on epithelial tissues and macrophages , so the levels of these components in the circulation might not be solely influenced by endothelial cells . in conclusion , in patients with dengue infection , elevated levels of syndecan-1 and chondroitin sulfate have a strong association with plasma leakage , and elevated levels of syndecan-1 and claudin-5 have a strong association with severe plasma leakage .

abstractbackground.the role of vascular endothelial ( ve ) components in dengue infection with plasma leakage is unknown . therefore , we conducted a study to determine the adjusted association of the endothelial glycocalyx layer ( egl ) and tight and adherens junction markers with plasma leakage . methods.a prospective observational study was conducted at cipto mangunkusumo hospital and persahabatan hospital , jakarta , indonesia . adult dengue patients admitted to the hospital on the third day of fever from november 2013 through august 2015 were included in the study . multiple regression analysis was used to determine the adjusted association of the ve biomarkers with the severity of the plasma leakage . results.a total of 103 dengue - infected patients participated in the study . in the critical phase , levels of syndecan-1 ( odds ratio [ or ] = 1.004 ; 95% confidence interval [ ci ] = 1.0011.007 ) and chondroitin sulfate ( or = 1.157 ; 95% ci = 1.0251.307 ) had an adjusted association with plasma leakage , whereas levels of syndecan-1 ( or = 1.004 ; 95% ci = 1.0001.008 ) and claudin-5 ( or = 1.038 ; 95% ci = 1.0041.074 ) had an adjusted association with severe plasma leakage.conclusions.in dengue - infected patients , elevated levels of syndecan-1 and chondroitin sulfate are strongly associated with plasma leakage , and elevated levels of syndecan-1 and claudin-5 are strongly associated with severe plasma leakage .

METHODS Study Design and Population Clinical and Laboratory Parameters Detection of the Biomarkers Using Enzyme-Linked Immunosorbent Assay Ethics Statistical Analysis RESULTS Clinical Characteristics Plasma Concentrations of the Vascular Endothelial Biomarkers Cytokine and Chemokine Plasma Concentrations DISCUSSION

we performed a prospective observational study at cipto mangunkusumo and persahabatan hospitals , jakarta , indonesia . patients with dengue infection who were aged > 14 years who were admitted to the hospital on the third day of fever from november 2013 through august 2015 were included in the study . subjects with plasma leakage and a serum albumin level 3 g / dl were categorized as having severe plasma leakage ( group 3 ) . biomarkers of the ve components were measured in duplicate using the following commercially available enzyme - linked immunosorbent assays ( elisas ) , performed according to the manufacturers recommendations : human scd138 ( syndecan-1 ) elisa ( diaclone ) , human heparan sulfate elisa ( mybiosource ) , human chondroitin sulfate elisa ( mybiosource ) , human hyaluronan quantikine elisa ( r&d systems ) , human claudin 5 elisa ( mybiosource ) , and human ve - cadherin quantikine elisa ( r&d systems ) . serum samples were diluted according to the manufacturers recommendation to obtain the best dilution factors to generate an optical density within the range of detection of the elisa reader . biomarkers of ve components in all 3 groups were measured twice : on the third day of fever ( febrile phase ) and 24 hours after defervescence ( critical phase ) . the study was approved by the faculty of medicine universitas indonesia ethics committee , and written informed consent was obtained from all patients . the sample size calculation was performed by estimation of the heparan sulfate population variance as 3.24 , which was the highest population variance among all of the ve biomarkers studied . the ve biomarker variables that significantly differed based on the severity of plasma leakage in the bivariate analysis were then entered into a multivariate analysis to determine the adjusted association . we performed a prospective observational study at cipto mangunkusumo and persahabatan hospitals , jakarta , indonesia . patients with dengue infection who were aged > 14 years who were admitted to the hospital on the third day of fever from november 2013 through august 2015 were included in the study . subjects with plasma leakage and a serum albumin level 3 g / dl were categorized as having severe plasma leakage ( group 3 ) . biomarkers of the ve components were measured in duplicate using the following commercially available enzyme - linked immunosorbent assays ( elisas ) , performed according to the manufacturers recommendations : human scd138 ( syndecan-1 ) elisa ( diaclone ) , human heparan sulfate elisa ( mybiosource ) , human chondroitin sulfate elisa ( mybiosource ) , human hyaluronan quantikine elisa ( r&d systems ) , human claudin 5 elisa ( mybiosource ) , and human ve - cadherin quantikine elisa ( r&d systems ) . serum samples were diluted according to the manufacturers recommendation to obtain the best dilution factors to generate an optical density within the range of detection of the elisa reader . biomarkers of ve components in all 3 groups were measured twice : on the third day of fever ( febrile phase ) and 24 hours after defervescence ( critical phase ) . the study was approved by the faculty of medicine universitas indonesia ethics committee , and written informed consent was obtained from all patients . the sample size calculation was performed by estimation of the heparan sulfate population variance as 3.24 , which was the highest population variance among all of the ve biomarkers studied . the ve biomarker variables that significantly differed based on the severity of plasma leakage in the bivariate analysis were then entered into a multivariate analysis to determine the adjusted association . a total of 30 patients ( 29.1% ) were classified as having df , 50 patients ( 48.5% ) were classified as having dhf , and 23 patients ( 22.3% ) were classified as having dengue with severe plasma leakage . dengue - infected patients : comparison of patients with dengue fever , dengue hemorrhagic fever , and severe plasma leakage abbreviations : denv , dengue virus ; df , dengue fever ; dhf , dengue hemorrhagic fever ; iqr , interquartile range . in the bivariate analysis in the febrile phase , there were significantly increased levels of syndecan-1 ( p = .002 ) , chondroitin sulfate ( p = .007 ) , and hyaluronan ( p = .03 ) in the dhf group compared with the df group . levels of syndecan-1 ( p < .001 ) , chondroitin sulfate ( p = .02 ) , and hyaluronan ( p = .002 ) were significantly higher in the group with severe plasma leakage compared with the df group as well . we also found a significantly increased level of claudin-5 ( p = .002 ) in the severe plasma leakage group . between the dhf and severe plasma leakage groups , there were significant differences in levels of syndecan-1 ( p = .04 ) and claudin-5 ( p = .04 ) ( table 2 ) . in the multivariate analysis of the biomarkers , there was an adjusted association for syndecan-1 between the df and dhf groups as well as between the df and severe plasma leakage groups . there was also an adjusted association for claudin-5 between the dhf and severe plasma leakage groups ( table 3 ) . plasma concentration of the endothelial glycocalyx layer and tight and adherens junction markers measured in the febrile and critical phases data presented as median ( interquartile range ) for each group . the mann multivariate analysis of the plasma concentrations of the endothelial glycocalyx layer and tight and adherens junction markers measured in the febrile phase between groups abbreviations : ci , confidence interval ; df , dengue fever ; dhf , dengue hemorrhagic fever . in the critical phase , we found significantly different levels of syndecan-1 , chondroitin sulfate , and hyaluronan ( p < .001 for each comparison ) between the df and dhf groups . between the df and severe plasma leakage groups , there were significantly different levels of syndecan-1 , chondroitin sulfate , hyaluronan , and claudin-5 ( p < .001 for each comparison ) . furthermore , between the dhf and severe plasma leakage groups , we found significantly different levels of syndecan-1 ( p = .03 ) and claudin-5 ( p < .001 ) ( table 2 ) . in the multivariate analysis between the df and dhf groups , we found an adjusted association for syndecan-1 and chondroitin sulfate . between the df and severe plasma leakage groups finally , between the dhf and severe plasma leakage groups , we found an adjusted association for claudin-5 ( table 4 ) . multivariate analysis of the plasma concentrations of the endothelial glycocalyx layer and tight and adherens junction markers measured in the critical phase between groups abbreviations : ci , confidence interval ; df , dengue fever ; dhf , dengue hemorrhagic fever . we found a significantly increased ( p = .03 ) il-10 in the dhf group ( median = 9.47 pg / ml ; interquartile range [ iqr ] = 7.65 - 13.83 ) compared with the df group ( median = 7.29 pg / ml ; iqr = 6.29 - 10.36 ) and also in the severe plasma leakage group ( median = 11.23 pg / ml ; iqr = 8.06 - 17.06 ) compared with the df group ( p = .02 ) ( figure 1 ) . finally , there was a significantly increased ( p = .006 ) level of cxcl10/ip10 in the severe plasma leakage group ( median = 61.39 pg / ml ; iqr = 22.24 - 108.13 ) compared with the df group ( figure 2 ) . scatter plot of the serum interleuking 10 ( il-10 ) levels in the dengue fever ( df ) , dengue hemorrhagic fever ( dhf ) , and severe plasma leakage groups . scatter plot of the serum chemokine ( c - x - c motif ) ligand 10/interferon -inducible protein 10 ( cxcl10/ip10 ) levels in the dengue fever ( df ) , dengue hemorrhagic fever ( dhf ) , and severe plasma leakage groups . a total of 30 patients ( 29.1% ) were classified as having df , 50 patients ( 48.5% ) were classified as having dhf , and 23 patients ( 22.3% ) were classified as having dengue with severe plasma leakage . dengue - infected patients : comparison of patients with dengue fever , dengue hemorrhagic fever , and severe plasma leakage abbreviations : denv , dengue virus ; df , dengue fever ; dhf , dengue hemorrhagic fever ; iqr , interquartile range . in the bivariate analysis in the febrile phase , there were significantly increased levels of syndecan-1 ( p = .002 ) , chondroitin sulfate ( p = .007 ) , and hyaluronan ( p = .03 ) in the dhf group compared with the df group . levels of syndecan-1 ( p < .001 ) , chondroitin sulfate ( p = .02 ) , and hyaluronan ( p = .002 ) were significantly higher in the group with severe plasma leakage compared with the df group as well . we also found a significantly increased level of claudin-5 ( p = .002 ) in the severe plasma leakage group . between the dhf and severe plasma leakage groups , there were significant differences in levels of syndecan-1 ( p = .04 ) and claudin-5 ( p = .04 ) ( table 2 ) . in the multivariate analysis of the biomarkers , there was an adjusted association for syndecan-1 between the df and dhf groups as well as between the df and severe plasma leakage groups . there was also an adjusted association for claudin-5 between the dhf and severe plasma leakage groups ( table 3 ) . plasma concentration of the endothelial glycocalyx layer and tight and adherens junction markers measured in the febrile and critical phases data presented as median ( interquartile range ) for each group . the mann multivariate analysis of the plasma concentrations of the endothelial glycocalyx layer and tight and adherens junction markers measured in the febrile phase between groups abbreviations : ci , confidence interval ; df , dengue fever ; dhf , dengue hemorrhagic fever . in the critical phase , we found significantly different levels of syndecan-1 , chondroitin sulfate , and hyaluronan ( p < .001 for each comparison ) between the df and dhf groups . between the df and severe plasma leakage groups , there were significantly different levels of syndecan-1 , chondroitin sulfate , hyaluronan , and claudin-5 ( p < .001 for each comparison ) . furthermore , between the dhf and severe plasma leakage groups , we found significantly different levels of syndecan-1 ( p = .03 ) and claudin-5 ( p < .001 ) ( table 2 ) . in the multivariate analysis between the df and dhf groups , we found an adjusted association for syndecan-1 and chondroitin sulfate . between the df and severe plasma leakage groups finally , between the dhf and severe plasma leakage groups , we found an adjusted association for claudin-5 ( table 4 ) . multivariate analysis of the plasma concentrations of the endothelial glycocalyx layer and tight and adherens junction markers measured in the critical phase between groups abbreviations : ci , confidence interval ; df , dengue fever ; dhf , dengue hemorrhagic fever . we found a significantly increased ( p = .03 ) il-10 in the dhf group ( median = 9.47 pg / ml ; interquartile range [ iqr ] = 7.65 - 13.83 ) compared with the df group ( median = 7.29 pg / ml ; iqr = 6.29 - 10.36 ) and also in the severe plasma leakage group ( median = 11.23 pg / ml ; iqr = 8.06 - 17.06 ) compared with the df group ( p = .02 ) ( figure 1 ) . finally , there was a significantly increased ( p = .006 ) level of cxcl10/ip10 in the severe plasma leakage group ( median = 61.39 pg / ml ; iqr = 22.24 - 108.13 ) compared with the df group ( figure 2 ) . scatter plot of the serum interleuking 10 ( il-10 ) levels in the dengue fever ( df ) , dengue hemorrhagic fever ( dhf ) , and severe plasma leakage groups . scatter plot of the serum chemokine ( c - x - c motif ) ligand 10/interferon -inducible protein 10 ( cxcl10/ip10 ) levels in the dengue fever ( df ) , dengue hemorrhagic fever ( dhf ) , and severe plasma leakage groups . this is the first indonesian study to assess the role of egl and endothelial cell junction components in determining the severity of plasma leakage , which is responsible for the life - threatening nature of dhf . previous studies reported that the level of serum albumin was associated with the severity of dengue infection and could be used as a surrogate indicator of severe plasma leakage [ 8 , 9 ] . bivariate analysis of egl components in the febrile phase showed significantly increased levels of syndecan-1 , chondroitin sulfate , and hyaluronan in the dhf and severe plasma leakage groups compared with the df group . an explanation for these findings is that there are several possible mechanisms of vascular permeability alteration due to egl degradation in dengue infection based on previous in vitro studies . the second mechanism is complement activation induced by either attachment of ns1 to the glycosaminoglycans heparan sulfate and chondroitin sulfate e or binding of denv to a specific syndecan-2 proteoglycan receptor [ 2729 ] . these 3 possible mechanisms may explain the degradation and shedding of the egl components in the circulation [ 26 , 29 ] . furthermore , in the multivariate analysis , the only egl component that had a strong association with the severity of plasma leakage was syndecan-1 . diminished levels of heparan sulfate chains on the syndecan-1 core protein cause a significant increase in shedding of syndecan-1 . in this study , both bivariate and multivariate analyses of tight junction markers showed an adjusted association for claudin-5 between the dhf and severe plasma leakage groups . bivariate analysis of adherens junction markers in the febrile phase showed there was no difference in ve - cadherin levels within the subject groups . in contrast with hus , plasma leakage in dengue infection occurs without morphological damage to the vascular endothelium . however , further studies are needed to determine the role of ve - cadherin in the most severe dengue infections , such as in dss . our findings regarding biomarker levels in the febrile phase suggest that syndecan-1 plays a dual role as a risk factor for plasma leakage occurring at 24 days after defervescence : first , as a risk factor for plasma leakage in general , and second , as a risk factor for severe plasma leakage together with claudin-5 . as has been discussed previously , syndecan-1 plays a vital role in the early phase , serving as a risk factor for plasma leakage , and this role has also been confirmed in the critical phase . we also found significantly elevated chondroitin sulfate in the dhf group compared with the df group . however , based on the multivariate analysis , the adjusted association of elevated chondroitin sulfate only persisted in the critical phase . this finding can be explained by the fact that plasma leakage is most commonly detected in the critical phase [ 2 , 3 ] . in addition , the median level of chondroitin sulfate was lower in the critical phase compared with the febrile phase . we postulate that the peak shedding of syndecan-1 is followed by a significant increase in the level of chondroitin sulfate . this finding may indicate a less significant role for hyaluronan , which is located on the apical surface of endothelial cells , compared with syndecan-1 and chondroitin sulfate in terms of the process of plasma leakage in dengue infection . we found significantly increased levels of the cytokine il-10 and the chemokine cxcl10/ip10 in the dhf group compared with the df group in the critical phase . a previous study in pediatric patients reported that circulating levels of il-10 and cxcl10/ip10 significantly increased in dengue patients with vascular leakage . a study in hemorrhagic shock patients reported a significantly increased level of syndecan-1 after injury and a positive correlation between the il-10 level and syndecan-1 shedding . we found no significant association between df and dhf and either tight or adherens junction components in the critical phase . consistent with our findings in the febrile phase , in the critical phase , there was an adjusted association for syndecan-1 and claudin-5 between the df and severe plasma leakage groups . we also found that claudin-5 had an adjusted association between the dhf and the severe plasma leakage groups . in addition , there were increased levels of il-10 and cxcl10/ip10 in the severe plasma leakage group compared with the df group . in sum , the results reflect major roles for syndecan-1 and claudin-5 in the mechanism of albumin leakage through transcellular and paracellular pathways during severe plasma leakage . a proposed mechanism of microvascular leakage in dengue - infected patients based on the overall results of our study is shown in figure 3 . plasma leakage : dengue virus ( denv ) nonstructural protein 1 ( ns1)induced degradation of syndecan-1 and chondroitin sulfate results in gp60 activation by albumin . a limitation of our study is the measurement of egl components and tight and adherens junction markers in the plasma . in particular , the predominant source of these components is endothelial cells , but these components are also present on epithelial tissues and macrophages , so the levels of these components in the circulation might not be solely influenced by endothelial cells . in conclusion , in patients with dengue infection , elevated levels of syndecan-1 and chondroitin sulfate have a strong association with plasma leakage , and elevated levels of syndecan-1 and claudin-5 have a strong association with severe plasma leakage .

myf5 and mrf4 ( also known as myf6 ) are upstream of myod in muscle precursor differentiation ; alternate tbx1 and pax3 pathways can initiate myod expression in pharyngeal muscles and in body and limb skeletal muscles , respectively , but not in eoms . regulatory elements for mrf4 reside within the myf5 locus , and myf5-targeted alleles have variable eom hypoplasia depending on the degree of residual mrf4 expression , with complete loss of both myf5 and myf4 resulting in a specific loss of eoms . we purchased myf5 mice from jackson laboratory ( b6.129s4-myf5/j , stock no . 007893 ; bar harbor , me , usa ) . this myf5 strain expresses cre recombinase at the endogenous myf5 locus , disrupting the myf5 basic helix - loop - helix domain , and is also predicted to disrupt transcription of the adjacent mrf4 gene because the myf5 strain , created by using a similar targeting vector , disrupts expression of both genes and lacks eoms ( but not other muscle types ) . myf5 mice were crossed to transgenic isl : gfp reporter mice ( mgi : j:132726 ; gift of sam pfaff , salk institute , san diego , ca , usa ) , which contain a farnesylated green fluorescent protein ( gfp ) that localizes to the membrane of motor neurons and axons and allows for visualization of the nerves during development . we refer to myf5::isl : gfp embryos as wild type and myf5::isl : gfp embryos as myf5 . all animal work was approved and performed in compliance with boston children 's hospital institutional animal care and use committee protocols and the arvo statement for the use of animals in ophthalmic and vision research . wild - type and myf5 embryos were taken at embryonic day ( e ) 11.5 and fixed in 4% paraformaldehyde ( pfa ) overnight ( o / n ) , washed , dehydrated , and placed in dent 's fixative ( 20% dmso , 80% methanol ) o / n , rehydrated , and placed in blocking solution ( 5% heat - inactivated goat serum , 20% dmso , 75% 1 pbs ) o / n . actin -smooth muscle - cy3 antibody ( anti-sma ; sigma - aldrich corp . , st . louis , mo , usa ) and anti - gfp ( invitrogen , carlsbad , ca , usa ) for 5 days at room temperature ( rt ) , washed , incubated in secondary antibody ( 488 goat anti - rabbit igg ; alexa fluor , life technologies , carlsbad , ca , usa ) for 3 days at rt , washed , dehydrated , and cleared o / n in babb ( one part benzyl alcohol and two parts benzyl benzoate ) . images were acquired with a zeiss lsm 710 laser scanning confocal microscope and zen software ( zeiss , oberkochen , germany ) and processed with imaris software ( bitplane , zurich , switzerland ) . onward , orbital dissections provide superior image detail and quality as compared to whole mount preparations . wild - type and myf5 embryos were taken at the appropriate age and fixed in 4% pfa o / cortex and olfactory bulbs above the orbit were removed under a dissecting microscope , leaving the distal cranial nerves and eoms ( if present ) intact . orbits were incubated with anti-sma for 3 days at rt or 4c , washed , and incubated in secondary antibody for 3 days at rt . after washing with pbs , orbits were further dissected sagitally at e12.5 or , from e13.5 onward , either from the superior aspect , looking down on the orbit from above ( superior view ) , or from the inferior aspect , looking up at the orbit from the palate ( inferior view ) . the oculomotor nerve was measured at its widest diameter , which occurs where the superior division of cn3 forms in control embryos , adjacent to where cn4 crosses above cn3 . the length of the cn3 branch to the io muscle was measured along its trajectory from the distal decision region to the distal nerve tip . statistical analyses of significance were performed by using unpaired t - tests in graphpad prism ( graph pad , san diego , ca , usa ) . whole embryos ( e11.5 ) or brain and brainstem ( e18.5 ) were fixed in 4% pfa o / n , washed in pbs , equilibrated in 30% sucrose , and frozen in optimal cutting temperature ( oct ) media . serial coronal sections of 20-m thickness were cut on a cryostat and stained with rabbit anti - islet1 ( isl1 ; abcam , cambridge , ma , usa ) . after blinding as to genotype , isl - positive motor neuron nuclei were counted in every fifth section through the rostral - caudal extent of the oculomotor and trochlear nuclei by using the cell - counter plug - in in fiji , and the total number of motor neurons counted was then multiplied by 5 . there were n = 3 mutant and n = 3 control embryos at each of the two ages . myf5 and mrf4 ( also known as myf6 ) are upstream of myod in muscle precursor differentiation ; alternate tbx1 and pax3 pathways can initiate myod expression in pharyngeal muscles and in body and limb skeletal muscles , respectively , but not in eoms . regulatory elements for mrf4 reside within the myf5 locus , and myf5-targeted alleles have variable eom hypoplasia depending on the degree of residual mrf4 expression , with complete loss of both myf5 and myf4 resulting in a specific loss of eoms . we purchased myf5 mice from jackson laboratory ( b6.129s4-myf5/j , stock no . 007893 ; bar harbor , me , usa ) . this myf5 strain expresses cre recombinase at the endogenous myf5 locus , disrupting the myf5 basic helix - loop - helix domain , and is also predicted to disrupt transcription of the adjacent mrf4 gene because the myf5 strain , created by using a similar targeting vector , disrupts expression of both genes and lacks eoms ( but not other muscle types ) . myf5 mice were crossed to transgenic isl : gfp reporter mice ( mgi : j:132726 ; gift of sam pfaff , salk institute , san diego , ca , usa ) , which contain a farnesylated green fluorescent protein ( gfp ) that localizes to the membrane of motor neurons and axons and allows for visualization of the nerves during development . we refer to myf5::isl : gfp embryos as wild type and myf5::isl : gfp embryos as myf5 . all animal work was approved and performed in compliance with boston children 's hospital institutional animal care and use committee protocols and the arvo statement for the use of animals in ophthalmic and vision research . wild - type and myf5 embryos were taken at embryonic day ( e ) 11.5 and fixed in 4% paraformaldehyde ( pfa ) overnight ( o / n ) , washed , dehydrated , and placed in dent 's fixative ( 20% dmso , 80% methanol ) o / n , rehydrated , and placed in blocking solution ( 5% heat - inactivated goat serum , 20% dmso , 75% 1 pbs ) o / n . actin -smooth muscle - cy3 antibody ( anti-sma ; sigma - aldrich corp . , st . louis , mo , usa ) and anti - gfp ( invitrogen , carlsbad , ca , usa ) for 5 days at room temperature ( rt ) , washed , incubated in secondary antibody ( 488 goat anti - rabbit igg ; alexa fluor , life technologies , carlsbad , ca , usa ) for 3 days at rt , washed , dehydrated , and cleared o / n in babb ( one part benzyl alcohol and two parts benzyl benzoate ) . images were acquired with a zeiss lsm 710 laser scanning confocal microscope and zen software ( zeiss , oberkochen , germany ) and processed with imaris software ( bitplane , zurich , switzerland ) . from e12.5 onward , orbital dissections provide superior image detail and quality as compared to whole mount preparations . wild - type and myf5 embryos were taken at the appropriate age and fixed in 4% pfa o / cortex and olfactory bulbs above the orbit were removed under a dissecting microscope , leaving the distal cranial nerves and eoms ( if present ) intact . orbits were incubated with anti-sma for 3 days at rt or 4c , washed , and incubated in secondary antibody for 3 days at rt . after washing with pbs , orbits were further dissected sagitally at e12.5 or , from e13.5 onward , either from the superior aspect , looking down on the orbit from above ( superior view ) , or from the inferior aspect , looking up at the orbit from the palate ( inferior view ) . the oculomotor nerve was measured at its widest diameter , which occurs where the superior division of cn3 forms in control embryos , adjacent to where cn4 crosses above cn3 . the length of the cn3 branch to the io muscle was measured along its trajectory from the distal decision region to the distal nerve tip . statistical analyses of significance were performed by using unpaired t - tests in graphpad prism ( graph pad , san diego , ca , usa ) . whole embryos ( e11.5 ) or brain and brainstem ( e18.5 ) were fixed in 4% pfa o / n , washed in pbs , equilibrated in 30% sucrose , and frozen in optimal cutting temperature ( oct ) media . serial coronal sections of 20-m thickness were cut on a cryostat and stained with rabbit anti - islet1 ( isl1 ; abcam , cambridge , ma , usa ) . after blinding as to genotype , isl - positive motor neuron nuclei were counted in every fifth section through the rostral - caudal extent of the oculomotor and trochlear nuclei by using the cell - counter plug - in in fiji , and the total number of motor neurons counted was then multiplied by 5 . there were n = 3 mutant and n = 3 control embryos at each of the two ages . in whole mount views of e11.5 wild - type embryos , the eom anlage can be seen adjacent to the globe ( figs . 1a , 1c ) and by e12.5 in contrast , homozygous myf5 embryos lack eoms but develop facial and skeletal muscles ( figs . 1b , 1d ) . sagittal views of cranial nerve and extraocular muscle development in e11.5 and e12.5 myf5::isl : gfp and myf5::isl : gfp embryos . ocular motor nerves are genetically labeled ( green ) , and eom and arteries are antibody labeled with -sma ( red ) . extraocular muscles are absent in myf5 embryos ( b , d , f ) , and thus the red muscle channel has been turned off in wild - type images ( a , c , e ) , to permit equivalent comparison of nerve trajectories in the presence and absence of eoms . ( a , a ) whole mount wild - type embryo and ( b ) whole mount myf5 embryo at e11.5 . the trajectories of the nerves from the brainstem appear nearly identical in mutant and wild - type embryos at e11.5 . the dotted line denotes the area of the orbit imaged in greater detail in ( c ) , ( c ) , and ( d ) . cn4 is defasciculated , while cn6 is fasciculated along their nerve trunks , and distal cn3 has formed a decision region . the developing muscle anlage stained with -sma can be seen in the wild type ( c ) , and an artery within the orbit stained with -sma is visible in both mutant and wild - type ( c , d ) . ( e , e , f ) orbital imaging at e12.5 . in the myf5 orbit , distinct extraocular muscles have formed and share a common origin at the annulus of zinn ( e , asterisk ) . the trunk of cn4 is now fasciculated , while the distal end forms a decision region adjacent to the so and sends terminal branches into the body of the muscle ( e , e , arrowhead ) . by contrast , the distal end of cn4 in the myf5 orbit fails to form an equivalent distal decision region and has no terminal branches ( f , arrowhead ) . in the wild - type orbit , the inferior division decision region has become more compact ( e , long arrow ) and extends a branch to the io muscle ( e , caret ) . the myf5 cn3 terminal decision region is less compacted ( f , long arrow ) and the branch to the io muscle appears hypoplastic ( f , caret ) . arrow in the lower left corner indicates direction relative to the eye . a , anterior ( toward front of eye ) ; s , superior ; isl , islet1 ; -sma , -smooth muscle actin . in e11.5 wild - type embryos , the ocular motor nerves follow stereotypical trajectories to reach the developing orbit ( figs . the main trunk of cn3 is fasciculated , and its distal end has formed a decision region ( figs . despite the absence of the eom anlage in the e11.5 myf5 embryos , the ocular motor nerves appear similar to wild - type ( figs . 1e , 1e ) ; the four rectus muscles and the so muscle ( whose tendon travels through the trochlea before forming its muscle body ) share a common origin at the annulus of zinn ( denoted by an asterisk in fig . 1e ) , while the io muscle is located nearer the front of the orbit ( see fig . the main trunk of cn4 is now fasciculated , and terminal axons are fanning out into the so muscle . the cn3 inferior division decision region is nestled adjacent to the mr and ir muscles , and the fasciculated branch to the io muscle is extending from or through it . thin branches extend from the cn3 superior and inferior divisions into the sr , mr , and ir muscles . the distal end of the abducens nerve crosses inferiorly to cn3 and extends toward the lr muscle . ( a ) schematic diagram showing semisagittal lateral view of mature mouse eye and orbital muscles . ( b ) horizontal top - down view showing orientation of mature mouse orbits relative to the head ; this orientation shifts during early development . ( c ) schematic of boxed area in ( b ) , showing the muscles and nerves visible in a top - down , superior view . ( d ) schematic showing the muscles and nerves visible in a bottom - up , inferior view . the views in ( c ) and ( d ) are those used in figure 3 . ( mr , ir , so , io ) are green , cn4 and the muscle it innervates ( so ) are blue , and cn6 and the muscles it innervates ( lr and rb ) are pink . beginning at e12.5 , differences are noted in development of ocular motor nerves in the presence and absence of eoms ( fig . distal cn4 does not form the extensive fan - like terminal branches seen in wild - type embryos . the cn3 inferior division decision region remains large ; its appearance is similar to wild - type at e11.5 . a branch of cn3 extends toward where the io muscle should be located , but it appears thinner , straighter , and blunter than in wild - type embryos ( fig . 1f , caret ) . in mouse , the orbits are oriented slightly laterally ( fig . 2b ) , and the position changes slightly as the embryo grows , so further anatomic descriptions are in reference to the eye . by e13.5 , orbit growth requires that orbital dissections be performed from either the superior ( looking down from above ) or inferior view ( looking up from below ; see schematic figs . the distal aspect of cn4 widens considerably , and terminal branches emanate into the so muscle from one - half of the decision region , while branches from the other half are oriented away from the muscle ( figs . multiple branches of the cn3 superior division extend from the main trunk and travel along and into the sr muscle ( fig . the cn3 inferior division decision region contains multiple nerve branches extending directly into the ir and mr muscles , while a long fasciculated branch extends to and sends terminal branches into the io muscle . cn6 crosses inferiorly to cn3 , extends along the lr muscle , and sends terminal branches into it ( figs . orbital dissections of e13.5 to e15.5 myf5:isl : gfp and myf5:isl : gfp embryos . nerves and muscles orbits are imaged from a superior ( a , a , b , e , e , f , i , i , j ) or inferior ( c , c , d , g , g , h , k , k , l ) view , as per figures 2b through 2d schematics . ( a d ) at e13.5 , myf5 eoms share a common origin at the annulus of zinn ( a , asterisk ; c ) . from the superior view ( a , a ) the so muscle begins to curve at its insertion onto the globe , and the rectus muscles have further differentiated from each other . cn3 superior division has multiple branches extending to the sr muscle ( a , triple - headed arrow ) . cn6 has crossed inferiorly to cn3 and extends terminal branches into the lr muscle . from the inferior view ( c , c ) , cn3 inferior division sends terminal branches to the ir and mr muscles ( c , long arrow ) , and a well - formed branch to the io muscle ( c , caret ) . at e13.5 , the myf5 orbit is devoid of eoms ( b , d ) . from the superior view ( b ) , cn4 has fasciculated but lacks an extensive distal decision region and terminal branches ( arrowhead ) , and cn3 superior division is not visible . from the inferior view ( d ) , cn3 distal decision region ( d , long arrow ) and the branch to the io muscle ( d , caret ) are hypoplastic and misoriented , and cn6 is stunted and barely crosses over cn3 ( d , double - headed arrow ) . ( e h ) at e14.5 , the myf5 eoms continue to grow and change in orientation ( e , g ) : the sr muscle is now a broad band inserting on the top of the globe , the lps muscle has appeared between the sr and so muscles , and the so muscle has obtained its stereotypic curved shape . there is extensive terminal branching of cn3 superior division into the sr muscle ( e , e ) , and cn4 has developed a blunt - ended branch that is not in association with an eom ( e , e ) . cn3 inferior division branch has continued extending terminal branches to the mr and ir muscles , and the branch to the io muscle extends into the muscle ( g , g ) . in the absence of eom , the myf5 cn4 appears thin and has developed multiple aberrant branches ( f ) , cn3 main trunk widens ( h ) where the absent superior branch should have formed ( h , triple arrowhead ) , the inferior division decision region is small , with no visible branches to the ir and mr muscles , and the branch to the io muscle is now hypoplastic and aberrant ( h ) . cn6 trajectory is aberrantly parallel to cn3 and its growth cones pathologically wander ( h , double arrowhead ) . l ) at e15.5 , myf5 orbits reveal the near - final ocular motor innervation patterns , and each nerve sends terminal and intramuscular branches into the appropriate eom . cn3 superior division branches have coalesced into a single trunk , and the blunted branch of cn4 is retracting ( i , i , k , k ) . by contrast , in myf5 orbits the nerves have failed to develop further since e14.5 and appear to be regressing , while the abducens nerve wanders ( double arrowhead ) . although more of the oculomotor nerve appears visible in figure 2j than in figure 2f , this is a staining and imaging artifact . in both images , the oculomotor nerve can be seen traveling inferiorly to the trochlear nerve and descending out of view near the stained artery ; the embryo in ( j ) was imaged slightly deeper . cn4 terminates in a single blunt end adjacent to where the so muscle should be ( fig . the cn3 superior division has not formed , and the nerve trunk is significantly wider where it should have formed , averaging 62.05 8.17 m in myf5 embryos and 35.92 8.43 m in wild type ( p = 0.0052 ) ( figs . the cn3 inferior division decision region is small and no terminal branches extend toward the region where the ir and mr should be . the branch extending toward where the io muscle should be is short , thin , and straight rather than arching as seen in wild - type embryos ( figs . cn6 appears thinner and shorter than in wild type and ends bluntly just after passing inferiorly to cn3 ( fig . the myf5 cn3 widens at the origin of the absent superior division and has a shorter branch to the io muscle as compared to the myf5 orbit . ( a ) orbit of an e13.5 myf5 from the inferior view as pictured in figure 2c with measurements illustrated . the diameter of cn3 was measured at the location where the superior division normally forms in control embryos . the length of the inferior division branch to the io muscle was defined as the distance from the termination of the distal decision region to the distal tip of the nerve and was measured in three dimensions by using two to three straight lines that approximated the curvature of the nerve . ( b ) cn3 superior division branch point is significantly wider in myf5 embryos from e13.5 to e15.5 ( average from 62.05 8.17 m at e13.5 to 63.96 2.08 m at e15.5 ) than in the wild - type embryos ( average 35.92 8.43 m at e13.5 to 38.26 2.89 m at e15.5 ) . this difference in width was significant at each age measured ( e13.5 : p = 0.0052 ; e14.5 : p < 0.0001 ; e15.5 : p < 0.0001 ) . ( c ) at e13.5 , cn3 inferior division branch to the io muscle is significantly longer in myf5 ( 373.8 138.7 m ) than in myf5 embryos ( 138.7 56.6 m ; p = 0.0017 ) . while this branch continues to lengthen in wt orbits , reaching an average length of 412.8 25.5 m at e14.5 and 477.6 57.9 m at e15.5 , it fails to lengthen substantially in mutant orbits ( 155.3 87.7 m at e14.5 and 106.7 44.0 m at e15.5 ) . the branch to the io muscle is significantly shorter in the mutant at each age ( e14.5 : p = 0.0007 ; e15.5 : p < 0.0001 ) . a second branch oriented away from the so terminates in a blunt end without visible muscle nearby ( figs . the cn3 superior division branches have grown in length and are running along the body of the sr muscle ( figs . 3e , 3e ) . the cn3 inferior division continues to send terminal branches into the mr and ir muscles , and the branch to the io muscle extends further into the muscle ( figs . cn4 is thin and forms multiple aberrant distal nerve branches that appear incorrectly oriented relative to the artery visible within the orbit ( fig . the cn3 superior division has not formed , and the nerve trunk widens where the superior division normally forms ( figs . 3h , 4b ) . the cn3 inferior division does not extend branches to the area of the ir or mr muscles . the branch to the io muscle is only a thin wisp extending toward where the muscle would normally be located ( figs . cn6 , which had crossed inferiorly to the oculomotor nerve at e13.5 , now has an aberrant trajectory parallel to the oculomotor nerve . at e15.5 , the ocular motor nerves in wild - type embryos are close to their mature configuration ( figs . so muscle has defined terminal branches associated with the muscle , while the second blunted branch appears thinner and less developed than at e14.5 ( figs . the cn3 superior division has formed a single fasciculated main trunk and developed more extensive intramuscular nerve branches into the sr ( figs . the cn3 inferior division sends longer terminal branches to the ir and mr muscles , and the io nerve branch has continued to elongate with its terminal branches in close association with the io muscle ( figs . cn6 continues to expand into the lr muscle , and branches to the rb muscle are now visible ( figs . cn4 maintains a few wandering branches that are mislocalized relative to the normal location of the so muscle ( fig . the cn3 trunk where the cn3 superior division should have formed remains significantly wider in myf5 embryos ( 63.96 2.08 m ) than in wild - type embryos ( 38.26 2.89 m , p < 0.001 ) ( figs . cn3 inferior division branches to the mr and ir muscles are not visible and the branch to the io muscle has failed to elongate ( 155.3 87.7 m at e14.5 , 106.7 44.0 m at e15.5 ; figs . this branch is significantly shorter than that in the wild type at e15.5 ( 477.6 57.9 m ; p < 0.001 ) . the cn6 trajectory remains aberrantly parallel to cn3 , and its terminal axons are defasciculated ( fig . 3l , double - headed arrow ) . to determine if eoms are required for motor neuron survival , we examined the number of motor neurons in oculomotor and trochlear nuclei at e11.5 and e18.5 . at e11.5 , myf5 and control embryos had a similar number of isl1-positive motor neurons in the oculomotor and trochlear nuclei ( 4285 214 control , 4477 182 myf5 , p = 0.84 ; figs . 5a c ) , indicating that there is normal initial proliferation of motor neurons . by e18.5 , there is substantial normal developmental cell death in the control embryos , as reported previously . myf5 embryos had greater levels of cell death , however , and have significantly fewer oculomotor and trochlear neurons than control embryos ( 2465 271 vs. 868.2 81.7 , p = 0.00049 , figs . f ) . myf5 oculomotor and trochlear neuron number and position are similar to control at e11.5 , but are significantly reduced in number by e18.5 . ( a , b ) oculomotor nucleus motor neurons were labeled with anti - isl1 in control ( a ) or myf5 ( b ) brainstems at e11.5 . ( c ) the number of motor neurons in the oculomotor and trochlear nuclei is equal in myf5 and control embryos at e11.5 ( 4285 214 , n = 3 , vs. 4477 182 , n = 3 , p = 0.84 ) , indicating that motor neurons are formed in equal numbers . ( d , e ) oculomotor nucleus motor neurons were labeled with anti - isl1 in control ( d ) or myf5 ( e ) brainstems at e18.5 . ( f ) the number of motor neurons is reduced by 2/3 in myf5 oculomotor nucleus ( 2465 271 , n = 3 , vs. 868.2 81.7 , n = 3 , p = 0.00049 ) , indicating that extraocular muscles are necessary for oculomotor neuron survival . in whole mount views of e11.5 wild - type embryos , the eom anlage can be seen adjacent to the globe ( figs . 1a , 1c ) and by e12.5 in contrast , homozygous myf5 embryos lack eoms but develop facial and skeletal muscles ( figs . 1b , 1d ) . sagittal views of cranial nerve and extraocular muscle development in e11.5 and e12.5 myf5::isl : gfp and myf5::isl : gfp embryos . ocular motor nerves are genetically labeled ( green ) , and eom and arteries are antibody labeled with -sma ( red ) . extraocular muscles are absent in myf5 embryos ( b , d , f ) , and thus the red muscle channel has been turned off in wild - type images ( a , c , e ) , to permit equivalent comparison of nerve trajectories in the presence and absence of eoms . ( a , a ) whole mount wild - type embryo and ( b ) whole mount myf5 embryo at e11.5 . the trajectories of the nerves from the brainstem appear nearly identical in mutant and wild - type embryos at e11.5 . the dotted line denotes the area of the orbit imaged in greater detail in ( c ) , ( c ) , and ( d ) . cn4 is defasciculated , while cn6 is fasciculated along their nerve trunks , and distal cn3 has formed a decision region . the developing muscle anlage stained with -sma can be seen in the wild type ( c ) , and an artery within the orbit stained with -sma is visible in both mutant and wild - type ( c , d ) . ( e , e , f ) orbital imaging at e12.5 . in the myf5 orbit , distinct extraocular muscles have formed and share a common origin at the annulus of zinn ( e , asterisk ) . the trunk of cn4 is now fasciculated , while the distal end forms a decision region adjacent to the so and sends terminal branches into the body of the muscle ( e , e , arrowhead ) . by contrast , the distal end of cn4 in the myf5 orbit fails to form an equivalent distal decision region and has no terminal branches ( f , arrowhead ) . in the wild - type orbit , the inferior division decision region has become more compact ( e , long arrow ) and extends a branch to the io muscle ( e , caret ) . the myf5 cn3 terminal decision region is less compacted ( f , long arrow ) and the branch to the io muscle appears hypoplastic ( f , caret ) . arrow in the lower left corner indicates direction relative to the eye . a , anterior ( toward front of eye ) in e11.5 wild - type embryos , the ocular motor nerves follow stereotypical trajectories to reach the developing orbit ( figs . 1a , 1c ) . the main trunk of cn3 is fasciculated , and its distal end has formed a decision region ( figs . despite the absence of the eom anlage in the e11.5 myf5 embryos , the ocular motor nerves appear similar to wild - type ( figs . by e12.5 , distinct eoms are recognizable ( figs . 1e , 1e ) ; the four rectus muscles and the so muscle ( whose tendon travels through the trochlea before forming its muscle body ) share a common origin at the annulus of zinn ( denoted by an asterisk in fig . 1e ) , while the io muscle is located nearer the front of the orbit ( see fig . the main trunk of cn4 is now fasciculated , and terminal axons are fanning out into the so muscle . the cn3 inferior division decision region is nestled adjacent to the mr and ir muscles , and the fasciculated branch to the io muscle is extending from or through it . thin branches extend from the cn3 superior and inferior divisions into the sr , mr , and ir muscles . the distal end of the abducens nerve crosses inferiorly to cn3 and extends toward the lr muscle . schematic diagrams of orbital anatomy . ( a ) schematic diagram showing semisagittal lateral view of mature mouse eye and orbital muscles . ( b ) horizontal top - down view showing orientation of mature mouse orbits relative to the head ; this orientation shifts during early development . ( c ) schematic of boxed area in ( b ) , showing the muscles and nerves visible in a top - down , superior view . ( d ) schematic showing the muscles and nerves visible in a bottom - up , inferior view . the views in ( c ) and ( d ) are those used in figure 3 . color coding : cn3 and the muscles it innervates ( mr , ir , so , io ) are green , cn4 and the muscle it innervates ( so ) are blue , and cn6 and the muscles it innervates ( lr and rb ) are pink . beginning at e12.5 , differences are noted in development of ocular motor nerves in the presence and absence of eoms ( fig . distal cn4 does not form the extensive fan - like terminal branches seen in wild - type embryos . the cn3 inferior division decision region remains large ; its appearance is similar to wild - type at e11.5 . a branch of cn3 extends toward where the io muscle should be located , but it appears thinner , straighter , and blunter than in wild - type embryos ( fig . 1f , caret ) . 2b ) , and the position changes slightly as the embryo grows , so further anatomic descriptions are in reference to the eye . by e13.5 , orbit growth requires that orbital dissections be performed from either the superior ( looking down from above ) or inferior view ( looking up from below ; see schematic figs . 2c , 2d ) . in e13.5 wild - type embryos , the eoms continue to grow and separate . the distal aspect of cn4 widens considerably , and terminal branches emanate into the so muscle from one - half of the decision region , while branches from the other half are oriented away from the muscle ( figs . multiple branches of the cn3 superior division extend from the main trunk and travel along and into the sr muscle ( fig . the cn3 inferior division decision region contains multiple nerve branches extending directly into the ir and mr muscles , while a long fasciculated branch extends to and sends terminal branches into the io muscle . cn6 crosses inferiorly to cn3 , extends along the lr muscle , and sends terminal branches into it ( figs . orbits are imaged from a superior ( a , a , b , e , e , f , i , i , j ) or inferior ( c , c , d , g , g , h , k , k , l ) view , as per figures 2b through 2d schematics . ( a d ) at e13.5 , myf5 eoms share a common origin at the annulus of zinn ( a , asterisk ; c ) . from the superior view ( a , a ) the so muscle begins to curve at its insertion onto the globe , and the rectus muscles have further differentiated from each other . cn3 superior division has multiple branches extending to the sr muscle ( a , triple - headed arrow ) . cn6 has crossed inferiorly to cn3 and extends terminal branches into the lr muscle . from the inferior view ( c , c ) , cn3 inferior division sends terminal branches to the ir and mr muscles ( c , long arrow ) , and a well - formed branch to the io muscle ( c , caret ) . at e13.5 , the myf5 orbit is devoid of eoms ( b , d ) . from the superior view ( b ) , cn4 has fasciculated but lacks an extensive distal decision region and terminal branches ( arrowhead ) , and cn3 superior division is not visible . from the inferior view ( d ) , cn3 distal decision region ( d , long arrow ) and the branch to the io muscle ( d , caret ) are hypoplastic and misoriented , and cn6 is stunted and barely crosses over cn3 ( d , double - headed arrow ) . ( e h ) at e14.5 , the myf5 eoms continue to grow and change in orientation ( e , g ) : the sr muscle is now a broad band inserting on the top of the globe , the lps muscle has appeared between the sr and so muscles , and the so muscle has obtained its stereotypic curved shape . there is extensive terminal branching of cn3 superior division into the sr muscle ( e , e ) , and cn4 has developed a blunt - ended branch that is not in association with an eom ( e , e ) . cn3 inferior division branch has continued extending terminal branches to the mr and ir muscles , and the branch to the io muscle extends into the muscle ( g , g ) . in the absence of eom , the myf5 cn4 appears thin and has developed multiple aberrant branches ( f ) , cn3 main trunk widens ( h ) where the absent superior branch should have formed ( h , triple arrowhead ) , the inferior division decision region is small , with no visible branches to the ir and mr muscles , and the branch to the io muscle is now hypoplastic and aberrant ( h ) . cn6 trajectory is aberrantly parallel to cn3 and its growth cones pathologically wander ( h , double arrowhead ) . ( i l ) at e15.5 , myf5 orbits reveal the near - final ocular motor innervation patterns , and each nerve sends terminal and intramuscular branches into the appropriate eom . cn3 superior division branches have coalesced into a single trunk , and the blunted branch of cn4 is retracting ( i , i , k , k ) . by contrast , in myf5 orbits the nerves have failed to develop further since e14.5 and appear to be regressing , while the abducens nerve wanders ( double arrowhead ) . although more of the oculomotor nerve appears visible in figure 2j than in figure 2f , this is a staining and imaging artifact . in both images , the oculomotor nerve can be seen traveling inferiorly to the trochlear nerve and descending out of view near the stained artery ; the embryo in ( j ) was imaged slightly deeper . cn4 terminates in a single blunt end adjacent to where the so muscle should be ( fig . the cn3 superior division has not formed , and the nerve trunk is significantly wider where it should have formed , averaging 62.05 8.17 m in myf5 embryos and 35.92 8.43 m in wild type ( p = 0.0052 ) ( figs . 4a , 4b ) . the cn3 inferior division decision region is small and no terminal branches extend toward the region where the ir and mr should be . the branch extending toward where the io muscle should be is short , thin , and straight rather than arching as seen in wild - type embryos ( figs . cn6 appears thinner and shorter than in wild type and ends bluntly just after passing inferiorly to cn3 ( fig . the myf5 cn3 widens at the origin of the absent superior division and has a shorter branch to the io muscle as compared to the myf5 orbit . ( a ) orbit of an e13.5 myf5 from the inferior view as pictured in figure 2c with measurements illustrated . the diameter of cn3 was measured at the location where the superior division normally forms in control embryos . the length of the inferior division branch to the io muscle was defined as the distance from the termination of the distal decision region to the distal tip of the nerve and was measured in three dimensions by using two to three straight lines that approximated the curvature of the nerve . ( b ) cn3 superior division branch point is significantly wider in myf5 embryos from e13.5 to e15.5 ( average from 62.05 8.17 m at e13.5 to 63.96 2.08 m at e15.5 ) than in the wild - type embryos ( average 35.92 8.43 m at e13.5 to 38.26 2.89 m at e15.5 ) . this difference in width was significant at each age measured ( e13.5 : p = 0.0052 ; e14.5 : p < 0.0001 ; e15.5 : p < 0.0001 ) . ( c ) at e13.5 , cn3 inferior division branch to the io muscle is significantly longer in myf5 ( 373.8 138.7 m ) than in myf5 embryos ( 138.7 56.6 m ; p = 0.0017 ) . while this branch continues to lengthen in wt orbits , reaching an average length of 412.8 25.5 m at e14.5 and 477.6 57.9 m at e15.5 , it fails to lengthen substantially in mutant orbits ( 155.3 87.7 m at e14.5 and 106.7 44.0 m at e15.5 ) . the branch to the io muscle is significantly shorter in the mutant at each age ( e14.5 : p = 0.0007 ; e15.5 : p < 0.0001 ) . , cn4 provides terminal innervation to the so muscle . a second branch oriented away from the cn3 superior division branches have grown in length and are running along the body of the sr muscle ( figs . the cn3 inferior division continues to send terminal branches into the mr and ir muscles , and the branch to the io muscle extends further into the muscle ( figs . cn6 crosses inferiorly to cn3 and extends terminal branches into the lr muscle ( figs . 3 g , 3g ) . in e14.5 myf5 embryos , eoms remain absent ( figs . 3f , 3h ) . cn4 is thin and forms multiple aberrant distal nerve branches that appear incorrectly oriented relative to the artery visible within the orbit ( fig . the cn3 superior division has not formed , and the nerve trunk widens where the superior division normally forms ( figs . 3h , 4b ) . the cn3 inferior division does not extend branches to the area of the ir or mr muscles . the branch to the io muscle is only a thin wisp extending toward where the muscle would normally be located ( figs . cn6 , which had crossed inferiorly to the oculomotor nerve at e13.5 , now has an aberrant trajectory parallel to the oculomotor nerve . at e15.5 , the ocular motor nerves in wild - type embryos are close to their mature configuration ( figs . so muscle has defined terminal branches associated with the muscle , while the second blunted branch appears thinner and less developed than at e14.5 ( figs . the cn3 superior division has formed a single fasciculated main trunk and developed more extensive intramuscular nerve branches into the sr ( figs . the cn3 inferior division sends longer terminal branches to the ir and mr muscles , and the io nerve branch has continued to elongate with its terminal branches in close association with the io muscle ( figs . 3k , 3k , 4c ) . cn6 continues to expand into the lr muscle , and branches to the rb muscle are now visible ( figs . cn4 maintains a few wandering branches that are mislocalized relative to the normal location of the so muscle ( fig . the cn3 trunk where the cn3 superior division should have formed remains significantly wider in myf5 embryos ( 63.96 2.08 m ) than in wild - type embryos ( 38.26 2.89 m , p < 0.001 ) ( figs . cn3 inferior division branches to the mr and ir muscles are not visible and the branch to the io muscle has failed to elongate ( 155.3 87.7 m at e14.5 , 106.7 44.0 m at e15.5 ; figs . this branch is significantly shorter than that in the wild type at e15.5 ( 477.6 57.9 m ; p < 0.001 ) . the cn6 trajectory remains aberrantly parallel to cn3 , and its terminal axons are defasciculated ( fig . to determine if eoms are required for motor neuron survival , we examined the number of motor neurons in oculomotor and trochlear nuclei at e11.5 and e18.5 . at e11.5 , myf5 and control embryos had a similar number of isl1-positive motor neurons in the oculomotor and trochlear nuclei ( 4285 214 control , 4477 182 myf5 , p = 0.84 ; figs . c ) , indicating that there is normal initial proliferation of motor neurons . by e18.5 , there is substantial normal developmental cell death in the control embryos , as reported previously . myf5 embryos had greater levels of cell death , however , and have significantly fewer oculomotor and trochlear neurons than control embryos ( 2465 271 vs. 868.2 81.7 , p = 0.00049 , figs . myf5 oculomotor and trochlear neuron number and position are similar to control at e11.5 , but are significantly reduced in number by e18.5 . ( a , b ) oculomotor nucleus motor neurons were labeled with anti - isl1 in control ( a ) or myf5 ( b ) brainstems at e11.5 . ( c ) the number of motor neurons in the oculomotor and trochlear nuclei is equal in myf5 and control embryos at e11.5 ( 4285 214 , n = 3 , vs. 4477 182 , n = 3 , p = 0.84 ) , indicating that motor neurons are formed in equal numbers . ( d , e ) oculomotor nucleus motor neurons were labeled with anti - isl1 in control ( d ) or myf5 ( e ) brainstems at e18.5 . ( f ) the number of motor neurons is reduced by 2/3 in myf5 oculomotor nucleus ( 2465 271 , n = 3 , vs. 868.2 81.7 , n = 3 , p = 0.00049 ) , indicating that extraocular muscles are necessary for oculomotor neuron survival . most prior studies of ocular cranial nerve development in mouse have relied on dii labeling or neurofilament staining , highlighting the initial nerve trajectories but not permitting visualization of their terminal processes in the orbit . by dissecting and visualizing the mouse orbit from timed embryos with genetically labeled motor axons , we characterized in greater detail the development of mouse oculomotor , trochlear , and abducens nerves , and showed that formation of the initial nerve trajectory does not require signals from the eoms , but formation of terminal branching does . ocular motor nerves arrive to the orbit and form distal decision regions in a spatially and temporally similar fashion in myf5 and wild - type embryos , demonstrating that axon growth and guidance from the brainstem to the proper position within the orbit is independent of eoms and likely occurs through cues from the mesenchyme and/or through cell autonomous processes . subsequent full nerve extension and terminal branching into the eom targets , maintenance of correct nerve position as the orbit expands , and survival of axons and their motor neurons , however , are dependent on the presence of eoms . the role of eoms in ocular motor nerve development is similar to the findings from previous reports of the contribution of skeletal muscle to spinal nerve development and survival . silver stains and histologic sectioning following surgical ablation or irradiation of developing chick limb muscles reveal normal development of the main motor nerve trunks and distal nerve branches adjacent to the missing muscles , but failure of the formation of terminal branches that normally would innervate the missing individual muscles . similarly , myod::myf5 mouse embryos lack all skeletal muscle and have normal initial growth and guidance of spinal motor nerves but fail to develop terminal nerve branches , followed by motor neuron apoptosis . together , these data support the influence of locally acting mesenchymal guidance cues on proximal nerve patterning and locally acting eom cues on terminal nerve branching into target muscles . we found minor differences in the relative timing of ocular motor nerve development in mouse compared to chick and zebrafish . in mouse we found that cn6 and cn3 reach the orbit simultaneously and before cn4 , which is similar to zebrafish but different from chick , in which the arrival of cn6 is followed by cn3 and then cn4 . in both mouse and chick , cn3 projects an initial nerve branch to the io muscle , followed by branches to the mr , ir , and sr muscles . by contrast , in zebrafish a branch is sent to the sr muscle many hours after branches are sent to inferior division muscles , correlating with the temporal development of the cn3 nucleus , in which zebrafish sr motor neurons are born after mr and ir motor neurons . first , we identified a trochlear branch in developing wild - type embryos that is not associated with a muscle , forms a blunt end , and subsequently retracts . consistent with its apparent lack of muscle target , the growth and regression of this wild - type trochlear branch resembles the growth and regression of ocular nerves in myf5 embryos . second , we found that the cn3 superior division , which in the mature state forms a single branch that then divides distally to innervate both the sr and lps muscles , fails to form in myf5 embryos . this suggests that , while axons within the main trunk of cn3 are guided in a cell - autonomous manner , by axon axon interactions , or by mesenchymal cues , the superior division axons exit from the main cn3 trunk in response to muscle cues . we have long been puzzled by the observation that the cn3 superior division first appears as multiple small branches extending away from the main cn3 trunk , which subsequently coalesce and elongate . our current imaging approaches now reveal that , at e11.5 to e12.5 , the sr / lps muscle anlage lies in close juxtaposition to the main trunk of cn3 , and these multiple small branches extend directly along and into the muscle anlage . as the orbital size expands over subsequent days , the distance between the origin and eom targets of these small branches increases and , by e15.5 , the branches have fasciculated into the superior division . myf5 embryos do not form these multiple small branches , and , at the position where they normally form , the cn3 main trunk is wider than in wild - type embryos . this is reminiscent of the pathology of kif21a mice . in these mutant mice that recapitulate a human ccdd , the superior division also fails to form , and a widened bulge is visible along cn3 that , by electron microscopy , contains convoluted axon trajectories and stalled growth cones . the kif21a bulge is located far proximally to the normal superior division branch - point and forms secondarily to attenuated kif21a autoinhibition . by contrast , we found the myf5 widening to be located at the position of the normal distal superior division branch - point . the parallels , however , suggest that the myf5 nerve widening may result from paused growth cones that fail to exit the main cn3 trunk secondary to the absence of eoms . ocular motor neurons , like other motor neurons , receive trophic support from their target muscles , and are responsive to neurotrophic factors , particularly bdnf and gdnf ( see ref . it is therefore not surprising that survival of ocular motor neurons depends on the presence of eoms . it is remarkable , however , that in the absence of target muscles , more than one - third of the oculomotor and trochlear neurons remain at e18.5 . by contrast , in myod::myf5 embryos , which lack all skeletal muscles , all somatic motor neurons in the spinal cord die by e17.5 , and most motor neurons in the facial nucleus are dead or dying . the ocular motor nerves are selectively spared until late in the course of motor neuron degenerative disorders such as amyotrophic lateral sclerosis and spinal motor atrophy . this selective sparing is theorized to result from increased resistance of the ocular motor neurons to environmental stressors or excitotoxicity . the retention of some motor neurons in cn3 and cn4 as late as e18.5 without trophic support from their target muscles may be another example of the intrinsic relative resistance of oculomotor neurons to pathologic conditions . using genetic tools , orbital dissection , and a developmental framework in timed mouse embryos , our findings catalogued the development of the ocular motor nerves and they implicate the eoms in the control of their terminal branching . the identities of the signals provided by eoms , whether they differ among different eoms , and whether they are mediated through short - range diffusible cues , direct contact , or both , remain to be determined .

purposeto spatially and temporally define ocular motor nerve development in the presence and absence of extraocular muscles ( eoms).methodsmyf5cre mice , which in the homozygous state lack eoms , were crossed to an islmn : gfp reporter line to fluorescently label motor neuron cell bodies and axons . embryonic day ( e ) 11.5 to e15.5 wild - type and myf5cre / cre : islmn : gfp whole mount embryos and dissected orbits were imaged by confocal microscopy to visualize the developing oculomotor , trochlear , and abducens nerves in the presence and absence of eoms . e11.5 and e18.5 brainstems were serially sectioned and stained for islet1 to determine the fate of ocular motor neurons.resultsat e11.5 , all three ocular motor nerves in mutant embryos approached the orbit with a trajectory similar to that of wild - type . subsequently , while wild - type nerves send terminal branches that contact target eoms in a stereotypical pattern , the myf5cre / cre ocular motor nerves failed to form terminal branches , regressed , and by e18.5 two - thirds of their corresponding motor neurons died . comparisons between mutant and wild - type embryos revealed novel aspects of trochlear and oculomotor nerve development.conclusionswe delineated mouse ocular motor nerve spatial and temporal development in unprecedented detail . moreover , we found that eoms are not necessary for initial outgrowth and guidance of ocular motor axons from the brainstem to the orbit but are required for their terminal branching and survival . these data suggest that intermediate targets in the mesenchyme provide cues necessary for appropriate targeting of ocular motor axons to the orbit , while eom cues are responsible for terminal branching and motor neuron survival .

Methods Generation of Mice Lacking Extraocular Muscles Whole Mount Preparation and Imaging Orbital Dissections and Imaging Oculomotor Nerve Measurements Motor Neuron Cell Counts Results EOMs Are Absent in EOMs Are Not Necessary for Initial Pathfinding of Ocular Motor Axons to the Orbit Axons of Ocular Motor Nerves Follow a Stereotypical Spatial and Temporal Trajectory in the Orbit in Both Wild-type and Ocular Motor Nerves Fail to Develop Terminal Branches in the Absence of EOMs Ocular Motor Neurons Die in the Absence of EOMs Discussion

myf5 mice were crossed to transgenic isl : gfp reporter mice ( mgi : j:132726 ; gift of sam pfaff , salk institute , san diego , ca , usa ) , which contain a farnesylated green fluorescent protein ( gfp ) that localizes to the membrane of motor neurons and axons and allows for visualization of the nerves during development . wild - type and myf5 embryos were taken at embryonic day ( e ) 11.5 and fixed in 4% paraformaldehyde ( pfa ) overnight ( o / n ) , washed , dehydrated , and placed in dent 's fixative ( 20% dmso , 80% methanol ) o / n , rehydrated , and placed in blocking solution ( 5% heat - inactivated goat serum , 20% dmso , 75% 1 pbs ) o / n . wild - type and myf5 embryos were taken at the appropriate age and fixed in 4% pfa o / cortex and olfactory bulbs above the orbit were removed under a dissecting microscope , leaving the distal cranial nerves and eoms ( if present ) intact . myf5 mice were crossed to transgenic isl : gfp reporter mice ( mgi : j:132726 ; gift of sam pfaff , salk institute , san diego , ca , usa ) , which contain a farnesylated green fluorescent protein ( gfp ) that localizes to the membrane of motor neurons and axons and allows for visualization of the nerves during development . wild - type and myf5 embryos were taken at embryonic day ( e ) 11.5 and fixed in 4% paraformaldehyde ( pfa ) overnight ( o / n ) , washed , dehydrated , and placed in dent 's fixative ( 20% dmso , 80% methanol ) o / n , rehydrated , and placed in blocking solution ( 5% heat - inactivated goat serum , 20% dmso , 75% 1 pbs ) o / n . wild - type and myf5 embryos were taken at the appropriate age and fixed in 4% pfa o / cortex and olfactory bulbs above the orbit were removed under a dissecting microscope , leaving the distal cranial nerves and eoms ( if present ) intact . in whole mount views of e11.5 wild - type embryos , the eom anlage can be seen adjacent to the globe ( figs . sagittal views of cranial nerve and extraocular muscle development in e11.5 and e12.5 myf5::isl : gfp and myf5::isl : gfp embryos . extraocular muscles are absent in myf5 embryos ( b , d , f ) , and thus the red muscle channel has been turned off in wild - type images ( a , c , e ) , to permit equivalent comparison of nerve trajectories in the presence and absence of eoms . ( a , a ) whole mount wild - type embryo and ( b ) whole mount myf5 embryo at e11.5 . the trajectories of the nerves from the brainstem appear nearly identical in mutant and wild - type embryos at e11.5 . the developing muscle anlage stained with -sma can be seen in the wild type ( c ) , and an artery within the orbit stained with -sma is visible in both mutant and wild - type ( c , d ) . in the myf5 orbit , distinct extraocular muscles have formed and share a common origin at the annulus of zinn ( e , asterisk ) . the trunk of cn4 is now fasciculated , while the distal end forms a decision region adjacent to the so and sends terminal branches into the body of the muscle ( e , e , arrowhead ) . by contrast , the distal end of cn4 in the myf5 orbit fails to form an equivalent distal decision region and has no terminal branches ( f , arrowhead ) . in the wild - type orbit , the inferior division decision region has become more compact ( e , long arrow ) and extends a branch to the io muscle ( e , caret ) . in e11.5 wild - type embryos , the ocular motor nerves follow stereotypical trajectories to reach the developing orbit ( figs . despite the absence of the eom anlage in the e11.5 myf5 embryos , the ocular motor nerves appear similar to wild - type ( figs . beginning at e12.5 , differences are noted in development of ocular motor nerves in the presence and absence of eoms ( fig . distal cn4 does not form the extensive fan - like terminal branches seen in wild - type embryos . the cn3 inferior division decision region remains large ; its appearance is similar to wild - type at e11.5 . a branch of cn3 extends toward where the io muscle should be located , but it appears thinner , straighter , and blunter than in wild - type embryos ( fig . the distal aspect of cn4 widens considerably , and terminal branches emanate into the so muscle from one - half of the decision region , while branches from the other half are oriented away from the muscle ( figs . orbital dissections of e13.5 to e15.5 myf5:isl : gfp and myf5:isl : gfp embryos . from the inferior view ( c , c ) , cn3 inferior division sends terminal branches to the ir and mr muscles ( c , long arrow ) , and a well - formed branch to the io muscle ( c , caret ) . ( e h ) at e14.5 , the myf5 eoms continue to grow and change in orientation ( e , g ) : the sr muscle is now a broad band inserting on the top of the globe , the lps muscle has appeared between the sr and so muscles , and the so muscle has obtained its stereotypic curved shape . there is extensive terminal branching of cn3 superior division into the sr muscle ( e , e ) , and cn4 has developed a blunt - ended branch that is not in association with an eom ( e , e ) . in the absence of eom , the myf5 cn4 appears thin and has developed multiple aberrant branches ( f ) , cn3 main trunk widens ( h ) where the absent superior branch should have formed ( h , triple arrowhead ) , the inferior division decision region is small , with no visible branches to the ir and mr muscles , and the branch to the io muscle is now hypoplastic and aberrant ( h ) . l ) at e15.5 , myf5 orbits reveal the near - final ocular motor innervation patterns , and each nerve sends terminal and intramuscular branches into the appropriate eom . the branch extending toward where the io muscle should be is short , thin , and straight rather than arching as seen in wild - type embryos ( figs . ( b ) cn3 superior division branch point is significantly wider in myf5 embryos from e13.5 to e15.5 ( average from 62.05 8.17 m at e13.5 to 63.96 2.08 m at e15.5 ) than in the wild - type embryos ( average 35.92 8.43 m at e13.5 to 38.26 2.89 m at e15.5 ) . cn6 , which had crossed inferiorly to the oculomotor nerve at e13.5 , now has an aberrant trajectory parallel to the oculomotor nerve . at e15.5 , the ocular motor nerves in wild - type embryos are close to their mature configuration ( figs . cn3 inferior division branches to the mr and ir muscles are not visible and the branch to the io muscle has failed to elongate ( 155.3 87.7 m at e14.5 , 106.7 44.0 m at e15.5 ; figs . to determine if eoms are required for motor neuron survival , we examined the number of motor neurons in oculomotor and trochlear nuclei at e11.5 and e18.5 . by e18.5 , there is substantial normal developmental cell death in the control embryos , as reported previously . myf5 oculomotor and trochlear neuron number and position are similar to control at e11.5 , but are significantly reduced in number by e18.5 . ( f ) the number of motor neurons is reduced by 2/3 in myf5 oculomotor nucleus ( 2465 271 , n = 3 , vs. 868.2 81.7 , n = 3 , p = 0.00049 ) , indicating that extraocular muscles are necessary for oculomotor neuron survival . in whole mount views of e11.5 wild - type embryos , the eom anlage can be seen adjacent to the globe ( figs . sagittal views of cranial nerve and extraocular muscle development in e11.5 and e12.5 myf5::isl : gfp and myf5::isl : gfp embryos . extraocular muscles are absent in myf5 embryos ( b , d , f ) , and thus the red muscle channel has been turned off in wild - type images ( a , c , e ) , to permit equivalent comparison of nerve trajectories in the presence and absence of eoms . ( a , a ) whole mount wild - type embryo and ( b ) whole mount myf5 embryo at e11.5 . the trajectories of the nerves from the brainstem appear nearly identical in mutant and wild - type embryos at e11.5 . the developing muscle anlage stained with -sma can be seen in the wild type ( c ) , and an artery within the orbit stained with -sma is visible in both mutant and wild - type ( c , d ) . in the myf5 orbit , distinct extraocular muscles have formed and share a common origin at the annulus of zinn ( e , asterisk ) . the trunk of cn4 is now fasciculated , while the distal end forms a decision region adjacent to the so and sends terminal branches into the body of the muscle ( e , e , arrowhead ) . by contrast , the distal end of cn4 in the myf5 orbit fails to form an equivalent distal decision region and has no terminal branches ( f , arrowhead ) . in the wild - type orbit , the inferior division decision region has become more compact ( e , long arrow ) and extends a branch to the io muscle ( e , caret ) . a , anterior ( toward front of eye ) in e11.5 wild - type embryos , the ocular motor nerves follow stereotypical trajectories to reach the developing orbit ( figs . despite the absence of the eom anlage in the e11.5 myf5 embryos , the ocular motor nerves appear similar to wild - type ( figs . beginning at e12.5 , differences are noted in development of ocular motor nerves in the presence and absence of eoms ( fig . distal cn4 does not form the extensive fan - like terminal branches seen in wild - type embryos . the cn3 inferior division decision region remains large ; its appearance is similar to wild - type at e11.5 . a branch of cn3 extends toward where the io muscle should be located , but it appears thinner , straighter , and blunter than in wild - type embryos ( fig . in e13.5 wild - type embryos , the eoms continue to grow and separate . the distal aspect of cn4 widens considerably , and terminal branches emanate into the so muscle from one - half of the decision region , while branches from the other half are oriented away from the muscle ( figs . from the inferior view ( c , c ) , cn3 inferior division sends terminal branches to the ir and mr muscles ( c , long arrow ) , and a well - formed branch to the io muscle ( c , caret ) . there is extensive terminal branching of cn3 superior division into the sr muscle ( e , e ) , and cn4 has developed a blunt - ended branch that is not in association with an eom ( e , e ) . in the absence of eom , the myf5 cn4 appears thin and has developed multiple aberrant branches ( f ) , cn3 main trunk widens ( h ) where the absent superior branch should have formed ( h , triple arrowhead ) , the inferior division decision region is small , with no visible branches to the ir and mr muscles , and the branch to the io muscle is now hypoplastic and aberrant ( h ) . ( i l ) at e15.5 , myf5 orbits reveal the near - final ocular motor innervation patterns , and each nerve sends terminal and intramuscular branches into the appropriate eom . ( b ) cn3 superior division branch point is significantly wider in myf5 embryos from e13.5 to e15.5 ( average from 62.05 8.17 m at e13.5 to 63.96 2.08 m at e15.5 ) than in the wild - type embryos ( average 35.92 8.43 m at e13.5 to 38.26 2.89 m at e15.5 ) . while this branch continues to lengthen in wt orbits , reaching an average length of 412.8 25.5 m at e14.5 and 477.6 57.9 m at e15.5 , it fails to lengthen substantially in mutant orbits ( 155.3 87.7 m at e14.5 and 106.7 44.0 m at e15.5 ) . cn6 , which had crossed inferiorly to the oculomotor nerve at e13.5 , now has an aberrant trajectory parallel to the oculomotor nerve . at e15.5 , the ocular motor nerves in wild - type embryos are close to their mature configuration ( figs . cn3 inferior division branches to the mr and ir muscles are not visible and the branch to the io muscle has failed to elongate ( 155.3 87.7 m at e14.5 , 106.7 44.0 m at e15.5 ; figs . to determine if eoms are required for motor neuron survival , we examined the number of motor neurons in oculomotor and trochlear nuclei at e11.5 and e18.5 . at e11.5 , myf5 and control embryos had a similar number of isl1-positive motor neurons in the oculomotor and trochlear nuclei ( 4285 214 control , 4477 182 myf5 , p = 0.84 ; figs . by e18.5 , there is substantial normal developmental cell death in the control embryos , as reported previously . myf5 oculomotor and trochlear neuron number and position are similar to control at e11.5 , but are significantly reduced in number by e18.5 . ( f ) the number of motor neurons is reduced by 2/3 in myf5 oculomotor nucleus ( 2465 271 , n = 3 , vs. 868.2 81.7 , n = 3 , p = 0.00049 ) , indicating that extraocular muscles are necessary for oculomotor neuron survival . most prior studies of ocular cranial nerve development in mouse have relied on dii labeling or neurofilament staining , highlighting the initial nerve trajectories but not permitting visualization of their terminal processes in the orbit . by dissecting and visualizing the mouse orbit from timed embryos with genetically labeled motor axons , we characterized in greater detail the development of mouse oculomotor , trochlear , and abducens nerves , and showed that formation of the initial nerve trajectory does not require signals from the eoms , but formation of terminal branching does . ocular motor nerves arrive to the orbit and form distal decision regions in a spatially and temporally similar fashion in myf5 and wild - type embryos , demonstrating that axon growth and guidance from the brainstem to the proper position within the orbit is independent of eoms and likely occurs through cues from the mesenchyme and/or through cell autonomous processes . subsequent full nerve extension and terminal branching into the eom targets , maintenance of correct nerve position as the orbit expands , and survival of axons and their motor neurons , however , are dependent on the presence of eoms . the role of eoms in ocular motor nerve development is similar to the findings from previous reports of the contribution of skeletal muscle to spinal nerve development and survival . similarly , myod::myf5 mouse embryos lack all skeletal muscle and have normal initial growth and guidance of spinal motor nerves but fail to develop terminal nerve branches , followed by motor neuron apoptosis . we found minor differences in the relative timing of ocular motor nerve development in mouse compared to chick and zebrafish . in mouse we found that cn6 and cn3 reach the orbit simultaneously and before cn4 , which is similar to zebrafish but different from chick , in which the arrival of cn6 is followed by cn3 and then cn4 . first , we identified a trochlear branch in developing wild - type embryos that is not associated with a muscle , forms a blunt end , and subsequently retracts . consistent with its apparent lack of muscle target , the growth and regression of this wild - type trochlear branch resembles the growth and regression of ocular nerves in myf5 embryos . second , we found that the cn3 superior division , which in the mature state forms a single branch that then divides distally to innervate both the sr and lps muscles , fails to form in myf5 embryos . this suggests that , while axons within the main trunk of cn3 are guided in a cell - autonomous manner , by axon axon interactions , or by mesenchymal cues , the superior division axons exit from the main cn3 trunk in response to muscle cues . our current imaging approaches now reveal that , at e11.5 to e12.5 , the sr / lps muscle anlage lies in close juxtaposition to the main trunk of cn3 , and these multiple small branches extend directly along and into the muscle anlage . myf5 embryos do not form these multiple small branches , and , at the position where they normally form , the cn3 main trunk is wider than in wild - type embryos . the parallels , however , suggest that the myf5 nerve widening may result from paused growth cones that fail to exit the main cn3 trunk secondary to the absence of eoms . it is therefore not surprising that survival of ocular motor neurons depends on the presence of eoms . by contrast , in myod::myf5 embryos , which lack all skeletal muscles , all somatic motor neurons in the spinal cord die by e17.5 , and most motor neurons in the facial nucleus are dead or dying . the ocular motor nerves are selectively spared until late in the course of motor neuron degenerative disorders such as amyotrophic lateral sclerosis and spinal motor atrophy . using genetic tools , orbital dissection , and a developmental framework in timed mouse embryos , our findings catalogued the development of the ocular motor nerves and they implicate the eoms in the control of their terminal branching .

adequate nutrition during infancy and early childhood is essential to ensure the growth , health , and development of children to their full potential . it has been recognized worldwide that breastfeeding is beneficial for both the mother and child , as breast milk is considered the best source of nutrition for an infant . the world health organization ( who ) recommends that infants be exclusively breastfed for the first six months , followed by breastfeeding along with complementary foods for up to two years of age or beyond . exclusive breastfeeding can be defined as a practice whereby the infants receive only breast milk and not even water , other liquids , tea , herbal preparations , or food during the first six months of life , with the exception of vitamins , mineral supplements , or medicines . the major advantage of exclusive breastfeeding from 4 to 6 months includes reduced morbidity due to gastrointestinal infection . however , many researchers are questioning if there is sufficient evidence to confidently recommend exclusive breastfeeding for 6 months for infants in developed countries due to the fact that breast milk may not meet the full energy requirements of the average infant at 6 months of age . nevertheless , there is scanty data that give estimation about the proportion of exclusively breastfed infants at risk of specific nutritional deficiencies . several studies have shown that mothers find it difficult to meet personal goals and to adhere to the expert recommendations for continued and exclusive breastfeeding despite increased rate of initiation . some of the major factors that affect exclusivity and duration of breastfeeding include breast problems such as sore nipples or mother 's perceptions that she is producing inadequate milk [ 4 , 8 , 9 ] ; societal barriers such as employment and length of maternity leave ; inadequate breastfeeding knowledge ; lack of familial and societal support ; lack of guidance and encouragement from health care professionals [ 2 , 9 ] . these factors in turn promote the early use of breast milk substitute . when breast milk or infant formula no longer supplies infants with required energy and nutrients to sustain normal growth and optimal health and development , complementary feeding should be introduced . according to the who recommendations , the appropriate age at which solids should be introduced is around 6 months owing to the immaturity of the gastrointestinal tract and the renal system as well as on the neurophysiological status of the infant . factors that influence the weaning process include infant feeding problems such as refusal to eat , colic , and vomiting among others . these factors represent challenges for mothers and in turn may either directly or indirectly influence the feeding pattern . hence , understanding the factors affecting infant nutrition in mauritius can help in developing strategies to promote breastfeeding and overcoming problems faced by mothers and children . urban or rural difference , age , breast problems , societal barriers , insufficient support from family , knowledge about good breastfeeding practices , mode of delivery , health system practices , and community beliefs have all been found to influence breastfeeding in different areas of developing countries [ 4 , 8 , 9 ] . information on the prevalence and factors influencing infant feeding practices is limited in mauritius and dates back to 1996 . this present study aims to determine infant feeding pattern and its predictors among mauritian mothers with the following objectives : ( 1 ) to elucidate breastfeeding practices , in terms of initiation , exclusivity , and termination , and the factors influencing them ; ( 2 ) to determine the time when weaning starts , the challenges met by mothers , and the type of weaning adopted . a survey - based study was conducted on a group of 500 mothers in 2011 ( from august 2011 to january 2012 ) to elicit information about infant feeding practices by the use of a properly designed questionnaire given to mothers in area health centres ( ahcs ) and community health centres ( chcs ) both in rural and urban areas of the island . research has been granted approval by the university research ethics committee , and prior consents were obtained from all participants . the questionnaire consisted primarily of a closed format including dichotomous questions ( e.g. , yes / no ) and multiple response for ease of completion and analysis . the resulting questionnaire consisted of 46 close - ended questions , all categorized in 4 sections as follows.section a : the first section elicited information on the participants in terms of age , place of residence , marital status , type of family , parity , lifestyle factors ( smoking and alcohol consumption ) , education , occupation , income , religion , and age of baby.section b : this section was sought to understand the main factors encouraging mothers to breastfeed , their awareness on colostrum , the practice of exclusive breastfeeding , the termination of breastfeeding , as well as the main problems encountered during breastfeeding . section c : multiple response questions were mainly used in this section to determine more information on the uptake of infant formula.section d : it consists of dichotomous and multiple response questions to find out more details on the weaning process . section a : the first section elicited information on the participants in terms of age , place of residence , marital status , type of family , parity , lifestyle factors ( smoking and alcohol consumption ) , education , occupation , income , religion , and age of baby . section b : this section was sought to understand the main factors encouraging mothers to breastfeed , their awareness on colostrum , the practice of exclusive breastfeeding , the termination of breastfeeding , as well as the main problems encountered during breastfeeding . section c : multiple response questions were mainly used in this section to determine more information on the uptake of infant formula . section d : it consists of dichotomous and multiple response questions to find out more details on the weaning process . a sample of the female population consisting of mothers aged 1845 years was considered since they are adults and are mature enough to participate in the study . in addition , the sampling was based on the following inclusion and exclusion criteria . mothers who already delivered their baby and those with a child who is below 5 years old were considered in this survey . pregnant women or mothers having a child with any kind of malformations . mothers with children who are above 5 years old . chi - square tests were used to evaluate relationships between different selected variables ( e.g. , to find association between breastfeeding initiation and mode of delivery ; association between breastfeeding duration and parity , alcohol consumption , education , and occupation of respondents ) . the critical value for significance was set at p < 0.05 for all analyses . a total of 500 respondents completed the questionnaire of which 216 were from urban areas and 284 were from rural areas , with 53% mothers having completed at least secondary level education . equal representation of mothers from rural and urban areas was achieved through a quota sampling technique based on place of residence . the age of the participants ranged from 18 to 45 years old whereby the majority of the participants ( 38.4% ) belonged to the age group 2531 years and most of them were married ( 92.6% ) living in a nuclear family ( 58.6% ) . a total of 93.4% of the mothers acknowledged that they breastfed their infants of which 64.7% stated that they were self - motivated to opt for the natural way of feeding their infant since they were aware of the health benefits of breast milk and claimed that breast milk is best . additionally , 60.6% of the participants initiated breastfeeding the same day after delivery , while 39.4% started to nurse their baby 24 hours after delivery . chi - square ( ) test confirmed that the timing of breastfeeding initiation was significantly associated with mode of delivery ( = 212 , p < 0.001 ) . it should be noted that there were a greater number of mothers , that is , 294 participants ( 58.8% ) delivered their infants by the normal vaginal method compared to 206 mothers ( 41.2% ) who delivered by the caesarean method . it has been observed that 42.6% who had a normal vaginal delivery initiated breastfeeding immediately or within minutes after birth compared to 23.9% of those who had a caesarean type of delivery . although 35.7% of the participants had adequate knowledge on the definition / meaning of ebf , the practice was relatively low compared to the who recommendation , whereby only 17.9% of the women gave their infants only breast milk during the first six months . the main deterrent of ebf is the early introduction of water ( table 1 ) and infant formula ( table 3 ) . it is worth noting that mothers stated during the survey that they started to give water around 2 months . other major barriers to ebf include employment ( 27.3% ) followed by milk insufficiency ( 22.6% ) as reported by the respondents . these factors in turn led to a very short mean duration of ebf that is 2.10 months . the majority of the women practiced exclusive breastfeeding for less than one month ( 34.3% ) , while only 17.9% of them breastfed their child exclusively for around 5 - 6 months . the majority of the mothers completely terminate breastfeeding around 1924 months ( 26.0% ) ; test confirms that there are associations between the duration of breastfeeding and parity , alcohol consumption , education , and occupation of the respondents , while age group , residence , type of family , and type of delivery were not statistically significant ( p > 0.05 ) . it has been found that more primiparous mothers would stop nursing their infants around 1924 months ( 34.5% ) compared to multiparous mothers ( 19.9% ) , and cessation of breastfeeding beyond 24 months is more prevalent among participants who never drink alcoholic beverages . as far as education is concerned , it has been seen that irrespective of the level of schooling attained , mothers usually stop breastfeeding their infants within 24 months . in addition , even if women are employed as professionals ( 28.3% ) or are housewives ( 26.3% ) , they are more likely to discontinue breastfeeding within 24 months . during the breastfeeding process , many mothers complained about the problems they encountered . it can be seen from figure 2 that the majority of the respondents ( 46.2% ) did not face any problems while breastfeeding , but among those having difficulties , breast engorgement was most prevalent ( 33.3% ) followed by fatigue ( 25.1% ) , back pain ( 24.9% ) , and soreness of nipple ( 23.2% ) , while pain due to caesarian section , reluctance of infant to suckle , or sickness were minor problems that mothers faced . with respect to the introduction and use of infant formula , results indicate that more participants ( 37.9% ) start to use breast milk substitute within one month after delivery , whereby 33.9% of participants who use infant formula highlighted milk insufficiency as being the major reason to bottle feed , while 32.5% reported that they had to resume work ; thus , they opted for formula feeding as shown in table 3 . though the majority of the mothers reported that they did not have any problems with the breast milk substitute , that is , they never had to change the type of formula milk used ( 80.8% ) , some reported that baby constipated ( 5.9% ) and fell sick ( 4.3% ) with the infant formula , respectively . complementary feeding was more commonly initiated around 46 months ( 75.2% ) and partial weaning ( when baby is breastfed once or twice per day while receiving complementary foods ) was the most common type of weaning practiced by mothers ( 62.8% ) . during complementary feeding , both home - made and commercially available foods ( cereals , ready - made pots ) are given to the infants ( 69.2% ) . it has been found that weaning started with mashed vegetables or fruits ( 66.9% ) and the main reasons are due to the freshness of home - made food and it is also more hygienic ( 93.5% ) . additionally , 86.4% of the participants reported that the nutritional quality of home - made food is superior to that of commercial food , while 84.9% of the women stated that food prepared at home provides room for more choices for a balanced meal . with regard to the commercially available baby foods , it was noted that mothers prefer cereals ( 34.1% ) to ready - made pots ( 7.80% ) . it has also been found that 68.7% of mothers did not encounter any difficulty with their infants during the weaning period . moreover , the other respondents ( 21.7% ) highlighted that their children were unwilling to take solid foods , while 19.6% of them reported that their infants prefer drinking to food . a higher standard of living coupled with a higher education level in mauritius during the last 20 years has resulted in more women in the working sector . however , this has not dramatically decreased breastfeeding practice as it has been noted that the prevalence of breastfeeding in mauritius has risen from 72% in 1991 to 93.4% as found in this study . this may reflect the success of health promotion campaigns reiterating that breast is best or breast milk is beneficial for babies and mothers which account for the fact that mothers were self - motivated to breastfeed . findings of this study are consistent with the one conducted in northern ireland which reported that mothers were encouraged to breastfeed only because they know that breast is best or owing to the benefits of breast milk . although who 's , global and national infant , and young child feeding guidelines recommend that all newborns should start breastfeeding immediately ( within the first hour after delivery ) , the current study showed that very few participants ( 27.2% ) started to breastfeed immediately / within minutes after delivery or within one hour after birth compared to 39.4% mothers who initiated breastfeeding later than 1 hour within the same day . it has been noted that 206 respondents delivered by caesarian section of which 76.1% began to breastfeed their infants after 24 hours of birth . the delayed initiation of breastfeeding was most probably related to ( 1 ) the physical condition of the mother after delivery , whereby some mothers claimed that they were not feeling well enough to be able to breastfeed ; ( 2 ) painful conditions associated with caesarian section ; ( 3 ) the absence of their infants who were kept in nursery . similarly , other studies also noted that the rate of breastfeeding initiation within 1 hour was low and the principal barrier to the initiation and even continuation of breastfeeding is due to the operative obstetrical intervention [ 1719 ] . it has also been reported that after the caesarean section , mothers and infants are separated for a long period of time owing to anesthesia , baby being kept in nursery , or mother being sedated for pain and unable to feed [ 19 , 20 ] . it has been found that although knowledge on ebf for the first 6 months as per who recommendation ( 35.7% ) was relatively high , only about half ( 17.9% ) actually practiced it . the mean duration of exclusive breastfeeding in mauritius is only 2.10 months , whereby there are 17.9% of mothers who practiced ebf for the first 6 months unlike in other developing countries such as east asia / pacific which have the highest rate of exclusive breastfeeding ( 43.0% ) followed by eastern / southern africa ( 41.0% ) ( unicef , the united nations children 's fund ) . therefore , it can be argued that mothers failed to adhere strictly to the who recommendation of ebf for the first 6 months owing to the introduction of water and infant formula much before 6 months . usually , female workers in mauritius are allowed 12 weeks of maternity leave which equals to approximately 3 months ( ssptw , social security programs throughout the world ) . under these circumstances , mothers are prompted to resort to the supplementation of infant formula before 3 months so that their infants familiarize to bottle feeding during their absence . this finding is consistent with other studies which highlighted employment and milk insufficiency as the major barriers to ebf [ 2 , 8 , 2325 ] , while another research pointed out that mothers stop ebf as they perceive that their infants feel hungry and unsatisfied with breast milk only . nevertheless , it has been argued that the exclusivity of breastfeeding is affected when mothers experience problems with the infant latching - on or sucking and they do not get assistance from some clinicians who do not feel intrepid in their skills to support breastfeeding and may have limited time to address the matter during preventive visits . additionally , 26.0% of the respondents cease breastfeeding within 2 years , while there are notably some mothers who breastfeed above 2 years . this implies that despite the fact that the majority of the participants adopt mixed feeding , they still adhere to the who recommendation which involves continued breastfeeding up to 2 years or beyond . this study reveals that factors including parity , alcohol consumption , education , and occupation are associated with the termination of breastfeeding . there are a greater number of women from the lower parity who terminate breastfeeding within 2 years as compared to their counterparts . primiparous women are less knowledgeable and skilful in breastfeeding ; hence , they will usually seek assistance , advice , and help from health care professionals who generally promote breastfeeding . furthermore , first time mothers are more likely to consider health promotion messages or be exposed to them in different ways . on the other side , higher parity leads to short birth intervals , hence , minimal time available for breastfeeding . in contrast to previous reviews , primiparity was associated with reduced risk for breastfeeding duration , while other studies done in the united kingdom and in bangladesh affirmed that breastfeeding duration increases with increasing parity which might be related to previous breastfeeding experiences . nevertheless , in another study , it was asserted that parity had no significant influence on duration of breastfeeding . there is an association between the frequency of intake of alcoholic beverages and duration of breastfeeding . mothers who never or seldom take alcoholic drinks are more apt to breastfeed longer than those who consume them occasionally . this might be because those who consume alcohol on a regular basis avoid breastfeeding owing to the fact that alcohol readily crosses into breast milk by simple diffusion , attaining levels approximately equal to that in the maternal blood stream . this finding agrees with those observed in other studies carried out in australia and in greece which stated that mothers stop breastfeeding their infants earlier because exposing the child to small amounts of alcohol through breast milk disrupts infant sleeping patterns . it was noted that the level of education did not have any influence on breastfeeding duration and mauritian mothers usually breastfeed at least for 12 months . in contrast to this study , it has been found in a previous research conducted in philippines that education plays a significant role in determining the duration of breastfeeding . increasing level of education also implies adoption of modern ideas while gradually leading to the dereliction of traditional practices regarding child care , thus , a decrease in the rate of breastfeeding . with regard to occupation of the mothers , it has been observed that regardless of the fact that the participants are housewives or employed as professionals , they would normally stop nursing their infants within 2 years . generally , housewives have unlimited time available to feed their infants while on the other hand , despite the fact that the participants work as professionals , they still breastfeed as long as housewives do . one most probable reason for this is that even though they work , they express their breast milk either manually or with pumps so that somebody else can still feed the baby or they are usually given flexible time at work to maintain breastfeeding . another study in malaysia reported that facility at workplace similar in mauritius such as allowing mothers a flexible time to express breast milk helps in maintaining lactation . conversely , other investigators observed that women having professional jobs especially in urban areas stop breastfeeding earlier than the recommended duration because they have reduced access to their children whereas those involved in traditional work have more time and maintain longer periods of lactation . although a greater part of the participants do not experience any difficulties while nursing their infants , there were still a significant number of the respondents who complain about breast engorgement , fatigue , back pain , and soreness of nipple . breast engorgement usually occurs when milk gets accumulated in the breast , while sore nipples arise because of the baby sucking the nipple area of the breast only . generally , nursing mothers breastfeed their children frequently during the day ( each 2 hours ) which leads to fatigue and back pain . this research affirmed that these difficulties result in a negative experience with breastfeeding which is followed by a decrease in mothers ' confidence to wet - nurse their infants , hence , causing early cessation of breastfeeding . these results are consistent with recent studies demonstrating that many women encounter problems such as cracked nipples , low milk supply , and breast engorgement [ 24 , 26 , 3741 ] . early termination of breastfeeding also implies early use of breast milk substitute and as pointed out above , factors such as work , milk insufficiency , and breastfeeding difficulties are the major reasons for adopting formula feeding . among the few participants who encountered minor feeding problems with the formula milk reported constipation and sickness such as vomiting , diarrhoea , colic , and regurgitation as the most common ones . the risk of constipation among formula - fed children is quite common and this has also been found in italy , whereby the authors reported that there is a prolonged gastrointestinal transit in formula - fed infants and the stool consistency is hard compared to breastfed infants . complementary foods are generally introduced between 4 and 6 months and partial weaning is the most common type of weaning adopted by mothers . generally , women who terminate breastfeeding within 2 years are more likely to adopt partial weaning because it involves nursing the infant as well as introducing complementary foods , while those who stop nursing their infants within 6 months adopt mother - led weaning . conversely , mother - led weaning occurs when the mother feels the need to introduce complementary foods . since , there is limited research on the type of weaning adopted by mothers during infant feeding practices , the results obtained in the present study are more suggestive than affirmative . results herein corroborate those carried out in switzerland which demonstrate that a greater number of women start to wean their infants with mashed vegetables or fruits followed by cereals . the main reason as pointed out by the participants in this study is that home - made food is more fresh , nutritious , and hygienic unlike commercially available cereal or baby foods . gradually , baby cereals or commercial purees are also used alongside home - made foods and more women prefer cereals ( 34.1% ) to ready - made pots ( 7.80% ) because they believe that commercial purees contain additives , high sugar content and salt content , compared to cereals . to date , there are no published data on the type of weaning food ( home - made versus commercially available food ) . a few mothers experience difficulties during complementary feeding which include unwillingness of the child to eat while exerting preferences to drink rather than eating . the minority of the participants affirmed that they encountered problems such as allergic reactions and health problems with the infant including vomiting , colic , and diarrhoea which may arise due to the feeding practices adopted by mothers . other possible barriers during complementary feeding found in other studies unlike the present study include food refusal , selective , picky or fussy eating , eating slowly , being less interested in food , and having a small appetite . this study shows that the prevalence of breastfeeding has increased over the past 20 years in mauritius . the who guidelines advise to breastfeed exclusively until 6 months of age . despite a high breastfeeding initiation rate of 61% , only 18% succeed to give exclusive breastfeeding until 5 - 6 months . the mean duration of exclusive breastfeeding is 2 months , with adding water as the main reason for not continuing exclusiveness . awareness of the health benefits of breastfeeding was noted in 65% , a percentage that may be increased by further breastfeeding education and support . the major barriers to breastfeeding practices in this study in terms of initiation , exclusivity , and duration are ( 1 ) type of delivery ; ( 2 ) parity ; ( 3 ) alcohol consumption ; ( 4 ) occupation and education ; ( 5 ) breast problems , mainly milk insufficiency . complementary foods are normally introduced around 4 to 6 months and mothers usually start with home - made food because of its freshness and for hygienic reasons . however , there are very few mothers who encountered difficulties during the weaning process as compared during breastfeeding practices such as refusal to eat followed by vomiting , colic , allergic reactions , and diarrhea which were rare . future studies along the same line should target children of 3 years as it has been suggested by khassawneh that this will reduce the risk of recall bias . to calculate the sampling size , , this data is not representative of the number of mothers aged between 18 and 45 years .

proper breastfeeding practices are effective ways for reducing childhood morbidity and mortality . while many mothers understand the importance of breastfeeding , others are less knowledgeable on the benefits of breastfeeding and weaning . the aim in here is to assess breastfeeding pattern , infant formula feeding pattern , and weaning introduction in mauritius and to investigate the factors that influence infant nutrition . 500 mothers were interviewed using a questionnaire which was designed to elicit information on infant feeding practices . statistical analyses were done using spss ( version 13.0 ) , whereby chi - square tests were used to evaluate relationships between different selected variables . the prevalence of breastfeeding practice in mauritius has risen from 72% in 1991 to 93.4% as found in this study , while only 17.9% breastfed their children exclusively for the first 6 months , and the mean duration of ebf ( exclusive breastfeeding ) is 2.10 months . complementary feeding was more commonly initiated around 46 months ( 75.2% ) . despite the fact that 60.6% of mothers initiate breastfeeding and 26.1% of mothers are found to breastfeed up to 2 years , the practice of ebf for the first 6 months is low ( 17.9% ) . factors found to influence infant feeding practices are type of delivery , parity , alcohol consumption , occupation , education , and breast problems .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion and Limitations

adequate nutrition during infancy and early childhood is essential to ensure the growth , health , and development of children to their full potential . the world health organization ( who ) recommends that infants be exclusively breastfed for the first six months , followed by breastfeeding along with complementary foods for up to two years of age or beyond . exclusive breastfeeding can be defined as a practice whereby the infants receive only breast milk and not even water , other liquids , tea , herbal preparations , or food during the first six months of life , with the exception of vitamins , mineral supplements , or medicines . the major advantage of exclusive breastfeeding from 4 to 6 months includes reduced morbidity due to gastrointestinal infection . however , many researchers are questioning if there is sufficient evidence to confidently recommend exclusive breastfeeding for 6 months for infants in developed countries due to the fact that breast milk may not meet the full energy requirements of the average infant at 6 months of age . several studies have shown that mothers find it difficult to meet personal goals and to adhere to the expert recommendations for continued and exclusive breastfeeding despite increased rate of initiation . some of the major factors that affect exclusivity and duration of breastfeeding include breast problems such as sore nipples or mother 's perceptions that she is producing inadequate milk [ 4 , 8 , 9 ] ; societal barriers such as employment and length of maternity leave ; inadequate breastfeeding knowledge ; lack of familial and societal support ; lack of guidance and encouragement from health care professionals [ 2 , 9 ] . when breast milk or infant formula no longer supplies infants with required energy and nutrients to sustain normal growth and optimal health and development , complementary feeding should be introduced . according to the who recommendations , the appropriate age at which solids should be introduced is around 6 months owing to the immaturity of the gastrointestinal tract and the renal system as well as on the neurophysiological status of the infant . factors that influence the weaning process include infant feeding problems such as refusal to eat , colic , and vomiting among others . these factors represent challenges for mothers and in turn may either directly or indirectly influence the feeding pattern . hence , understanding the factors affecting infant nutrition in mauritius can help in developing strategies to promote breastfeeding and overcoming problems faced by mothers and children . urban or rural difference , age , breast problems , societal barriers , insufficient support from family , knowledge about good breastfeeding practices , mode of delivery , health system practices , and community beliefs have all been found to influence breastfeeding in different areas of developing countries [ 4 , 8 , 9 ] . information on the prevalence and factors influencing infant feeding practices is limited in mauritius and dates back to 1996 . this present study aims to determine infant feeding pattern and its predictors among mauritian mothers with the following objectives : ( 1 ) to elucidate breastfeeding practices , in terms of initiation , exclusivity , and termination , and the factors influencing them ; ( 2 ) to determine the time when weaning starts , the challenges met by mothers , and the type of weaning adopted . a survey - based study was conducted on a group of 500 mothers in 2011 ( from august 2011 to january 2012 ) to elicit information about infant feeding practices by the use of a properly designed questionnaire given to mothers in area health centres ( ahcs ) and community health centres ( chcs ) both in rural and urban areas of the island . research has been granted approval by the university research ethics committee , and prior consents were obtained from all participants . the resulting questionnaire consisted of 46 close - ended questions , all categorized in 4 sections as follows.section a : the first section elicited information on the participants in terms of age , place of residence , marital status , type of family , parity , lifestyle factors ( smoking and alcohol consumption ) , education , occupation , income , religion , and age of baby.section b : this section was sought to understand the main factors encouraging mothers to breastfeed , their awareness on colostrum , the practice of exclusive breastfeeding , the termination of breastfeeding , as well as the main problems encountered during breastfeeding . section c : multiple response questions were mainly used in this section to determine more information on the uptake of infant formula.section d : it consists of dichotomous and multiple response questions to find out more details on the weaning process . section a : the first section elicited information on the participants in terms of age , place of residence , marital status , type of family , parity , lifestyle factors ( smoking and alcohol consumption ) , education , occupation , income , religion , and age of baby . section b : this section was sought to understand the main factors encouraging mothers to breastfeed , their awareness on colostrum , the practice of exclusive breastfeeding , the termination of breastfeeding , as well as the main problems encountered during breastfeeding . section c : multiple response questions were mainly used in this section to determine more information on the uptake of infant formula . section d : it consists of dichotomous and multiple response questions to find out more details on the weaning process . a sample of the female population consisting of mothers aged 1845 years was considered since they are adults and are mature enough to participate in the study . in addition , the sampling was based on the following inclusion and exclusion criteria . mothers who already delivered their baby and those with a child who is below 5 years old were considered in this survey . chi - square tests were used to evaluate relationships between different selected variables ( e.g. , to find association between breastfeeding initiation and mode of delivery ; association between breastfeeding duration and parity , alcohol consumption , education , and occupation of respondents ) . a total of 93.4% of the mothers acknowledged that they breastfed their infants of which 64.7% stated that they were self - motivated to opt for the natural way of feeding their infant since they were aware of the health benefits of breast milk and claimed that breast milk is best . additionally , 60.6% of the participants initiated breastfeeding the same day after delivery , while 39.4% started to nurse their baby 24 hours after delivery . chi - square ( ) test confirmed that the timing of breastfeeding initiation was significantly associated with mode of delivery ( = 212 , p < 0.001 ) . it should be noted that there were a greater number of mothers , that is , 294 participants ( 58.8% ) delivered their infants by the normal vaginal method compared to 206 mothers ( 41.2% ) who delivered by the caesarean method . it has been observed that 42.6% who had a normal vaginal delivery initiated breastfeeding immediately or within minutes after birth compared to 23.9% of those who had a caesarean type of delivery . although 35.7% of the participants had adequate knowledge on the definition / meaning of ebf , the practice was relatively low compared to the who recommendation , whereby only 17.9% of the women gave their infants only breast milk during the first six months . the main deterrent of ebf is the early introduction of water ( table 1 ) and infant formula ( table 3 ) . these factors in turn led to a very short mean duration of ebf that is 2.10 months . the majority of the women practiced exclusive breastfeeding for less than one month ( 34.3% ) , while only 17.9% of them breastfed their child exclusively for around 5 - 6 months . the majority of the mothers completely terminate breastfeeding around 1924 months ( 26.0% ) ; test confirms that there are associations between the duration of breastfeeding and parity , alcohol consumption , education , and occupation of the respondents , while age group , residence , type of family , and type of delivery were not statistically significant ( p > 0.05 ) . it has been found that more primiparous mothers would stop nursing their infants around 1924 months ( 34.5% ) compared to multiparous mothers ( 19.9% ) , and cessation of breastfeeding beyond 24 months is more prevalent among participants who never drink alcoholic beverages . in addition , even if women are employed as professionals ( 28.3% ) or are housewives ( 26.3% ) , they are more likely to discontinue breastfeeding within 24 months . during the breastfeeding process , many mothers complained about the problems they encountered . it can be seen from figure 2 that the majority of the respondents ( 46.2% ) did not face any problems while breastfeeding , but among those having difficulties , breast engorgement was most prevalent ( 33.3% ) followed by fatigue ( 25.1% ) , back pain ( 24.9% ) , and soreness of nipple ( 23.2% ) , while pain due to caesarian section , reluctance of infant to suckle , or sickness were minor problems that mothers faced . with respect to the introduction and use of infant formula , results indicate that more participants ( 37.9% ) start to use breast milk substitute within one month after delivery , whereby 33.9% of participants who use infant formula highlighted milk insufficiency as being the major reason to bottle feed , while 32.5% reported that they had to resume work ; thus , they opted for formula feeding as shown in table 3 . though the majority of the mothers reported that they did not have any problems with the breast milk substitute , that is , they never had to change the type of formula milk used ( 80.8% ) , some reported that baby constipated ( 5.9% ) and fell sick ( 4.3% ) with the infant formula , respectively . complementary feeding was more commonly initiated around 46 months ( 75.2% ) and partial weaning ( when baby is breastfed once or twice per day while receiving complementary foods ) was the most common type of weaning practiced by mothers ( 62.8% ) . during complementary feeding , both home - made and commercially available foods ( cereals , ready - made pots ) are given to the infants ( 69.2% ) . it has been found that weaning started with mashed vegetables or fruits ( 66.9% ) and the main reasons are due to the freshness of home - made food and it is also more hygienic ( 93.5% ) . additionally , 86.4% of the participants reported that the nutritional quality of home - made food is superior to that of commercial food , while 84.9% of the women stated that food prepared at home provides room for more choices for a balanced meal . it has also been found that 68.7% of mothers did not encounter any difficulty with their infants during the weaning period . moreover , the other respondents ( 21.7% ) highlighted that their children were unwilling to take solid foods , while 19.6% of them reported that their infants prefer drinking to food . however , this has not dramatically decreased breastfeeding practice as it has been noted that the prevalence of breastfeeding in mauritius has risen from 72% in 1991 to 93.4% as found in this study . this may reflect the success of health promotion campaigns reiterating that breast is best or breast milk is beneficial for babies and mothers which account for the fact that mothers were self - motivated to breastfeed . findings of this study are consistent with the one conducted in northern ireland which reported that mothers were encouraged to breastfeed only because they know that breast is best or owing to the benefits of breast milk . although who 's , global and national infant , and young child feeding guidelines recommend that all newborns should start breastfeeding immediately ( within the first hour after delivery ) , the current study showed that very few participants ( 27.2% ) started to breastfeed immediately / within minutes after delivery or within one hour after birth compared to 39.4% mothers who initiated breastfeeding later than 1 hour within the same day . the delayed initiation of breastfeeding was most probably related to ( 1 ) the physical condition of the mother after delivery , whereby some mothers claimed that they were not feeling well enough to be able to breastfeed ; ( 2 ) painful conditions associated with caesarian section ; ( 3 ) the absence of their infants who were kept in nursery . similarly , other studies also noted that the rate of breastfeeding initiation within 1 hour was low and the principal barrier to the initiation and even continuation of breastfeeding is due to the operative obstetrical intervention [ 1719 ] . it has been found that although knowledge on ebf for the first 6 months as per who recommendation ( 35.7% ) was relatively high , only about half ( 17.9% ) actually practiced it . the mean duration of exclusive breastfeeding in mauritius is only 2.10 months , whereby there are 17.9% of mothers who practiced ebf for the first 6 months unlike in other developing countries such as east asia / pacific which have the highest rate of exclusive breastfeeding ( 43.0% ) followed by eastern / southern africa ( 41.0% ) ( unicef , the united nations children 's fund ) . therefore , it can be argued that mothers failed to adhere strictly to the who recommendation of ebf for the first 6 months owing to the introduction of water and infant formula much before 6 months . usually , female workers in mauritius are allowed 12 weeks of maternity leave which equals to approximately 3 months ( ssptw , social security programs throughout the world ) . under these circumstances , mothers are prompted to resort to the supplementation of infant formula before 3 months so that their infants familiarize to bottle feeding during their absence . this finding is consistent with other studies which highlighted employment and milk insufficiency as the major barriers to ebf [ 2 , 8 , 2325 ] , while another research pointed out that mothers stop ebf as they perceive that their infants feel hungry and unsatisfied with breast milk only . nevertheless , it has been argued that the exclusivity of breastfeeding is affected when mothers experience problems with the infant latching - on or sucking and they do not get assistance from some clinicians who do not feel intrepid in their skills to support breastfeeding and may have limited time to address the matter during preventive visits . additionally , 26.0% of the respondents cease breastfeeding within 2 years , while there are notably some mothers who breastfeed above 2 years . this implies that despite the fact that the majority of the participants adopt mixed feeding , they still adhere to the who recommendation which involves continued breastfeeding up to 2 years or beyond . this study reveals that factors including parity , alcohol consumption , education , and occupation are associated with the termination of breastfeeding . primiparous women are less knowledgeable and skilful in breastfeeding ; hence , they will usually seek assistance , advice , and help from health care professionals who generally promote breastfeeding . furthermore , first time mothers are more likely to consider health promotion messages or be exposed to them in different ways . nevertheless , in another study , it was asserted that parity had no significant influence on duration of breastfeeding . there is an association between the frequency of intake of alcoholic beverages and duration of breastfeeding . this might be because those who consume alcohol on a regular basis avoid breastfeeding owing to the fact that alcohol readily crosses into breast milk by simple diffusion , attaining levels approximately equal to that in the maternal blood stream . in contrast to this study , it has been found in a previous research conducted in philippines that education plays a significant role in determining the duration of breastfeeding . increasing level of education also implies adoption of modern ideas while gradually leading to the dereliction of traditional practices regarding child care , thus , a decrease in the rate of breastfeeding . with regard to occupation of the mothers , it has been observed that regardless of the fact that the participants are housewives or employed as professionals , they would normally stop nursing their infants within 2 years . generally , housewives have unlimited time available to feed their infants while on the other hand , despite the fact that the participants work as professionals , they still breastfeed as long as housewives do . another study in malaysia reported that facility at workplace similar in mauritius such as allowing mothers a flexible time to express breast milk helps in maintaining lactation . although a greater part of the participants do not experience any difficulties while nursing their infants , there were still a significant number of the respondents who complain about breast engorgement , fatigue , back pain , and soreness of nipple . breast engorgement usually occurs when milk gets accumulated in the breast , while sore nipples arise because of the baby sucking the nipple area of the breast only . generally , nursing mothers breastfeed their children frequently during the day ( each 2 hours ) which leads to fatigue and back pain . this research affirmed that these difficulties result in a negative experience with breastfeeding which is followed by a decrease in mothers ' confidence to wet - nurse their infants , hence , causing early cessation of breastfeeding . these results are consistent with recent studies demonstrating that many women encounter problems such as cracked nipples , low milk supply , and breast engorgement [ 24 , 26 , 3741 ] . early termination of breastfeeding also implies early use of breast milk substitute and as pointed out above , factors such as work , milk insufficiency , and breastfeeding difficulties are the major reasons for adopting formula feeding . among the few participants who encountered minor feeding problems with the formula milk reported constipation and sickness such as vomiting , diarrhoea , colic , and regurgitation as the most common ones . the risk of constipation among formula - fed children is quite common and this has also been found in italy , whereby the authors reported that there is a prolonged gastrointestinal transit in formula - fed infants and the stool consistency is hard compared to breastfed infants . complementary foods are generally introduced between 4 and 6 months and partial weaning is the most common type of weaning adopted by mothers . generally , women who terminate breastfeeding within 2 years are more likely to adopt partial weaning because it involves nursing the infant as well as introducing complementary foods , while those who stop nursing their infants within 6 months adopt mother - led weaning . since , there is limited research on the type of weaning adopted by mothers during infant feeding practices , the results obtained in the present study are more suggestive than affirmative . the main reason as pointed out by the participants in this study is that home - made food is more fresh , nutritious , and hygienic unlike commercially available cereal or baby foods . to date , there are no published data on the type of weaning food ( home - made versus commercially available food ) . a few mothers experience difficulties during complementary feeding which include unwillingness of the child to eat while exerting preferences to drink rather than eating . the minority of the participants affirmed that they encountered problems such as allergic reactions and health problems with the infant including vomiting , colic , and diarrhoea which may arise due to the feeding practices adopted by mothers . other possible barriers during complementary feeding found in other studies unlike the present study include food refusal , selective , picky or fussy eating , eating slowly , being less interested in food , and having a small appetite . this study shows that the prevalence of breastfeeding has increased over the past 20 years in mauritius . the who guidelines advise to breastfeed exclusively until 6 months of age . despite a high breastfeeding initiation rate of 61% , only 18% succeed to give exclusive breastfeeding until 5 - 6 months . the mean duration of exclusive breastfeeding is 2 months , with adding water as the main reason for not continuing exclusiveness . awareness of the health benefits of breastfeeding was noted in 65% , a percentage that may be increased by further breastfeeding education and support . the major barriers to breastfeeding practices in this study in terms of initiation , exclusivity , and duration are ( 1 ) type of delivery ; ( 2 ) parity ; ( 3 ) alcohol consumption ; ( 4 ) occupation and education ; ( 5 ) breast problems , mainly milk insufficiency . complementary foods are normally introduced around 4 to 6 months and mothers usually start with home - made food because of its freshness and for hygienic reasons . however , there are very few mothers who encountered difficulties during the weaning process as compared during breastfeeding practices such as refusal to eat followed by vomiting , colic , allergic reactions , and diarrhea which were rare .

lung cancer is considered the second most frequent cancer for men after prostate cancer and breast cancer for women . 1 non - specific cytotoxic agents are currently used for the treatment of non - small cell lung cancer ( nsclc ) and small cell lung cancer ( sclc).2 - 4 however ; side effects are the reason for some patients to postpone their treatment and in some cases to be hospitalized increasing the cost of treatment.5 in the recent years targeted therapies have emerged and based on the genome of cancer specific oral agents are being administered . 2 epidermal growth factor receptor status and anaplastic lymphoma kinase status are the two most investigated pathways regarding adenocarcinoma . 6 - 8 erlotinib , gefinitib , afatinib and crizotinib are the most frequently used targeted agents.7 , 9 erlotinib , gefinitib and afatinib have presented superiority in terms of overall survival and disease control when compared to doublet chemotherapy agents in a specific group of patients.10 however ; resistance to tyrosine kinase inhibitors has been observed and therefore new or alternative treatment approaches are being investigated.11 these agents are considered safer in the sense that the patients have less side effects , however ; they still have side effects which in some cases are dose dependent and directly compared with the positive disease control.12 regarding sclc chemotherapy is still the considered the best first line treatment , however ; several novel therapies are being investigated.13 - 16 the role of tyrosine kinase inhibitors as neo - adjuvant or adjuvant treatment is still under investigation . one observation has to be taken into serious consideration that if chemotherapy is administered in egfr positive patients as neo - adjuvant treatment then the mutation status will change along with the gene status of several other involved pathways with an unknown treatment effect in the future if treatment will become necessary ( disease relapse ) . 18 it is suggested that there are no clear evidence that a patient should receive neo - adjuvant tyrosine kinase inhibitors for early stage nsclc , however ; there are evidence that patients will benefit from this oral drug administration . in the case presented by funakoshi y et al . 19 a patient received gefinitib as neo - adjuvant treatment , downstaging of the disease was observed and the patient underwent pneumonectomy . in the study by li n et al . 20 it is suggested that as adjuvant treatment gefinitib can be administered with or without other chemotherapy agents . in the editorial by martinez p 21 it is suggested that tyrosine kinase inhibitors could be used as adjuvant treatment , however ; because of the few trials which have small patient samples and with unselected patients more studies are still needed . regarding afatinib a meta - analysis presented data where this drug could be considered as more toxic than erlotinib and gefitinib.7 there are no data regarding afatinib as neo - adjuvant treatment , however ; regarding adjuvant treatment there is one study by burtness b et al . maybe this study could be used for further investigation of afatinib as adjuvant treatment for nsclc . there is one group of patients that tyrosine kinase inhibitors are still under investigation whether they would benefit or not . if one could set the age for this group of patients it would probably be > 75 years of age which is more or less recognized by the international medical community the cut off age that a patient should receive chemotherapy . although in several patient cases an individualized approach is necessary if the patient has could performance status . the elderly are not considered could candidates for trials since they tend not to complete them due to side effects.23 in the current mini review we will focus on the group of patients > 70 years of age . we will present up to date studies and comment on the targeted treatment for this group of patients . we performed an electronic article search through pubmed , google scholar , medscape and scopus databases , using combinations of the following keywords : tyrosine kinase inhobitors , erlotinib , gefitinib , afatinib , and combination of these words with the word elderly . all types of articles ( randomised controlled trials , clinical observational cohort studies , review articles , case reports ) were included . selected references from identified articles were searched for further consideration indifferent of the language . in the study by yoshioka h. et al . 24 a very well designed study the patients and adverse effects of the tkis were stratified according to age in three different groups as follows : a ) < 75 , b ) 75 -84 and c ) 85. the incidence of interstitial lung disease ( ild ) ( all grades ) was 4.2% ( < 75 years ) , 5.1% ( 75 - 84 years ) , and 3.4% ( 85 years ) . therefore it was concluded that erlotinib is safe and efficient for patients > 75 years of age . in the study by kurishima k et al . 25 patients receiving erlotinib were divided into two groups : a ) 75 and b ) > 75 . it was observed that adverse effects were the same in both groups and the overall survival did not differ between the elderly and younger groups ( median , 170 days ; 95% ci : 142239 days vs. median , 146 days ; 95% ci : 114185 days , respectively ) ( p=0.7642 ) . therefore it was concluded that targeted treatment is safe and efficient for this group of patients . in the study by jackman dm . the most common toxicities were acneiform rash ( 79% ) and diarrhea ( 69% ) . four patients developed interstitial lung disease of grade 3 or higher , with one treatment - related death . it was concluded that erlotinib merits consideration for further investigation as a first - line therapeutic option in elderly patients . in the study by chen ym . 27 patients of 70 years of age were included and received either erlotinib or vinorelbine . overall survival was longer for egfr - mutated patients than for egfr wild - type patients ( p < 0.0001 ) . it was concluded that erlotinib is highly effective compared with oral vinorelbine in elderly , chemonaive , taiwanese patients with nsclc . egfr - mutated patients had better survival than those with egfr wild - type disease , regardless of the treatment received . in the study by wheatley - price p. et al . 28 two groups were formed : a ) < 70 years of age and b ) > 70 years of age ( elderly ) . elderly patients , compared with young patients , had significantly more overall and severe ( grade 3 and 4 ) toxicity ( 35% v 18% ; p _ .001 ) , were more likely to discontinue treatment as a result of treatment - related toxicity ( 12% v 3% ; p _ .0001 ) , and had lower relative dose - intensity ( 64% v 82% received _ it was concluded that elderly patients treated with erlotinib gain similar survival and qol benefits as younger patients but experience greater toxicity . in the study by platania m. et al . grade 3 - 4 adverse events were observed in 16 cases ( 37% ) . it was concluded that the use of erlotinib after chemotherapy failure in an unselected elderly population affected by nsclc showed moderate efficacy and a moderate safety profile and therefore erlotinib represents a valid option in this setting . it was observed that elevation of aspartate transaminase and/or alanine transaminase ( 18.3% ) was the most common adverse event , and one treatment - related death ( pneumonitis ) occurred . it was concluded that first - line gefitinib is efficacious with acceptable toxicity in relatively fit elderly patients with advanced nsclc harboring an egfr mutation . in the study by des 31 patients were divided in two major groups : a ) < 70 years of age and b ) > 70 years of age . patients were subdivided into three groups receiving three different drugs ( gefitinib ; gemcitabine ; and docetaxel ) . it was observed that older patients had a decreased risk of progression / death compared to younger patients . single - agent chemotherapy can be considered for patients aged 70years with a ps of 2 . in the study by takahashi k. et al . 32 patients between 72 - 90 years of age were included specifically to access the safety and effectiveness of gefitinib in the elderly ( median age was 79.5 years ) . the functional assessment of cancer therapy - lung cancer subscale ( fact - lcs ) scores improved significantly 4 weeks after the initiation of gefitinib ( p = 0.037 ) and maintained favorably over a 12-week assessment period . the most common adverse events were rash and liver dysfunction . although grade 1 pneumonitis developed in one patient , no treatment - related death was observed . it was observed that first - line gefitinib therapy is effective and feasible for elderly patients harboring egfr mutation , and improves disease - related symptoms , especially pulmonary symptoms like shortness of breath and cough . in the study by inoue a. et al . 33 patients > 74 years of age were included with poor performance status and with the administration of gefitinib no treatment - related deaths were observed . this was the first study that verified safety and efficacy of first - line treatment with gefitinib in elderly patients having advanced nsclc with egfr mutation . it was concluded that due to its strong antitumor activity and mild toxicity , first - line gefitinib may be preferable to standard chemotherapy for this population . 35 patients of 80 years of age were included and the tkis gefitinib and erlotinib were administered . it was observed that the administration of these agents to this group of age was acceptable , however ; the authors suggest that a careful dose selection according to the overall medical condition should be made . in the study by yoshioka h. et al . 24 a very well designed study the patients and adverse effects of the tkis were stratified according to age in three different groups as follows : a ) < 75 , b ) 75 -84 and c ) 85. the incidence of interstitial lung disease ( ild ) ( all grades ) was 4.2% ( < 75 years ) , 5.1% ( 75 - 84 years ) , and 3.4% ( 85 years ) therefore it was concluded that erlotinib is safe and efficient for patients > 75 years of age . in the study by kurishima k et al . 25 patients receiving erlotinib were divided into two groups : a ) 75 and b ) > 75 . it was observed that adverse effects were the same in both groups and the overall survival did not differ between the elderly and younger groups ( median , 170 days ; 95% ci : 142239 days vs. median , 146 days ; 95% ci : 114185 days , respectively ) ( p=0.7642 ) . therefore it was concluded that targeted treatment is safe and efficient for this group of patients . in the study by jackman dm . the most common toxicities were acneiform rash ( 79% ) and diarrhea ( 69% ) . four patients developed interstitial lung disease of grade 3 or higher , with one treatment - related death . it was concluded that erlotinib merits consideration for further investigation as a first - line therapeutic option in elderly patients . in the study by chen ym . 27 patients of 70 years of age were included and received either erlotinib or vinorelbine . overall survival was longer for egfr - mutated patients than for egfr wild - type patients ( p < 0.0001 ) . it was concluded that erlotinib is highly effective compared with oral vinorelbine in elderly , chemonaive , taiwanese patients with nsclc . egfr - mutated patients had better survival than those with egfr wild - type disease , regardless of the treatment received . in the study by wheatley - price p. et al . 28 two groups were formed : a ) < 70 years of age and b ) > 70 years of age ( elderly ) . elderly patients , compared with young patients , had significantly more overall and severe ( grade 3 and 4 ) toxicity ( 35% v 18% ; p _ .001 ) , were more likely to discontinue treatment as a result of treatment - related toxicity ( 12% v 3% ; p _ .0001 ) , and had lower relative dose - intensity ( 64% v 82% received _ it was concluded that elderly patients treated with erlotinib gain similar survival and qol benefits as younger patients but experience greater toxicity . in the study by platania m. et al . grade 3 - 4 adverse events were observed in 16 cases ( 37% ) . it was concluded that the use of erlotinib after chemotherapy failure in an unselected elderly population affected by nsclc showed moderate efficacy and a moderate safety profile and therefore erlotinib represents a valid option in this setting . it was observed that elevation of aspartate transaminase and/or alanine transaminase ( 18.3% ) was the most common adverse event , and one treatment - related death ( pneumonitis ) occurred . it was concluded that first - line gefitinib is efficacious with acceptable toxicity in relatively fit elderly patients with advanced nsclc harboring an egfr mutation . in the study by des 31 patients were divided in two major groups : a ) < 70 years of age and b ) > 70 years of age . patients were subdivided into three groups receiving three different drugs ( gefitinib ; gemcitabine ; and docetaxel ) . it was observed that older patients had a decreased risk of progression / death compared to younger patients . single - agent chemotherapy can be considered for patients aged 70years with a ps of 2 . in the study by takahashi k. et al . 32 patients between 72 - 90 years of age were included specifically to access the safety and effectiveness of gefitinib in the elderly ( median age was 79.5 years ) . the functional assessment of cancer therapy - lung cancer subscale ( fact - lcs ) scores improved significantly 4 weeks after the initiation of gefitinib ( p = 0.037 ) and maintained favorably over a 12-week assessment period . the most common adverse events were rash and liver dysfunction . although grade 1 pneumonitis developed in one patient , no treatment - related death was observed . it was observed that first - line gefitinib therapy is effective and feasible for elderly patients harboring egfr mutation , and improves disease - related symptoms , especially pulmonary symptoms like shortness of breath and cough . in the study by inoue a. 33 patients > 74 years of age were included with poor performance status and with the administration of gefitinib no treatment - related deaths were observed . this was the first study that verified safety and efficacy of first - line treatment with gefitinib in elderly patients having advanced nsclc with egfr mutation . it was concluded that due to its strong antitumor activity and mild toxicity , first - line gefitinib may be preferable to standard chemotherapy for this population . 35 patients of 80 years of age were included and the tkis gefitinib and erlotinib were administered . it was observed that the administration of these agents to this group of age was acceptable , however ; the authors suggest that a careful dose selection according to the overall medical condition should be made . in the study by yoshioka h. et al . 24 a very well designed study the patients and adverse effects of the tkis were stratified according to age in three different groups as follows : a ) < 75 , b ) 75 -84 and c ) 85. the incidence of interstitial lung disease ( ild ) ( all grades ) was 4.2% ( < 75 years ) , 5.1% ( 75 - 84 years ) , and 3.4% ( 85 years ) therefore it was concluded that erlotinib is safe and efficient for patients > 75 years of age . in the study by kurishima k et al . 25 patients receiving erlotinib were divided into two groups : a ) 75 and b ) > 75 . it was observed that adverse effects were the same in both groups and the overall survival did not differ between the elderly and younger groups ( median , 170 days ; 95% ci : 142239 days vs. median , 146 days ; 95% ci : 114185 days , respectively ) ( p=0.7642 ) . therefore it was concluded that targeted treatment is safe and efficient for this group of patients . in the study by jackman dm . the most common toxicities were acneiform rash ( 79% ) and diarrhea ( 69% ) . four patients developed interstitial lung disease of grade 3 or higher , with one treatment - related death . it was concluded that erlotinib merits consideration for further investigation as a first - line therapeutic option in elderly patients . in the study by chen ym . 27 patients of 70 years of age were included and received either erlotinib or vinorelbine . overall survival was longer for egfr - mutated patients than for egfr wild - type patients ( p < 0.0001 ) . it was concluded that erlotinib is highly effective compared with oral vinorelbine in elderly , chemonaive , taiwanese patients with nsclc . egfr - mutated patients had better survival than those with egfr wild - type disease , regardless of the treatment received . in the study by wheatley - price p. et al . 28 two groups were formed : a ) < 70 years of age and b ) > 70 years of age ( elderly ) . elderly patients , compared with young patients , had significantly more overall and severe ( grade 3 and 4 ) toxicity ( 35% v 18% ; p _ .001 ) , were more likely to discontinue treatment as a result of treatment - related toxicity ( 12% v 3% ; p _ .0001 ) , and had lower relative dose - intensity ( 64% v 82% received _ it was concluded that elderly patients treated with erlotinib gain similar survival and qol benefits as younger patients but experience greater toxicity . in the study by platania m. et al . grade 3 - 4 adverse events were observed in 16 cases ( 37% ) . it was concluded that the use of erlotinib after chemotherapy failure in an unselected elderly population affected by nsclc showed moderate efficacy and a moderate safety profile and therefore erlotinib represents a valid option in this setting . it was observed that elevation of aspartate transaminase and/or alanine transaminase ( 18.3% ) was the most common adverse event , and one treatment - related death ( pneumonitis ) occurred . it was concluded that first - line gefitinib is efficacious with acceptable toxicity in relatively fit elderly patients with advanced nsclc harboring an egfr mutation . in the study by des 31 patients were divided in two major groups : a ) < 70 years of age and b ) > 70 years of age . patients were subdivided into three groups receiving three different drugs ( gefitinib ; gemcitabine ; and docetaxel ) . it was observed that older patients had a decreased risk of progression / death compared to younger patients . single - agent chemotherapy can be considered for patients aged 70years with a ps of 2 . in the study by takahashi k. et al . 32 patients between 72 - 90 years of age were included specifically to access the safety and effectiveness of gefitinib in the elderly ( median age was 79.5 years ) . the functional assessment of cancer therapy - lung cancer subscale ( fact - lcs ) scores improved significantly 4 weeks after the initiation of gefitinib ( p = 0.037 ) and maintained favorably over a 12-week assessment period . the most common adverse events were rash and liver dysfunction . although grade 1 pneumonitis developed in one patient , no treatment - related death was observed . it was observed that first - line gefitinib therapy is effective and feasible for elderly patients harboring egfr mutation , and improves disease - related symptoms , especially pulmonary symptoms like shortness of breath and cough . in the study by inoue a. et al . 33 patients > 74 years of age were included with poor performance status and with the administration of gefitinib no treatment - related deaths were observed . this was the first study that verified safety and efficacy of first - line treatment with gefitinib in elderly patients having advanced nsclc with egfr mutation . it was concluded that due to its strong antitumor activity and mild toxicity , first - line gefitinib may be preferable to standard chemotherapy for this population . 35 patients of 80 years of age were included and the tkis gefitinib and erlotinib were administered . it was observed that the administration of these agents to this group of age was acceptable , however ; the authors suggest that a careful dose selection according to the overall medical condition should be made . possibly a new group of patients including > 75 years of age could be considered in the treatment of lung cancer since targeted treatment with tkis surpass the major obstacle of chemotherapy side effects . in the study by costa gj et al . 36 patients were divided in two groups : a ) < 70 years of age and b ) > 70 years of age . > 70 years of age for the first two years of drug administration had the same side effects when compared to patients of < 70 and treatment was well tolerated > 70 years of age could choose non - specific cytotoxic agents for treatment of nsclc . in the study by yellen sb . 37 patients were divided into two groups : a ) < 65 years of age and b ) > 65 . it was observed that most patients in group b were willing to have chemotherapy treatment , however ; when they were asked to choose between survival and quality of life , most of them chose quality of life . in all the studies included patients were harboring epidermal growth factor receptor ( egfr ) mutations . in the study by wheatley - price p. 28 severe toxicity was observed in a number of patients and therefore dose modification was required . in the study by bai h. et al . a major issue that we identified during our search firstly is that there is no clear definition which patient is considered elder . in the u.s.a elderly were considered mostly patients > 70 , while in europe > 75 years of age . secondly very few studies were designed especially with the purpose to identify whether tkis should be definitely administered to the elderly . one of the reasons is that in general studies conducted with patients more than 70 years of age are difficult to recruit and maintain the follow up of the patients.23 thirdly in a number of studies the first line treatment of choice was chemotherapy and the second line was a tki . however ; as previously presented the status of the egfr changes after chemotherapy , therefore we need more studies without previously treated elderly.17 moreover ; there are no data concerning afatinib and the elderly patients . afatinib is a very new drug in the market and current data indicate that is slightly more toxic than erlotinib and gefitinib.39 based on the current information we suggest that tkis should administered in the elderly , and with the word elderly we propose the age of 75 . the treating medical doctor has to evaluate the performance status of a patient and decide the best treatment in several cases indifferent of the age . tkis in most studies presented safety and efficiency and of course dose modification should be made when necessary . comorbidities should be considered in any case especially in this group of patients and the treating physician should act accordingly .

until few years ago non - specific cytotoxic agents were considered the tip of the arrow as first line treatment for lung cancer . however ; age > 75 was considered a major drawback for this kind of therapy . few exceptions were made by doctors based on the performance status of the patient . the side effects of these agents are still severe for several patients . in the recent years further investigation of the cancer genome has led to targeted therapies . there have been numerous publications regarding novel agents such as ; erlotinib , gefitinib and afatinib . in specific populations these agents have demonstrated higher efficiency and this observation is explained by the overexpression of the egfr pathway in these populations . we suggest that tkis should administered in the elderly , and with the word elderly we propose the age of 75 . the treating medical doctor has to evaluate the performance status of a patient and decide the best treatment in several cases indifferent of the age . tkis in most studies presented safety and efficiency and of course dose modification should be made when necessary . comorbidities should be considered in any case especially in this group of patients and the treating physician should act accordingly .

Introduction Search methods Tyrosine Kinase in the elderly (TKIs) Erlotinib Gefitinib Erlotinb/Gefitinb Discussion

lung cancer is considered the second most frequent cancer for men after prostate cancer and breast cancer for women . 1 non - specific cytotoxic agents are currently used for the treatment of non - small cell lung cancer ( nsclc ) and small cell lung cancer ( sclc).2 - 4 however ; side effects are the reason for some patients to postpone their treatment and in some cases to be hospitalized increasing the cost of treatment.5 in the recent years targeted therapies have emerged and based on the genome of cancer specific oral agents are being administered . 6 - 8 erlotinib , gefinitib , afatinib and crizotinib are the most frequently used targeted agents.7 , 9 erlotinib , gefinitib and afatinib have presented superiority in terms of overall survival and disease control when compared to doublet chemotherapy agents in a specific group of patients.10 however ; resistance to tyrosine kinase inhibitors has been observed and therefore new or alternative treatment approaches are being investigated.11 these agents are considered safer in the sense that the patients have less side effects , however ; they still have side effects which in some cases are dose dependent and directly compared with the positive disease control.12 regarding sclc chemotherapy is still the considered the best first line treatment , however ; several novel therapies are being investigated.13 - 16 the role of tyrosine kinase inhibitors as neo - adjuvant or adjuvant treatment is still under investigation . one observation has to be taken into serious consideration that if chemotherapy is administered in egfr positive patients as neo - adjuvant treatment then the mutation status will change along with the gene status of several other involved pathways with an unknown treatment effect in the future if treatment will become necessary ( disease relapse ) . 18 it is suggested that there are no clear evidence that a patient should receive neo - adjuvant tyrosine kinase inhibitors for early stage nsclc , however ; there are evidence that patients will benefit from this oral drug administration . in the case presented by funakoshi y et al . 19 a patient received gefinitib as neo - adjuvant treatment , downstaging of the disease was observed and the patient underwent pneumonectomy . in the study by li n et al . in the editorial by martinez p 21 it is suggested that tyrosine kinase inhibitors could be used as adjuvant treatment , however ; because of the few trials which have small patient samples and with unselected patients more studies are still needed . regarding afatinib a meta - analysis presented data where this drug could be considered as more toxic than erlotinib and gefitinib.7 there are no data regarding afatinib as neo - adjuvant treatment , however ; regarding adjuvant treatment there is one study by burtness b et al . maybe this study could be used for further investigation of afatinib as adjuvant treatment for nsclc . there is one group of patients that tyrosine kinase inhibitors are still under investigation whether they would benefit or not . if one could set the age for this group of patients it would probably be > 75 years of age which is more or less recognized by the international medical community the cut off age that a patient should receive chemotherapy . although in several patient cases an individualized approach is necessary if the patient has could performance status . the elderly are not considered could candidates for trials since they tend not to complete them due to side effects.23 in the current mini review we will focus on the group of patients > 70 years of age . we will present up to date studies and comment on the targeted treatment for this group of patients . we performed an electronic article search through pubmed , google scholar , medscape and scopus databases , using combinations of the following keywords : tyrosine kinase inhobitors , erlotinib , gefitinib , afatinib , and combination of these words with the word elderly . selected references from identified articles were searched for further consideration indifferent of the language . in the study by yoshioka h. et al . 24 a very well designed study the patients and adverse effects of the tkis were stratified according to age in three different groups as follows : a ) < 75 , b ) 75 -84 and c ) 85. the incidence of interstitial lung disease ( ild ) ( all grades ) was 4.2% ( < 75 years ) , 5.1% ( 75 - 84 years ) , and 3.4% ( 85 years ) . therefore it was concluded that erlotinib is safe and efficient for patients > 75 years of age . in the study by kurishima k et al . 25 patients receiving erlotinib were divided into two groups : a ) 75 and b ) > 75 . it was observed that adverse effects were the same in both groups and the overall survival did not differ between the elderly and younger groups ( median , 170 days ; 95% ci : 142239 days vs. median , 146 days ; 95% ci : 114185 days , respectively ) ( p=0.7642 ) . therefore it was concluded that targeted treatment is safe and efficient for this group of patients . in the study by jackman dm . it was concluded that erlotinib merits consideration for further investigation as a first - line therapeutic option in elderly patients . in the study by chen ym . it was concluded that erlotinib is highly effective compared with oral vinorelbine in elderly , chemonaive , taiwanese patients with nsclc . egfr - mutated patients had better survival than those with egfr wild - type disease , regardless of the treatment received . in the study by wheatley - price p. et al . elderly patients , compared with young patients , had significantly more overall and severe ( grade 3 and 4 ) toxicity ( 35% v 18% ; p _ .001 ) , were more likely to discontinue treatment as a result of treatment - related toxicity ( 12% v 3% ; p _ .0001 ) , and had lower relative dose - intensity ( 64% v 82% received _ it was concluded that elderly patients treated with erlotinib gain similar survival and qol benefits as younger patients but experience greater toxicity . in the study by platania m. et al . it was concluded that the use of erlotinib after chemotherapy failure in an unselected elderly population affected by nsclc showed moderate efficacy and a moderate safety profile and therefore erlotinib represents a valid option in this setting . it was observed that elevation of aspartate transaminase and/or alanine transaminase ( 18.3% ) was the most common adverse event , and one treatment - related death ( pneumonitis ) occurred . in the study by des 31 patients were divided in two major groups : a ) < 70 years of age and b ) > 70 years of age . single - agent chemotherapy can be considered for patients aged 70years with a ps of 2 . in the study by takahashi k. et al . 32 patients between 72 - 90 years of age were included specifically to access the safety and effectiveness of gefitinib in the elderly ( median age was 79.5 years ) . the functional assessment of cancer therapy - lung cancer subscale ( fact - lcs ) scores improved significantly 4 weeks after the initiation of gefitinib ( p = 0.037 ) and maintained favorably over a 12-week assessment period . it was observed that first - line gefitinib therapy is effective and feasible for elderly patients harboring egfr mutation , and improves disease - related symptoms , especially pulmonary symptoms like shortness of breath and cough . in the study by inoue a. et al . 33 patients > 74 years of age were included with poor performance status and with the administration of gefitinib no treatment - related deaths were observed . this was the first study that verified safety and efficacy of first - line treatment with gefitinib in elderly patients having advanced nsclc with egfr mutation . it was concluded that due to its strong antitumor activity and mild toxicity , first - line gefitinib may be preferable to standard chemotherapy for this population . 35 patients of 80 years of age were included and the tkis gefitinib and erlotinib were administered . it was observed that the administration of these agents to this group of age was acceptable , however ; the authors suggest that a careful dose selection according to the overall medical condition should be made . in the study by yoshioka h. et al . 24 a very well designed study the patients and adverse effects of the tkis were stratified according to age in three different groups as follows : a ) < 75 , b ) 75 -84 and c ) 85. the incidence of interstitial lung disease ( ild ) ( all grades ) was 4.2% ( < 75 years ) , 5.1% ( 75 - 84 years ) , and 3.4% ( 85 years ) therefore it was concluded that erlotinib is safe and efficient for patients > 75 years of age . in the study by kurishima k et al . 25 patients receiving erlotinib were divided into two groups : a ) 75 and b ) > 75 . it was observed that adverse effects were the same in both groups and the overall survival did not differ between the elderly and younger groups ( median , 170 days ; 95% ci : 142239 days vs. median , 146 days ; 95% ci : 114185 days , respectively ) ( p=0.7642 ) . therefore it was concluded that targeted treatment is safe and efficient for this group of patients . in the study by jackman dm . it was concluded that erlotinib merits consideration for further investigation as a first - line therapeutic option in elderly patients . in the study by chen ym . it was concluded that erlotinib is highly effective compared with oral vinorelbine in elderly , chemonaive , taiwanese patients with nsclc . egfr - mutated patients had better survival than those with egfr wild - type disease , regardless of the treatment received . in the study by wheatley - price p. et al . elderly patients , compared with young patients , had significantly more overall and severe ( grade 3 and 4 ) toxicity ( 35% v 18% ; p _ .001 ) , were more likely to discontinue treatment as a result of treatment - related toxicity ( 12% v 3% ; p _ .0001 ) , and had lower relative dose - intensity ( 64% v 82% received _ it was concluded that elderly patients treated with erlotinib gain similar survival and qol benefits as younger patients but experience greater toxicity . in the study by platania m. et al . it was concluded that the use of erlotinib after chemotherapy failure in an unselected elderly population affected by nsclc showed moderate efficacy and a moderate safety profile and therefore erlotinib represents a valid option in this setting . it was observed that elevation of aspartate transaminase and/or alanine transaminase ( 18.3% ) was the most common adverse event , and one treatment - related death ( pneumonitis ) occurred . in the study by des 31 patients were divided in two major groups : a ) < 70 years of age and b ) > 70 years of age . single - agent chemotherapy can be considered for patients aged 70years with a ps of 2 . in the study by takahashi k. et al . 32 patients between 72 - 90 years of age were included specifically to access the safety and effectiveness of gefitinib in the elderly ( median age was 79.5 years ) . the functional assessment of cancer therapy - lung cancer subscale ( fact - lcs ) scores improved significantly 4 weeks after the initiation of gefitinib ( p = 0.037 ) and maintained favorably over a 12-week assessment period . it was observed that first - line gefitinib therapy is effective and feasible for elderly patients harboring egfr mutation , and improves disease - related symptoms , especially pulmonary symptoms like shortness of breath and cough . in the study by inoue a. 33 patients > 74 years of age were included with poor performance status and with the administration of gefitinib no treatment - related deaths were observed . this was the first study that verified safety and efficacy of first - line treatment with gefitinib in elderly patients having advanced nsclc with egfr mutation . it was concluded that due to its strong antitumor activity and mild toxicity , first - line gefitinib may be preferable to standard chemotherapy for this population . 35 patients of 80 years of age were included and the tkis gefitinib and erlotinib were administered . it was observed that the administration of these agents to this group of age was acceptable , however ; the authors suggest that a careful dose selection according to the overall medical condition should be made . in the study by yoshioka h. et al . 24 a very well designed study the patients and adverse effects of the tkis were stratified according to age in three different groups as follows : a ) < 75 , b ) 75 -84 and c ) 85. the incidence of interstitial lung disease ( ild ) ( all grades ) was 4.2% ( < 75 years ) , 5.1% ( 75 - 84 years ) , and 3.4% ( 85 years ) therefore it was concluded that erlotinib is safe and efficient for patients > 75 years of age . 25 patients receiving erlotinib were divided into two groups : a ) 75 and b ) > 75 . it was observed that adverse effects were the same in both groups and the overall survival did not differ between the elderly and younger groups ( median , 170 days ; 95% ci : 142239 days vs. median , 146 days ; 95% ci : 114185 days , respectively ) ( p=0.7642 ) . therefore it was concluded that targeted treatment is safe and efficient for this group of patients . in the study by jackman dm . it was concluded that erlotinib merits consideration for further investigation as a first - line therapeutic option in elderly patients . in the study by chen ym . it was concluded that erlotinib is highly effective compared with oral vinorelbine in elderly , chemonaive , taiwanese patients with nsclc . egfr - mutated patients had better survival than those with egfr wild - type disease , regardless of the treatment received . in the study by wheatley - price p. et al . elderly patients , compared with young patients , had significantly more overall and severe ( grade 3 and 4 ) toxicity ( 35% v 18% ; p _ .001 ) , were more likely to discontinue treatment as a result of treatment - related toxicity ( 12% v 3% ; p _ .0001 ) , and had lower relative dose - intensity ( 64% v 82% received _ it was concluded that elderly patients treated with erlotinib gain similar survival and qol benefits as younger patients but experience greater toxicity . in the study by platania m. et al . it was concluded that the use of erlotinib after chemotherapy failure in an unselected elderly population affected by nsclc showed moderate efficacy and a moderate safety profile and therefore erlotinib represents a valid option in this setting . it was observed that elevation of aspartate transaminase and/or alanine transaminase ( 18.3% ) was the most common adverse event , and one treatment - related death ( pneumonitis ) occurred . in the study by des 31 patients were divided in two major groups : a ) < 70 years of age and b ) > 70 years of age . single - agent chemotherapy can be considered for patients aged 70years with a ps of 2 . in the study by takahashi k. et al . 32 patients between 72 - 90 years of age were included specifically to access the safety and effectiveness of gefitinib in the elderly ( median age was 79.5 years ) . the functional assessment of cancer therapy - lung cancer subscale ( fact - lcs ) scores improved significantly 4 weeks after the initiation of gefitinib ( p = 0.037 ) and maintained favorably over a 12-week assessment period . it was observed that first - line gefitinib therapy is effective and feasible for elderly patients harboring egfr mutation , and improves disease - related symptoms , especially pulmonary symptoms like shortness of breath and cough . in the study by inoue a. et al . 33 patients > 74 years of age were included with poor performance status and with the administration of gefitinib no treatment - related deaths were observed . this was the first study that verified safety and efficacy of first - line treatment with gefitinib in elderly patients having advanced nsclc with egfr mutation . it was concluded that due to its strong antitumor activity and mild toxicity , first - line gefitinib may be preferable to standard chemotherapy for this population . 35 patients of 80 years of age were included and the tkis gefitinib and erlotinib were administered . it was observed that the administration of these agents to this group of age was acceptable , however ; the authors suggest that a careful dose selection according to the overall medical condition should be made . possibly a new group of patients including > 75 years of age could be considered in the treatment of lung cancer since targeted treatment with tkis surpass the major obstacle of chemotherapy side effects . in the study by costa gj et al . > 70 years of age for the first two years of drug administration had the same side effects when compared to patients of < 70 and treatment was well tolerated > 70 years of age could choose non - specific cytotoxic agents for treatment of nsclc . in the study by yellen sb . it was observed that most patients in group b were willing to have chemotherapy treatment , however ; when they were asked to choose between survival and quality of life , most of them chose quality of life . in the study by wheatley - price p. 28 severe toxicity was observed in a number of patients and therefore dose modification was required . in the study by bai h. et al . a major issue that we identified during our search firstly is that there is no clear definition which patient is considered elder . in the u.s.a elderly were considered mostly patients > 70 , while in europe > 75 years of age . secondly very few studies were designed especially with the purpose to identify whether tkis should be definitely administered to the elderly . one of the reasons is that in general studies conducted with patients more than 70 years of age are difficult to recruit and maintain the follow up of the patients.23 thirdly in a number of studies the first line treatment of choice was chemotherapy and the second line was a tki . however ; as previously presented the status of the egfr changes after chemotherapy , therefore we need more studies without previously treated elderly.17 moreover ; there are no data concerning afatinib and the elderly patients . afatinib is a very new drug in the market and current data indicate that is slightly more toxic than erlotinib and gefitinib.39 based on the current information we suggest that tkis should administered in the elderly , and with the word elderly we propose the age of 75 . the treating medical doctor has to evaluate the performance status of a patient and decide the best treatment in several cases indifferent of the age . tkis in most studies presented safety and efficiency and of course dose modification should be made when necessary . comorbidities should be considered in any case especially in this group of patients and the treating physician should act accordingly .

alterations in the dna structure that hinder the progression of rna polymerases during transcription are highly toxic for the cell and , if not properly resolved , can lead to cellular apoptosis or senescence ( ljungman and zhang 1996 ; ljungman and lane 2004 ) . helix - distorting lesions located in the transcribed strand of active genes initiate the transcription - coupled nucleotide excision repair ( tc - ner ) pathway to resolve the transcription - blocking dna damage ( hanawalt and spivak 2008 ) . lesion stalled elongating rna polymerase ii ( rna pol ii ) triggers the recruitment of several tc - ner - specific factors to form a functional tc - ner complex , including the cockayne syndrome a and b ( csa , csb ) proteins ( fousteri et al . 2006 ) ( fig . i during transcription , uvssa , usp7 , and csb interact transiently with elongating rna pol ii . ii upon encountering a lesion ( indicated by star ) , stalled rna pol ii stabilizes its binding with these proteins and triggers the recruitment of several other dna repair factors , including csa . since rna pol ii shields the dna lesion in its active pocket , the stalled transcription complex must be remodeled to enable access of repair proteins to the lesion . in the case of helix - distorting lesions ( iiia ) , tc - ner is initiated for which extensive modulation of stalled rna pol ii is needed , causing a prolonged transcriptional arrest . during this slow process , csb assembles a functional tc - ner complex and assists in the remodeling of rna pol ii . in response to dna damage , csb is ubiquitinated and eventually degraded . in order to allow sufficient time for csb to perform its function in tc - ner , its presence is protected by the concerted action of uvssa and usp7 by counteracting the ubiquitination - dependent degradation . when the required remodeling of stalled rna pol ii is completed , the tc - ner complex is destabilized and csb is degraded by the proteasome . following further helix unwinding and lesion verification , xpg and xpf are required for the double incision of the damaged dna strand . the repair reaction is completed with dna polymerase gap filling of the repair patch and sealing of the nicks . oxidative lesions ( iiib ) have a minor effect on the dna helix structure , which therefore requires relatively little remodeling of rna pol ii for efficient repair . consequently , rna pol ii is only slowed down or transiently arrested in response to these lesions resulting in a faster response as compared to iiia . csb remodeling helps to efficiently remove oxidative lesions likely via the ber pathway or to pass stalled complexes over the lesion ( lesion bypass ) . however , due to the faster response , protection of csb from ubiquitination - dependent degradation by uvssa / usp7 is not necessary . after repair is completed ( iv ) , transcription is resumed ( v ) model for resolving transcription - blocking lesions . i during transcription , uvssa , usp7 , and csb interact transiently with elongating rna pol ii . ii upon encountering a lesion ( indicated by star ) , stalled rna pol ii stabilizes its binding with these proteins and triggers the recruitment of several other dna repair factors , including csa . since rna pol ii shields the dna lesion in its active pocket , the stalled transcription complex must be remodeled to enable access of repair proteins to the lesion . in the case of helix - distorting lesions ( iiia ) , tc - ner is initiated for which extensive modulation of stalled rna pol ii is needed , causing a prolonged transcriptional arrest . during this slow process , csb assembles a functional tc - ner complex and assists in the remodeling of rna pol ii . in response to dna damage , csb is ubiquitinated and eventually degraded . in order to allow sufficient time for csb to perform its function in tc - ner , its presence is protected by the concerted action of uvssa and usp7 by counteracting the ubiquitination - dependent degradation . when the required remodeling of stalled rna pol ii is completed , the tc - ner complex is destabilized and csb is degraded by the proteasome . following further helix unwinding and lesion verification , xpg and xpf are required for the double incision of the damaged dna strand . the repair reaction is completed with dna polymerase gap filling of the repair patch and sealing of the nicks . oxidative lesions ( iiib ) have a minor effect on the dna helix structure , which therefore requires relatively little remodeling of rna pol ii for efficient repair . consequently , rna pol ii is only slowed down or transiently arrested in response to these lesions resulting in a faster response as compared to iiia . csb remodeling helps to efficiently remove oxidative lesions likely via the ber pathway or to pass stalled complexes over the lesion ( lesion bypass ) . however , due to the faster response , protection of csb from ubiquitination - dependent degradation by uvssa / usp7 is not necessary . in humans , defective tc - ner is associated with two autosomal recessive dna repair deficiency disorders : cockayne syndrome ( cs ) and uv - sensitive syndrome ( uvs ) ( nance and berry 1992 ; spivak 2005 ) . although patient - derived cells are equally deficient in uv - induced tc - ner in vitro , the patients exhibit strikingly distinct clinical symptoms : cs individuals display severe developmental , neurological , and premature aging features , whereas uvs patients express much milder features , mostly restricted to uv hypersensitivity . cs is caused by mutations in two genes indispensable for tc - ner , csa and csb . uvs comprises three complementation groups , which are defined by specific mutations in csa , csb , and in the recently identified gene encoding for uv - stimulated scaffold protein a ( uvssa ) ( table 1 ) . intriguingly , neither the site nor the nature of the mutations in csa or csb seems to correlate with the clinical differences observed among patients with cs and uvs ( nance and berry 1992 ; laugel et al . thus , an important question remains : how do molecular defects within the same tc - ner pathway and even different mutations within the same genes lead to such diverse pathologies?table 1overview of uvs mutations in csa , csb , and uvssapatient / cell linegene affectedmutationreferenceskps2uvssap.lys123*1 , 3 , 4 , 5kps3uvssap.lys123*1 , 2 , 3 , 4 , 5uvs24tauvssap.ile31phefs*91 , 2 , 6xp24kouvssap.lys123*1 , 2 , 7 , 8xp70touvssap.cys32arg1 , 7 , 9uvss1vicsap.trp361cys10uvs1kocsbp.arg77*3 , 4 , 6 , 11 , 12cs3amcsbp.arg77*13references : 1 . itoh et al . ( 1994 ) , 6 . cleaver and thomas ( 1993 ) , 7 . miyauchi - hashimoto et al . ( 1998 ) overview of uvs mutations in csa , csb , and uvssa references : 1 . miyauchi - hashimoto et al . ( 1998 ) several models are proposed that try to explain the underlying molecular reason for the wide variety in tc - ner - deficient phenotype ( sarasin 2012 ; cleaver 2012 ) . most of these hypotheses are based on additional functions of the cs proteins outside of their role in tc - ner while assuming uvssa is not implicated in these processes , hence explaining the milder phenotype in uvs ( spivak and hanawalt 2006 ) ( fig . 2involvement of csa , csb , and uvssa in transcription , ber , and tc - ner . while the cs proteins have functions outside of tc - ner , a role for uvssa in these other processes has presently not been shown involvement of csa , csb , and uvssa in transcription , ber , and tc - ner . while the cs proteins have functions outside of tc - ner , a role for uvssa in these other processes has presently not been shown one of these models , the transcription defect model , is mainly based on data obtained for the csb protein . csb is a member of the snf2/swi2 family of dna - dependent atpases ( troelstra et al . 1990 ) and is suggested to have chromatin remodeling abilities , possibly through recruitment of the p300 histone acetyltransferase ( citterio et al . 2000 ; newman et al . 2006 ; frontini and proietti - de - santis 2009 ) . csb transiently interacts with elongating rna pol ii and stimulates transcription ( balajee et al . this suggests that when csb is absent or mutated , transcription would be affected even in the absence of dna damage . there is a growing list of human diseases which are associated with defects in transcription , and many of these diseases are characterized by congenital defects ( villard 2004 ) , as also observed in cs patients . this suggests that the severe developmental and premature aging features of cs could partially be caused by a defect in transcription , while the sun sensitivity in these patients is most likely caused by the dna repair deficiency of the tc - ner pathway . another hypothesis involves a role of the cs proteins in repairing endogenous dna damage from reactive oxygen species ( ros ) , which is usually repaired by the base excision repair ( ber ) machinery ( hegde et al . both human and mouse cell lines deficient in the cs proteins indeed display enhanced sensitivity to agents that produce oxidative dna damage ( dianov et al . 1999 ; tuo et al . 2003 ; de waard et al . 2004 ; gorgels et al . 2007 ; derrico et al . 2007 ; stevnsner et al . 2008 ) . 2012 ) recently showed recruitment of csb to local oxidative dna lesions in living cells without an accumulation of downstream ner factors , indicating the involvement of csb in the response to oxidative dna lesions independent of the downstream ner reaction . importantly , csb binding to oxidative damage was reduced upon transcription inhibition which suggests a role of csb in transcription - associated repair of oxidative lesions . in cs patients , the accumulation of unrepaired oxidative dna damage and the subsequent prolonged transcription arrest at these lesions could result in apoptosis and consequentially tissue degeneration . since active metabolism during development and a high level of oxidative metabolism in neural tissues may generate a considerable amount of oxidative damage , the consequences of the transcription arrest are mainly disclosed by developmental and neurological symptoms which are a hallmark of cs ( hoeijmakers 2007 ) . this model is further supported by transcriptomic analysis of csb - deficient mice which revealed systematic suppression of growth and oxidative metabolism , an altered glycolysis and an upregulated antioxidant defense , collectively referred to as survival response ( van der pluijm et al . ( 2012 ) recently observed a disturbed redox balance in patient - derived cs cells . in addition , alterations in cellular metabolism including the glycolysis pathway and oxidative metabolism were observed . addition of antioxidants to the cells reduced ros levels and at least partially reverted the alterations in cellular metabolism , suggesting that oxidative stress plays a causative role in cs pathology . although there is compelling evidence supporting this hypothesis , no direct proof is currently present and it needs further exploration . within a third model , a role for the cs proteins in repair of oxidative dna damage specifically in mitochondrial dna ( mtdna ) has been reported ( osenbroch et al . 2009 ; kamenisch et al . mitochondria possess an independent repair machinery for oxidative lesions , which is thought to play a crucial role in protecting the integrity of mtdna from the relatively high levels of ros generated in this organelle . certain clinical symptoms associated with mitochondrial dysfunction are also observed in cs patients , including the severe neurological and premature aging features ( stevnsner et al . this would suggest that the cause of these symptoms in cs results in part from the loss of mitochondrial function as a consequence of unrepaired oxidative mtdna damage due to mutations in the cs proteins . the abovementioned hypotheses to explain the severe phenotypical consequences of cs do not uphold , however , for the intriguing case of two unrelated patients with null mutations in csb associated with mild uvs symptoms ( miyauchi - hashimoto et al . in contrast , two other patients with a deletion of the promoter sequence of csb ( resulting in no detectable csb protein ) have been found , which showed the most severe cs symptoms ( laugel et al . thus , the absence of csb alone can not explain the striking variability in phenotype between cs and uvs . a factor possibly involved in this variability might be the expression of an evolutionary conserved csb - pgbd3 fusion protein , containing exons 15 of csb joined in frame with the piggybac transposase . reports have indicated that this fusion protein might play a role in the cs phenotype ( bailey et al . recently , three laboratories identified uvssa as the causative gene for the uvs - a complementation group . microcell - mediated chromosome transfer ( zhang et al . 2012 ) , whole - exome sequencing ( nakazawa et al . 2012 ) , and quantitative proteomics ( schwertman et al . 2012 ) were used to identify kiaa1530 as a new tc - ner factor , which was subsequently renamed uvssa . sequencing of this gene in five uvs - a patient cell lines revealed three different inactivating mutations in the uvssa gene ( table 1 ) . within uvssa , two conserved , though poorly characterized , domains are identified with homology to the vps27-hrs - stam ( vhs ) domain and the duf2043 domain . uvs - a cells expressing uvssa mutants without either of these domains fail to complement uvs - a deficiency ( nakazawa et al . the uvssa protein was shown to interact with the tc - ner factors rna pol ii , csa , csb , and tfiih ( schwertman et al . a specialized chromatin immunoprecipitation ( chip ) procedure showed that uvssa resides in active , chromatin - bound tc - ner complexes upon uv damage . furthermore , gfp - tagged uvssa accumulated at local uv damage in living cells with similar recruitment kinetics as the tc - ner factor csb ( schwertman et al . there are two , though not mutually exclusive , models explaining how uvssa is recruited to uv lesions : ( 1 ) as an rna pol ii interaction partner ( schwertman et al . 2012 ) and ( 2 ) as a csa interaction partner ( zhang et al . 2012 ; fei and chen 2012 ) . we ( schwertman et al . 2012 ) observed a uv - independent interaction between rna pol ii and uvssa in chip experiments . this interaction was also observed in csb - deficient patient cells , indicative of a csb - independent binding of uvssa to tc - ner complexes . in line with this , a csa- and csb - independent accumulation of gfp - uvssa was observed at sites of local uv damage using live cell imaging . rna pol ii interaction and showed a csa - dependent interaction with uvssa in the chromatin fraction using non - cross - linking ips ; the latter was also observed by fei and chen ( 2012 ) . an explanation for this apparent discrepancy in uvssa recruitment could be that the cs proteins may be required for stable integration into a functional tc - ner complex rather than for the recruitment of uvssa . transient or low - affinity interactions could appear csa / csb independent if interactions are fixed by cross - linking as in chip experiments ( schwertman et al . 2012 ) and csa / csb dependent as observed in non - cross - linking ips ( zhang et al . 2012 ; fei and chen 2012 ) . the observed csa - dependent stable association of uvssa into tc - ner complexes provides the basis for a possible molecular explanation of the uvs / cs phenotypic difference . the csa protein is a subunit of an e3 ubiquitin ligase complex ( groisman et al . 2003 ) that is recruited to the site of damage by csb ( fousteri et al . although essential for tc - ner , the precise role of csa remains largely unknown . ( 2009 ) described a patient with a specific missense csa mutation , which causes the mild uv - sensitive phenotype in the absence of the severe premature aging features common in cs . cells of this uvs patient were hypersensitive to uv light , but not to oxidative damaging agents . this finding thus implies a separable role for csa in response to uv and oxidative dna damage . interestingly , fei and chen ( 2012 ) showed that this same csa mutant disrupts the interaction of csa with uvssa . this might suggest that an impaired interaction of uvssa with mutated csa at sites of dna damage results in uvs in this patient , while an unaffected role of csa in other processes such as in oxidative dna damage repair prevents the additional cs symptoms . in addition to its interaction with csa and rna pol ii , uvssa forms a uv - independent protein complex with the deubiquitinating enzyme ( dub ) ubiquitin - specific protease 7 ( usp7 ) . usp7 ( also known as hausp ) has multiple roles in the dna damage response , as illustrated by the wide variety of substrates including mdm2 , p53 , claspin , chfr , and histone h2b ( li et al . 2002 ; meulmeester et al . 2005 ; faustrup et al . 2009 ; khoronenkova et al . additionally , its diverse activity also includes targeting tumor suppressors , immune responders , viral proteins , and epigenetic modulators ( nicholson and suresh kumar 2011 ) . through its interaction with uvssa , usp7 is recruited to active tc - ner complexes upon uv damage ( schwertman et al . depletion of usp7 leads to a similar tc - ner deficiency as seen with uvssa depletion , such as a decrease in uv survival and rna synthesis recovery after uv ( schwertman et al . , it is known that csb is ubiquitinated and degraded by the proteasome in response to uv ( groisman et al . it is therefore suggested that uvssa stabilizes csb after uv by targeting the pleiotropic dub usp7 to tc - ner complexes , which subsequently removes uv - induced ubiquitin chains . the purpose of this could be to provide an increased time frame for csb to orchestrate tc - ner complex formation or to efficiently complete repair before csb is degraded . the stabilizing function of uvssa / usp7 on csb might be restricted to uv - induced tc - ner , since the reduced csb levels in uvss - a cells do not result in developmental and neurological symptoms in uvs patients . interestingly , overexpression of csb in uvs - a cells did not correct the tc - ner defect ( schwertman et al . this implies that the reduced level of csb in uvs - a cells alone is not sufficient to explain the uvs phenotype . therefore , the ubiquitination state of csb itself might have an additional function for proper tc - ner , for example to mediate a ubiquitin - mediated functional change in csb , or uvssa / usp7 might have additional substrates that can cause the uvs phenotype . several transcription - associated factors recognize the c - terminal domain ( ctd ) of rna pol ii by means of a conserved ctd - interacting domain ( cid ) . 2002 ; meinhart and cramer 2004 ) , suggesting that the observed interaction between uvssa and rna pol ii is mediated via the vhs domain of uvssa . this interaction might be important for an additional function of uvssa , as nakazawa et al . ( 2012 ) showed that uvssa is needed for processing stalled rna pol ii at sites of uv damage . during tc - ner , elongating rna pol ii can be recycled for a new round of transcription by means of dephosphorylating the elongating form of rna pol ii into the hypophosphorylated initiating form ( rockx et al . 2000 ) . without the uvssa protein , this dephosphorylation step is substantially inhibited ( nakazawa et al . next to its possible involvement in rna pol ii binding , the vhs domain has also been implicated in ubiquitin binding ( mizuno et al . 2003 ) , raising the possibility that uvssa binds ubiquitinated tc - ner proteins . upon uv irradiation , rna pol ii is ubiquitinated and , under specific conditions , degraded ( bregman et al . ( 2012 ) identified a uvssa - dependent ubiquitination of rna pol ii , which however is not subject to proteasomal degradation . interestingly , this new ubiquitinated form of stalled elongating rna pol ii is uv specific , as it was not observed for oxidative dna damage induced by hydrogen peroxide . interaction of uvssa with ubiquitinated rna pol ii was indeed observed by nakazawa et al . we ( schwertman et al . 2012 ) identified uvssa in a stable isotope labeling with amino acids in cell culture ( silac)-based proteomic screen for differentially ubiquitinated proteins following uv irradiation . while the ubiquitination status of uvssa was not changed after uv , a similar silac ratio for uvssa and rna pol ii was observed . this would suggest that uvssa was co - purified by virtue of the enhanced ubiquitination of rna pol ii in response to uv . the vhs domain of uvssa is suggested to be also involved in the interaction between uvssa and csb , since a uvss - a patient missense mutation ( cys32arg ) within the vhs domain disrupts the interaction with csb in response to uv . interestingly , uvs - a cells expressing uvssa with this mutation were able to restore csb stability to wild - type levels upon uv , while rna pol ii ubiquitination and dephosphorylation remained absent ( nakazawa et al . this might indicate that while the vhs domain of uvssa is dispensable for csb stabilization , it is important for rna pol ii processing . apparently , the action of uvssa on both csb and rna pol ii contributes to the uvs phenotype . although the precise involvement of uvssa in processing these rna pol ii modifications remains to be elucidated , it is hypothesized that the modifications themselves contribute to the coordination of the removal of stalled rna pol ii and formation of the tc - ner complex and the subsequent resumption of transcription . mutations in uvssa were recently identified to cause uvs , a previously unresolved tc - ner deficiency disorder with rather mild clinical manifestations as compared to cs . uvssa and its interaction partner usp7 were identified as new factors involved in tc - ner repair efficiency and cell survival after uv damage . additionally , uvssa is also important for the fate and ubiquitination state of both rna pol ii and csb . the identification of these new transcription - coupled dna repair factors presents us with the opportunity to gain more insight into the molecular mechanisms underlying the clinical differences between uvs and cs . the transcription defect model discussed previously is based on a role for the cs proteins in transcription , while assuming uvssa is not involved in this process . the uv - independent transient interaction between uvssa and rna pol ii may suggest a role for uvssa in transcription . however , no other data are currently available to conclude whether uvssa is as equally important as csb for transcription on undamaged dna . therefore , further studies are needed to determine the possible involvement of uvssa in transcription . one of the models including uvssa involves the aberrant processing of elongating rna pol ii when stalled at a lesion ( fig . 3 ) . for efficient repair to take place , the rna pol ii complex must be remodeled since it will likely shield the dna lesion and consequently prevent accessibility of repair proteins ( donahue et al . 1994 ; tornaletti et al . 2001 ; brueckner et al . 2007 ; fousteri and mullenders 2008 ; wilson et al . 2013 ) additionally , when a lesion can not be repaired or bypassed , stalled rna pol ii is thought to be ubiquitinated and degraded in order to prevent persistent transcriptional arrest ( wilson et al . ii can still be ubiquitinated in a csa / csb - dependent way , which results in proteasomal degradation . in contrast , stalled rna pol ii can not be degraded by the proteasome in cs cells ( hanawalt and spivak 2008 ) . this would suggest that the lack of dna repair combined with persistent arrest of rna pol ii at dna lesions in cs cells , which results in apoptosis or senescence , might be causative for the severe cs phenotype . in uvss - a cells , prolonged arrest is prevented and the lesion is made accessible for alternative dna repair by degradation of rna pol ii , hence resulting in the milder uvs phenotype ( fig . 3differential aberrant processing of stalled elongating rna pol ii is responsible for the differences in uvs / cs phenotype . in wild - type cells , lesion stalled rna pol ii initiates tc - ner to remove the transcription - blocking damage . in the infrequent cases that repair can not take place , rna pol ii is ubiquitinated and degraded by the proteasome to make the lesion accessible for alternative dna repair . in uvs - a cells since the prolonged stalled rna pol ii can still be ubiquitinated in a csa / csb dependent way , rna pol ii is degraded and the lesion is made accessible for alternative dna repair . in cs cells , in addition , the ubiquitination and degradation of rna pol ii does not take place . the resultant persistent transcription arrest is more harmful for the cell , since it leads to apoptosis or senescence and might contribute to the more severe phenotype in cs patients differential aberrant processing of stalled elongating rna pol ii is responsible for the differences in uvs / cs phenotype . in wild - type cells , lesion stalled rna pol ii initiates tc - ner to remove the transcription - blocking damage . in the infrequent cases that repair can not take place , rna pol ii is ubiquitinated and degraded by the proteasome to make the lesion accessible for alternative dna repair . in uvs - a cells ii can still be ubiquitinated in a csa / csb dependent way , rna pol ii is degraded and the lesion is made accessible for alternative dna repair . in cs cells , in addition , the ubiquitination and degradation of rna pol ii does not take place . the resultant persistent transcription arrest is more harmful for the cell , since it leads to apoptosis or senescence and might contribute to the more severe phenotype in cs patients the cs proteins play important roles in both tc - ner and oxidative dna damage repair , while the precise function of uvssa in these processes remains unclear . the previously discussed oxidative damage model assumes that uvssa does not play a role in oxidative dna damage repair ( spivak and hanawalt 2006 ) , hence explaining the milder phenotype of uvs . we would like to postulate a model including uvssa and its role in both repair pathways ( fig . 1 ) . we hypothesize that based on the nature of the dna damage , distinct modulations of lesion stalled rna pol ii are required and that this involves a remodeling function of csb . while oxidative lesions only slow down or transiently arrest rna pol ii , more helix - distorting lesions ( such as those induced by uv ) would lead to a prolonged transcriptional arrest . in the case of transient transcription arrest , csb remodeling assists in the efficient removal of a lesion or in bypassing the lesion ( doetsch 2002 ; charlet - berguerand et al . in contrast , for a prolonged blocked transcription , more time would be required for csb to assemble a functional tc - ner complex and to assist in remodeling of the stalled rna pol ii . the presence of csb should , in the latter case , be protected by the concerted action of uvssa / usp7 to allow sufficient time for csb to perform its remodeling function . whether the ubiquitination of csb takes place at all after transient transcription arrest at oxidative dna lesions or if csb is ubiquitinated but not degraded before the repair of these lesions is completed remains to be elucidated . in this proposed model , mutations in uvssa would lead to dysfunctional tc - ner only , since the lack of csb protection would not affect repair or restart of transcription blocked by oxidative lesions . the discovery of uvssa enables us to test the proposed models in greater detail and whether or not they are mutually exclusive . to further substantiate the hypothesis that the phenotypical difference between uvs and cs is derived from different sensitivities for endogenous levels of oxidative dna damage , dedicated lesion - specific dna repair assays are required ( spivak et al . 2009 ) . furthermore , dissecting the differences in molecular composition between repair complexes stalled at uv - induced lesions or oxidative lesions will help us to better understand the molecular mechanism of transcription - coupled repair for different types of dna damage . characterization of repair complexes present on the different lesions using immunoprecipitation combined with mass spectrometry would be a powerful tool to study this . another interesting area for further investigation is the differential ubiquitination of several dna repair factors , which is likely an important driver of protein hand over and passing of repair intermediates through successive steps during tc - ner . especially the actual and/or precise involvement of usp7 in changing the ubiquitination state of csb and rna pol ii upon uv is of great interest , as is the establishment of identified or new e3 ubiquitin ligases responsible for the ubiquitin modifications . finally , with the identification of uvssa , a uvs - a mouse model can now be generated for a more extensive study of the uvs phenotype and molecular defect . also , an overall comparison of the uvs - a mouse with the already existing cs mouse models in an isogenic background will be a valuable source of information . phenotypic consequences and possible differential responses to various genotoxic agents in the whole organism will contribute to our understanding of the clinical differences of cs versus uvs on a molecular level .

transcription - coupled nucleotide excision repair ( tc - ner ) specifically removes transcription - blocking lesions from our genome . defects in this pathway are associated with two human disorders : cockayne syndrome ( cs ) and uv - sensitive syndrome ( uvss ) . despite a similar cellular defect in the uv dna damage response , patients with these syndromes exhibit strikingly distinct symptoms ; cs patients display severe developmental , neurological , and premature aging features , whereas the phenotype of uvss patients is mostly restricted to uv hypersensitivity . the exact molecular mechanism behind these clinical differences is still unknown ; however , they might be explained by additional functions of cs proteins beyond tc - ner . a short overview of the current hypotheses addressing possible molecular mechanisms and the proteins involved are presented in this review . in addition , we will focus on two new players involved in tc - ner which were recently identified : uv - stimulated scaffold protein a ( uvssa ) and ubiquitin - specific protease 7 ( usp7 ) . uvssa has been found to be the causative gene for uvss and , together with usp7 , is implicated in regulating tc - ner activity . we will discuss the function of uvssa and usp7 and how the discovery of these proteins contributes to a better understanding of the molecular mechanisms underlying the clinical differences between uvss and the more severe cs .

Introduction Cockayne syndrome versus UV-sensitive syndrome The UVSSA factor UVSSA recruitment UVSSA function: a role in CSB stability UVSSA function: a role in the processing of stalled RNA Pol II New insights in transcription-coupled DNA repair from a UVSSA perspective

alterations in the dna structure that hinder the progression of rna polymerases during transcription are highly toxic for the cell and , if not properly resolved , can lead to cellular apoptosis or senescence ( ljungman and zhang 1996 ; ljungman and lane 2004 ) . helix - distorting lesions located in the transcribed strand of active genes initiate the transcription - coupled nucleotide excision repair ( tc - ner ) pathway to resolve the transcription - blocking dna damage ( hanawalt and spivak 2008 ) . lesion stalled elongating rna polymerase ii ( rna pol ii ) triggers the recruitment of several tc - ner - specific factors to form a functional tc - ner complex , including the cockayne syndrome a and b ( csa , csb ) proteins ( fousteri et al . i during transcription , uvssa , usp7 , and csb interact transiently with elongating rna pol ii . ii upon encountering a lesion ( indicated by star ) , stalled rna pol ii stabilizes its binding with these proteins and triggers the recruitment of several other dna repair factors , including csa . in the case of helix - distorting lesions ( iiia ) , tc - ner is initiated for which extensive modulation of stalled rna pol ii is needed , causing a prolonged transcriptional arrest . during this slow process , csb assembles a functional tc - ner complex and assists in the remodeling of rna pol ii . in order to allow sufficient time for csb to perform its function in tc - ner , its presence is protected by the concerted action of uvssa and usp7 by counteracting the ubiquitination - dependent degradation . when the required remodeling of stalled rna pol ii is completed , the tc - ner complex is destabilized and csb is degraded by the proteasome . however , due to the faster response , protection of csb from ubiquitination - dependent degradation by uvssa / usp7 is not necessary . after repair is completed ( iv ) , transcription is resumed ( v ) model for resolving transcription - blocking lesions . i during transcription , uvssa , usp7 , and csb interact transiently with elongating rna pol ii . ii upon encountering a lesion ( indicated by star ) , stalled rna pol ii stabilizes its binding with these proteins and triggers the recruitment of several other dna repair factors , including csa . in the case of helix - distorting lesions ( iiia ) , tc - ner is initiated for which extensive modulation of stalled rna pol ii is needed , causing a prolonged transcriptional arrest . during this slow process , csb assembles a functional tc - ner complex and assists in the remodeling of rna pol ii . in order to allow sufficient time for csb to perform its function in tc - ner , its presence is protected by the concerted action of uvssa and usp7 by counteracting the ubiquitination - dependent degradation . when the required remodeling of stalled rna pol ii is completed , the tc - ner complex is destabilized and csb is degraded by the proteasome . however , due to the faster response , protection of csb from ubiquitination - dependent degradation by uvssa / usp7 is not necessary . in humans , defective tc - ner is associated with two autosomal recessive dna repair deficiency disorders : cockayne syndrome ( cs ) and uv - sensitive syndrome ( uvs ) ( nance and berry 1992 ; spivak 2005 ) . although patient - derived cells are equally deficient in uv - induced tc - ner in vitro , the patients exhibit strikingly distinct clinical symptoms : cs individuals display severe developmental , neurological , and premature aging features , whereas uvs patients express much milder features , mostly restricted to uv hypersensitivity . cs is caused by mutations in two genes indispensable for tc - ner , csa and csb . uvs comprises three complementation groups , which are defined by specific mutations in csa , csb , and in the recently identified gene encoding for uv - stimulated scaffold protein a ( uvssa ) ( table 1 ) . intriguingly , neither the site nor the nature of the mutations in csa or csb seems to correlate with the clinical differences observed among patients with cs and uvs ( nance and berry 1992 ; laugel et al . thus , an important question remains : how do molecular defects within the same tc - ner pathway and even different mutations within the same genes lead to such diverse pathologies?table 1overview of uvs mutations in csa , csb , and uvssapatient / cell linegene affectedmutationreferenceskps2uvssap.lys123*1 , 3 , 4 , 5kps3uvssap.lys123*1 , 2 , 3 , 4 , 5uvs24tauvssap.ile31phefs*91 , 2 , 6xp24kouvssap.lys123*1 , 2 , 7 , 8xp70touvssap.cys32arg1 , 7 , 9uvss1vicsap.trp361cys10uvs1kocsbp.arg77*3 , 4 , 6 , 11 , 12cs3amcsbp.arg77*13references : 1 . ( 1998 ) overview of uvs mutations in csa , csb , and uvssa references : 1 . ( 1998 ) several models are proposed that try to explain the underlying molecular reason for the wide variety in tc - ner - deficient phenotype ( sarasin 2012 ; cleaver 2012 ) . most of these hypotheses are based on additional functions of the cs proteins outside of their role in tc - ner while assuming uvssa is not implicated in these processes , hence explaining the milder phenotype in uvs ( spivak and hanawalt 2006 ) ( fig . 2involvement of csa , csb , and uvssa in transcription , ber , and tc - ner . while the cs proteins have functions outside of tc - ner , a role for uvssa in these other processes has presently not been shown involvement of csa , csb , and uvssa in transcription , ber , and tc - ner . while the cs proteins have functions outside of tc - ner , a role for uvssa in these other processes has presently not been shown one of these models , the transcription defect model , is mainly based on data obtained for the csb protein . 1990 ) and is suggested to have chromatin remodeling abilities , possibly through recruitment of the p300 histone acetyltransferase ( citterio et al . this suggests that when csb is absent or mutated , transcription would be affected even in the absence of dna damage . there is a growing list of human diseases which are associated with defects in transcription , and many of these diseases are characterized by congenital defects ( villard 2004 ) , as also observed in cs patients . this suggests that the severe developmental and premature aging features of cs could partially be caused by a defect in transcription , while the sun sensitivity in these patients is most likely caused by the dna repair deficiency of the tc - ner pathway . another hypothesis involves a role of the cs proteins in repairing endogenous dna damage from reactive oxygen species ( ros ) , which is usually repaired by the base excision repair ( ber ) machinery ( hegde et al . both human and mouse cell lines deficient in the cs proteins indeed display enhanced sensitivity to agents that produce oxidative dna damage ( dianov et al . 2012 ) recently showed recruitment of csb to local oxidative dna lesions in living cells without an accumulation of downstream ner factors , indicating the involvement of csb in the response to oxidative dna lesions independent of the downstream ner reaction . in cs patients , the accumulation of unrepaired oxidative dna damage and the subsequent prolonged transcription arrest at these lesions could result in apoptosis and consequentially tissue degeneration . since active metabolism during development and a high level of oxidative metabolism in neural tissues may generate a considerable amount of oxidative damage , the consequences of the transcription arrest are mainly disclosed by developmental and neurological symptoms which are a hallmark of cs ( hoeijmakers 2007 ) . in addition , alterations in cellular metabolism including the glycolysis pathway and oxidative metabolism were observed . within a third model , a role for the cs proteins in repair of oxidative dna damage specifically in mitochondrial dna ( mtdna ) has been reported ( osenbroch et al . certain clinical symptoms associated with mitochondrial dysfunction are also observed in cs patients , including the severe neurological and premature aging features ( stevnsner et al . this would suggest that the cause of these symptoms in cs results in part from the loss of mitochondrial function as a consequence of unrepaired oxidative mtdna damage due to mutations in the cs proteins . the abovementioned hypotheses to explain the severe phenotypical consequences of cs do not uphold , however , for the intriguing case of two unrelated patients with null mutations in csb associated with mild uvs symptoms ( miyauchi - hashimoto et al . in contrast , two other patients with a deletion of the promoter sequence of csb ( resulting in no detectable csb protein ) have been found , which showed the most severe cs symptoms ( laugel et al . a factor possibly involved in this variability might be the expression of an evolutionary conserved csb - pgbd3 fusion protein , containing exons 15 of csb joined in frame with the piggybac transposase . recently , three laboratories identified uvssa as the causative gene for the uvs - a complementation group . 2012 ) were used to identify kiaa1530 as a new tc - ner factor , which was subsequently renamed uvssa . the uvssa protein was shown to interact with the tc - ner factors rna pol ii , csa , csb , and tfiih ( schwertman et al . a specialized chromatin immunoprecipitation ( chip ) procedure showed that uvssa resides in active , chromatin - bound tc - ner complexes upon uv damage . furthermore , gfp - tagged uvssa accumulated at local uv damage in living cells with similar recruitment kinetics as the tc - ner factor csb ( schwertman et al . this interaction was also observed in csb - deficient patient cells , indicative of a csb - independent binding of uvssa to tc - ner complexes . an explanation for this apparent discrepancy in uvssa recruitment could be that the cs proteins may be required for stable integration into a functional tc - ner complex rather than for the recruitment of uvssa . the observed csa - dependent stable association of uvssa into tc - ner complexes provides the basis for a possible molecular explanation of the uvs / cs phenotypic difference . ( 2009 ) described a patient with a specific missense csa mutation , which causes the mild uv - sensitive phenotype in the absence of the severe premature aging features common in cs . this finding thus implies a separable role for csa in response to uv and oxidative dna damage . this might suggest that an impaired interaction of uvssa with mutated csa at sites of dna damage results in uvs in this patient , while an unaffected role of csa in other processes such as in oxidative dna damage repair prevents the additional cs symptoms . in addition to its interaction with csa and rna pol ii , uvssa forms a uv - independent protein complex with the deubiquitinating enzyme ( dub ) ubiquitin - specific protease 7 ( usp7 ) . usp7 ( also known as hausp ) has multiple roles in the dna damage response , as illustrated by the wide variety of substrates including mdm2 , p53 , claspin , chfr , and histone h2b ( li et al . through its interaction with uvssa , usp7 is recruited to active tc - ner complexes upon uv damage ( schwertman et al . depletion of usp7 leads to a similar tc - ner deficiency as seen with uvssa depletion , such as a decrease in uv survival and rna synthesis recovery after uv ( schwertman et al . it is therefore suggested that uvssa stabilizes csb after uv by targeting the pleiotropic dub usp7 to tc - ner complexes , which subsequently removes uv - induced ubiquitin chains . the purpose of this could be to provide an increased time frame for csb to orchestrate tc - ner complex formation or to efficiently complete repair before csb is degraded . the stabilizing function of uvssa / usp7 on csb might be restricted to uv - induced tc - ner , since the reduced csb levels in uvss - a cells do not result in developmental and neurological symptoms in uvs patients . interestingly , overexpression of csb in uvs - a cells did not correct the tc - ner defect ( schwertman et al . therefore , the ubiquitination state of csb itself might have an additional function for proper tc - ner , for example to mediate a ubiquitin - mediated functional change in csb , or uvssa / usp7 might have additional substrates that can cause the uvs phenotype . 2002 ; meinhart and cramer 2004 ) , suggesting that the observed interaction between uvssa and rna pol ii is mediated via the vhs domain of uvssa . this interaction might be important for an additional function of uvssa , as nakazawa et al . during tc - ner , elongating rna pol ii can be recycled for a new round of transcription by means of dephosphorylating the elongating form of rna pol ii into the hypophosphorylated initiating form ( rockx et al . 2003 ) , raising the possibility that uvssa binds ubiquitinated tc - ner proteins . while the ubiquitination status of uvssa was not changed after uv , a similar silac ratio for uvssa and rna pol ii was observed . this would suggest that uvssa was co - purified by virtue of the enhanced ubiquitination of rna pol ii in response to uv . the vhs domain of uvssa is suggested to be also involved in the interaction between uvssa and csb , since a uvss - a patient missense mutation ( cys32arg ) within the vhs domain disrupts the interaction with csb in response to uv . apparently , the action of uvssa on both csb and rna pol ii contributes to the uvs phenotype . although the precise involvement of uvssa in processing these rna pol ii modifications remains to be elucidated , it is hypothesized that the modifications themselves contribute to the coordination of the removal of stalled rna pol ii and formation of the tc - ner complex and the subsequent resumption of transcription . mutations in uvssa were recently identified to cause uvs , a previously unresolved tc - ner deficiency disorder with rather mild clinical manifestations as compared to cs . uvssa and its interaction partner usp7 were identified as new factors involved in tc - ner repair efficiency and cell survival after uv damage . the identification of these new transcription - coupled dna repair factors presents us with the opportunity to gain more insight into the molecular mechanisms underlying the clinical differences between uvs and cs . the transcription defect model discussed previously is based on a role for the cs proteins in transcription , while assuming uvssa is not involved in this process . the uv - independent transient interaction between uvssa and rna pol ii may suggest a role for uvssa in transcription . this would suggest that the lack of dna repair combined with persistent arrest of rna pol ii at dna lesions in cs cells , which results in apoptosis or senescence , might be causative for the severe cs phenotype . in uvss - a cells , prolonged arrest is prevented and the lesion is made accessible for alternative dna repair by degradation of rna pol ii , hence resulting in the milder uvs phenotype ( fig . in wild - type cells , lesion stalled rna pol ii initiates tc - ner to remove the transcription - blocking damage . in cs cells , in addition , the ubiquitination and degradation of rna pol ii does not take place . the resultant persistent transcription arrest is more harmful for the cell , since it leads to apoptosis or senescence and might contribute to the more severe phenotype in cs patients differential aberrant processing of stalled elongating rna pol ii is responsible for the differences in uvs / cs phenotype . in wild - type cells , lesion stalled rna pol ii initiates tc - ner to remove the transcription - blocking damage . in cs cells , in addition , the ubiquitination and degradation of rna pol ii does not take place . the resultant persistent transcription arrest is more harmful for the cell , since it leads to apoptosis or senescence and might contribute to the more severe phenotype in cs patients the cs proteins play important roles in both tc - ner and oxidative dna damage repair , while the precise function of uvssa in these processes remains unclear . the previously discussed oxidative damage model assumes that uvssa does not play a role in oxidative dna damage repair ( spivak and hanawalt 2006 ) , hence explaining the milder phenotype of uvs . we hypothesize that based on the nature of the dna damage , distinct modulations of lesion stalled rna pol ii are required and that this involves a remodeling function of csb . in contrast , for a prolonged blocked transcription , more time would be required for csb to assemble a functional tc - ner complex and to assist in remodeling of the stalled rna pol ii . the presence of csb should , in the latter case , be protected by the concerted action of uvssa / usp7 to allow sufficient time for csb to perform its remodeling function . whether the ubiquitination of csb takes place at all after transient transcription arrest at oxidative dna lesions or if csb is ubiquitinated but not degraded before the repair of these lesions is completed remains to be elucidated . in this proposed model , mutations in uvssa would lead to dysfunctional tc - ner only , since the lack of csb protection would not affect repair or restart of transcription blocked by oxidative lesions . the discovery of uvssa enables us to test the proposed models in greater detail and whether or not they are mutually exclusive . to further substantiate the hypothesis that the phenotypical difference between uvs and cs is derived from different sensitivities for endogenous levels of oxidative dna damage , dedicated lesion - specific dna repair assays are required ( spivak et al . furthermore , dissecting the differences in molecular composition between repair complexes stalled at uv - induced lesions or oxidative lesions will help us to better understand the molecular mechanism of transcription - coupled repair for different types of dna damage . another interesting area for further investigation is the differential ubiquitination of several dna repair factors , which is likely an important driver of protein hand over and passing of repair intermediates through successive steps during tc - ner . finally , with the identification of uvssa , a uvs - a mouse model can now be generated for a more extensive study of the uvs phenotype and molecular defect . phenotypic consequences and possible differential responses to various genotoxic agents in the whole organism will contribute to our understanding of the clinical differences of cs versus uvs on a molecular level .

calcium ions ( ca ) participate in a diverse range of biological activities ranging from ventricular contractions to cell motility . mass movement of ca across membranes causes electrical polarization and underlies neuronal synaptic transmission and cardiovascular contractions . as a second messenger in signaling pathways , ca activates regulatory proteins such as calmodulin , which then act on other proteins to cause large - scale physiological changes . ca is also directly involved in the activities of many enzymes , where the metal ion functions as a cofactor ( prothrombinase ) or plays a role in thermostability ( mammalian trypsin ) . the involvement of ca in a range of biological activities has led to interest in identifying calcium - binding proteins and also in designing proteins that can bind to calcium . although the ef - hand motif is common in many calcium - binding proteins , there exist a significant number of calcium binding sites with differing coordinating residues and structures . prediction of calcium binding sites has therefore been mostly reliant on structure - based methods rather than 2d sequence motifs . used hydrophobicity to identify metal - binding sites and noted that long - range properties of the protein , such as electrostatics , did not work as well . nayal and di cera subsequently presented a method to predict calcium sites with higher accuracy using the estimated valence of a point as a predictor of local presence of calcium . wei and altman suggested the use of protein microenvironments as statistical predictors of calcium sites ( feature ) and showed that a nave bayes model trained on just 16 sites and 100 nonsites had a high recall on a small test set of 33 sites . developing further along the idea of a feature - based model , liang et al . incorporated information from binding motifs to increase the classification power , while halperin et al . increased the computational efficiency to make the feature method tractable . glazer et al . and liu et al . used molecular dynamics and loop - modeling , respectively , to improve the performance of previous feature models . additionally , sodhi et al . developed a neural network classifier to detect calcium sites ( metsite ) using sequence profile information and approximate structural data . a random forest classifier was trained by bordner et al . on various protein sequences and structure properties ( sitepredict ) but explicitly avoided the reliance on exact positions of residue atoms . deng et al . also demonstrated the use of graph theory in a geometry - based approach to the site - prediction problem . many of these methods returned protein regions that are likely to contain sites , rather than specific site positions within the protein . although the prediction of site regions may be sufficient to infer protein function , prediction of specific site locations is often desirable when designing peptides or re - engineering calcium - binding sites . there is unfortunately no standard method to directly infer exact site locations from predicted regions . the difficulty in doing so is that unless a perfect scoring function ( where a high score is given to the exact site location and nowhere else nearby ) is used , significant regions around each site would also have relatively high scores , leading to a large number of false positives when aggregation is performed . while varying levels of success have been reported with earlier methods , many of them do not take full advantage of the large number of calcium sites available in current structure repositories . the exponential growth in the number of crystal structures deposited in the protein data bank ( pdb ) repository over the past two decades has provided an opportunity to build models with better predictive power . furthermore , the increase in computing power over the years has allowed previously computationally intensive methods to now be feasibly accomplished on a personal computer . in this paper , we revisit the feature - based calcium model first introduced by wei and altman , using a 20-fold increase in training size and performing a more rigorous analysis of the model . we evaluated our model s performance against a range of data sets used in other papers and also on curated alternative ion data sets and show that our current model performs significantly better at site classification than the earlier feature - based calcium model . we also studied our model s sensitivity as a function of distance of a query point from the location of the known calcium sites and showed that the relationship to distance is similar to a logistic function . finally , we develop an algorithm that utilizes the feature scores obtained from a grid scan of a protein to predict exact site positions . we applied the algorithm on a set of 91 pdb structures with 190 sites and were able to recover more than 90% of the sites , with more than half of the predictions made within 0.5 from actual sites . our training set for the calcium model consisted of 314 calcium sites and 2735 nonsites from 312 protein structures taken from the rcsb protein data bank ( pdb ) , and the full list of calcium sites is provided in table s1 of the supporting information . we first queried uniprot for only reviewed protein entries that had an annotation for a calcium ligand and restricted the results to those that had an associated pdb and chain identifier . the protein chains were assigned to clusters based on precomputed blastclust results downloaded from the rcsb pdb server ( ftp://resources.rcsb.org/sequence/clusters/ ; 11 january 2013 snapshot ) , with the sequence similarity threshold set to 70% . the pdb structures with the best resolution in each cluster were chosen , and structures with a resolution of more than 3.5 were rejected . pdb s blastclust results do not cluster proteins with significantly different lengths together and may therefore over - represent the actual number of clusters . to address this , we recomputed the blast score between the protein chains using blast+ , with an identity cutoff of 70% for the best scoring local alignment between each pair . the pairs of chains with more than 70% identity were then aligned structurally , and only the chain from the structure with the highest resolution was retained if the associated calcium site for both chains were found to be in the same relative position . in the case where the associated calcium sites in both chains were in different relative positions , we characterized two physical properties of the sites in the training set , specifically the number of protein atoms within 1.25 and 5 of a site , in order to generate matched nonsites . we consider points in protein regions more than 10 from metal ions as potential candidates for nonsites . the nonsites were obtained by first generating protein surface points with pymol from the 312 calcium - binding protein structures , excluding points that were within 10 from any calcium , zinc , sodium , and magnesium ions . for each of the pdb structures , a subset of 1000 surface points were selected randomly , to which vectors of length 1.5 were then added in a random direction to generate nonsites with diverse distances from protein atoms . the perturbed points were retained if the number of protein atoms found within 1.25 and 5 matched that of the training sites . to avoid any possible sequence - based bias in the nonsites , no more than 10 out of the 1000 points generated were accepted for each protein structure . to evaluate the performance of the model , we used three different data sets ( table s2 ) from previous studies as test sets , similar to work done by deng et al . data set i consist of 62 calcium sites from 32 pdbs and was previously compiled and studied by nayal and di cera . one of the protein structures in data set i ( pdb i d : 2msb ) was also used in our training set . consist of 14 noncalcium - binding proteins and 92 calcium sites from 40 calcium - binding proteins . four of the calcium - binding structures were repeated in data set i ( pdb ids : 1ova , 1snc , 2por , 4sbv ) , containing a total of eight calcium sites . twelve of the calcium - binding protein structures were also used in our training set ( pdb ids : 1axn , 1clc , 1kit , 1kuh , 1mhl , 1poc , 1scm , 1sra , 1tco , 2aaa , 2scp , 3dni ) , containing a total of 33 calcium sites . data set iii consists of 60 calcium sites from 44 pdbs and was compiled and studied by pidcock and moore . six of the protein structures ( pdb ids : 1kit , 2fib , 1mmq , 1bjr , 1sra , 1bf2 ) were also used in our training set , containing a total of nine calcium sites . four of the calcium - binding structures in data set iii also appeared in data set ii ( pdb ids : 1cel , 1esl , 1kit , 1sra ) . the three data sets yielded a total of 154 unique calcium sites from 91 calcium - binding protein structures , excluding all duplicated structures ( and their sites ) from our training set . in reporting the performance metrics , we refer to this set of 154 calcium sites as the a total of 788 nonsites were generated from the 91 calcium - binding protein structures in the combined data set , using the same method that generated the nonsites for the training set . additionally , alternative ( noncalcium ) ion test sets were similarly obtained by querying uniprot for desired ion - binding sites and then clustering the results using blastclust . using a threshold of 70% sequence similarity , we obtained 651 zinc sites , 349 magnesium sites , 59 copper sites , 14 chloride sites , and 19 potassium sites for the test sets . the feature software was used to characterize the microenvironment of a given point . briefly , the microenvironment of the point is partitioned into six 1.25 - thick concentric shell ; 80 physiochemical properties such as hydrophobicity , electrostatic and the number of protein atoms are evaluated within each of those six shells , ultimately returning a combined numeric vector of length 480 for the given point ( 6 shells 80 properties / shell ) . all heteroatoms , such as water molecules , are ignored . all sites and nonsites a nave bayesian classifier was built using the feature software , with the feature vectors corresponding to the calcium sites and nonsites used as the training data . the prior probability for calcium site classification was set at 0.01 , and for each of the 480 properties in the vector , the range of values was divided into five bins ( query values beyond the range obtained from the training data were assigned to the nearest bin ) , as per a previous version of the calcium model . for each property p , the conditional probability of a value vp falling in bin bp , given that the query was a site , is simply : we then used bayes rule to calculate the posterior probability that vp was drawn from the distribution of the site values rather than the nonsite values : the reported feature score of a model is the summation of the log - odds ratio across all 480 properties and is an indication of the likelihood that a given query is a site . the full set of training data ( 314 calcium sites and 2735 nonsites ) was used to build the calcium model . to determine the score cutoffs corresponding to the desired precision thresholds ( 95% and 99% ) for the calcium model briefly , the full training data set was partitioned into 10 nonoverlapping , equal - sized subsets , and in each fold , a model was trained on nine subsets and tested on the left - out subset . the precision was collected over a range of cutoff values on each left - out subset . the lowest score at which both the mean and median precision ( of the 10 models ) reached the desired threshold was designated the score cutoff ( figure s1 ) . the precision - recall curves for each of the 10 models were plotted , and the area under the curves ( auc ) computed in order to detect the presence of anomalous data . in order to determine the sensitivity of the model s scoring function to distance from actual sites , we studied the distribution of scores at various distances from the sites . a series of spherical grid shells were generated for each calcium site in our training data with 0.05 spacing ( up to 5 ) , where and angles were sampled at 22.5 degree increments . this yielded a total of 16 angles 16 angles 100 shells = 25,600 grid points per calcium site . the set of all grid points from the various calcium sites were scored using the calcium model and then grouped together based on distance from the calcium sites . for a given distance , we computed the proportion of scores that were above the score cutoff corresponding to the model s precision threshold , i.e. , the fraction of points ( at that distance from actual calcium sites ) that would still have been classified as sites . to evaluate the performance of the calcium model independently , we applied the calcium model on data sets i iii and an alternative ions data set . for data sets i iii and the combined data set , a true positive ( tp ) was defined as a calcium site that had a score above the determined score cutoff ( based on the model s precision threshold ) . conversely , a calcium site that scored below the score cutoff was determined as a false negative ( fn ) . a nonsite that scored above the score cutoff was determined as a false positive ( fp ) and a true negative ( tn ) otherwise . the precision and recall of the calcium model were calculated as for the alternative ion test sets , a true negative ( tnion ) was defined as a point that scored below the score cutoff , and a false positive ( fpion ) was defined as a point that scored above the score cutoff . the specificity of the calcium model in each ion test set was calculated as as a simulation of actual use scenario , we scanned across the structures from data set i iii and scored the points in order to predict the location of calcium sites . a cubic grid with a spacing of 0.48 was constructed for each pdb structure , extending by 1 beyond the extreme cartesian coordinates of the structure . the grid spacing was determined by the evaluating the score function s sensitivity to distance from actual site . the grid points were converted into feature vectors and then scored using the calcium model . we retained only the points that were above the score cutoff determined by the model s precision threshold . the following was then done recursively to obtain aggregate numerous high scoring grid points into a more compact set of site predictions : while ( number of remaining points > 0):(i)take the top scoring point as an initial guess of the site s position.(ii)weigh all points within 3.5 of the initial guess by the exponential of their feature score , and refine the site position by taking the weighted mean of those points.(iii)return the refined position as a predicted site , and remove all points within 3.5 of the refined predicted site . take the top scoring point as an initial guess of the site s position . weigh all points within 3.5 of the initial guess by the exponential of their feature score , and refine the site position by taking the weighted mean of those points . return the refined position as a predicted site , and remove all points within 3.5 of the refined predicted site . a true prediction ( tpred ) is defined as a prediction that lies within 3.5 ( similar to comparable work by deng et al . ) from an actual calcium site , and a predicted site ( ps ) is defined as a calcium site with at least one prediction within 3.5 . note that for site predictions , all annotated calcium sites in a structure are retained as actual sites since the scanning was done across the entire structure . the performance of the site prediction algorithm was determined by the site recall ( sr ) , redundancy ( re ) , median relative rank ( mrr ) , first rank percentage ( frp ) , and the median distances between tpred and the actual calcium sites ( md ) . the site recall represents the proportion of true calcium sites that were detected by our method and is calculated as the proportion of calcium sites that had at least one prediction within 3.5 . the redundancy is the average number of predictions found within 3.5 in the ps . due to the design of the algorithm , each prediction is associated with a cluster of points within 3.5 , and in nearly all cases , the predictions are ordered by the magnitude of the highest score found within each respective cluster . this natural ranking of predicted sites lends itself to the notion of a relative rank , whereby the relative rank of a particular true prediction , going down the list of prediction in descending order , is the difference between the ranks of the previous true prediction and the current prediction . that is , if only the second and fifth predictions for a protein structure were found to be tpred , the second and fifth predictions have relative ranks 2 and 3 , respectively . we note that it is possible for a structure with multiple ps to have the respective predictions all ranked sequentially so that the relative rank for all except the first ps ( the site with the highest absolute rank prediction in the structure ) is 1 but with the highest - ranked tpred having an absolute rank much greater than 1 . if this pathological example was sufficiently common , the mrr would be 1 even though a large number of false positives would be made before the first tpred is encountered . to account for this , we also report the frp , which is the percentage of highest rank tpred for a structure , out of all highest rank tpred for structures in the data set , which has an absolute rank of 1 . to calculate the md , we consider the distance between the ps and the highest - ranked prediction for that site . we found that there were no protein atoms within 1.25 in all 283 calcium sites . the number of protein atoms within 5 of a site ranged from 10 to 50 , with a median of around 30 protein atoms . the number of water molecules found in a resolved crystal structure depended on the crystallization techniques employed and was thus not a good physical property to generate matched nonsites . nonetheless , we found that the sites generally had less than eight water molecules within 5 , with most sites having none or less than three water molecules . we initially trained a model using only 70% of the full training set ( 220 sites and 1914 nonsites ) , leaving the remaining 94 sites and 821 nonsites as the test set . we observed a clear separation in feature scores between the test sites and nonsites and an area under precision - recall curve of 0.98 ( figure s2 ) . the full model ( hereafter referred to as the calcium model ) , which was trained on all 314 sites and 2735 nonsites , was used to perform the site predictions and also scoring of the validation data sets . the calcium model discriminates well between the sites and nonsites ( figure 1 ) in the training set . the 10 cross - validation models were also in good agreement with one another , with a mean area under the precision - recall curve ( auprc ) of 0.989 and a standard deviation of 0.01 . the feature score cutoffs for the calcium model , as determined by the 10-fold cross - validation ( figure s1 ) , were 2.08 and 25.67 at the 95% and 99% precision thresholds , respectively . we refer to these two score cutoffs as low and high , respectively . the corresponding recalls estimated for those two precision levels were 96% and 88% ( figure 2 ) . among the 480 feature properties , we find some trends that are well expected ( figure s3 ) . oxygen atoms are enriched in shells 2 and 6 ( 1.252.5 and 6.257.5 from sites , respectively ) , which is within the ligand distance ( 1.63.3 ) previously reported by nayal and di cera . polar and charged residues that are known to ligate calcium , such as aspartic acid , asparagine , and glutamic acid , are also found to be enriched in those shells . the training calcium sites showed significantly lower hydrophobicity between shells 2 to 6 ( 1.257.5 from sites ) than the nonsites , which concurs with the observations made by yamashita et al . a generally high recall was obtained across the data sets i iii and the combined data set , with a recall of 94% in the latter when the low score cutoff was used ( table 1 ) . at that cutoff , the precision and specificity obtain on the combined data set was 98% and 99.6% , respectively . when the high score cutoff ( corresponding to the model s precision threshold of 99% ) was used , the recall on the combined data set decreased to 84% , while the precision and specificity both attained a full 100% . the model also displayed generally high specificity when applied to the alternative ion test sets , with the exception of mg and k ( table 2 ) . notably , the specificity reported on the k data set improves sharply to 95% when the model s precision threshold was increased from 95% to 99% . ( left ) histogram of the feature scores corresponding to the training set when the full calcium model was applied . the red bars correspond to the scores of the sites , while the blue bars correspond to the scores of nonsites . ( right ) precision - recall curves obtained by using a range of score cutoffs in each of the 10-fold cv test set . note that while recall is a strictly increasing function with respect to decreasing score cutoff , precision is not . for each of the two score cutoffs , the corresponding precision and recall values in each of the 10-fold models is plotted . triangular points ( both upward- and downward - pointing ) represent two circular points at that position of the same color . top and right margins : boxplots for the precision and recall of both score cutoffs are plotted using the corresponding colors . the diamond peg and line segment inside the box are the mean and median of the values , respectively . the whiskers extend 1.5 interquartile - ranges beyond the first and third quartiles . any points beyond the whiskers are represented by empty circles . after establishing the performance of the calcium model in classifying sites and nonsites , we evaluated the distribution of feature scores with respect to distance from documented calcium sites . as shown in figure 3 , the proportion of points classified as a site decreases sharply with distance from the actual calcium sites , exhibiting a logistic behavior . the greatest decrease in the site classification occurs after 1 from the actual calcium site , and at 2 away , less than 50% of the points were still classified as sites . at a distance of 0.414 from a calcium site , a point has a 95% chance of being classified as a site under the score cutoff for 99% precision . this is the maximum distance that a grid point can be from an actual site in order to still pick up the site information reliably . we therefore determined the appropriate grid spacing for the scanning box to be ( 4 0.414/3 ) = 0.48 . our site prediction algorithm reports a very high site recall of more than 90% , even when the high score cutoff ( model s 99% precision threshold ) was used for the calcium model ( table 3 ) . the algorithm has a slightly better site recall when the precision threshold is set lower at 95% . the median relative rank across all three data sets is 1 , and approximately 80% of the highest ranked tpred for each structure in the data sets was also rank 1 ( table 4 ) . an average of less than two predictions was made for each site ( table 5 ) , and the median distance between the best prediction and the site was less than 0.5 in the combined data set ( table 6 ) . grid points that were more than 4 from protein atoms had a generally uniform negative score , and points that were in steric clashes with protein atoms scored even more negatively ( figures 4 and 5 ) . ( left ) proportion of points classified as sites , at various distances from actual calcium sites for 95% and 99% precision cutoffs . ( right ) zoom - in image of the brown box in the left plot . n here is the total number of calcium ions annotated in the crystal structures from the data set . a sectional - plane through three calcium sites in a sarcoplasmic calcium - binding protein from nereis diversicolor ( pdb i d : 2scp ) . the three calcium sites are located in ( b ) and ( c ) with red and black dots , respectively . ( b ) minimum distance ( in ) to protein atoms at each grid point in the plane . ( c ) feature scores for the corresponding grid points when our model is applied . sectional - plane through three calcium sites in calmodulin from rattus rattus ( pdb i d : 3cln ) . three calcium sites are located in ( b ) and ( c ) with red and black dots , respectively . ( b ) minimum distance ( in ) to protein atoms at each grid point in the plane . ( c ) feature scores for the corresponding grid points when our model is applied . n here is the total number of calcium ions annotated in the crystal structures from the data set . each prediction has a relative rank , and the mrr is the median of these relative ranks across the data set . the frp calculates the percentage of predictions in the data set with relative rank 1 . n here is the total number of calcium ions annotated in the crystal structures from the data set . the re is the ratio of true predictions to the number of predicted sites . n here is the total number of calcium ions annotated in the crystal structures from the data set . in this work , we built a feature - based calcium model for scoring points in 3d structures and also developed an algorithm that utilizes the model for automated calcium site prediction in protein structures . we evaluated the feature - based calcium model on various test sets and showed that the model exhibited high recall for calcium ions across all data sets i iii , with a recall of 94% and 84% on the combined data set when the low and high score cutoff were used , respectively . this is a significant improvement from the 75% recall obtained when wei s calcium model was applied to the same data set . using random points close to the protein surface as nonsites for evaluation , the model achieved a precision of 98% and 100% at the low and high score cutoffs , respectively . the improved performance is expected given that nave bayes models tend to perform better with increased training set size , and our current training set is approximately 20-fold larger than the one used in wei s feature calcium model . in order to determine if our calcium model was sensitive to the choice of sequence similarity threshold used to define the training set , we also built an additional model with identical methods , but using 30% sequence similarity threshold to create the training sets . smaller model had comparable performance with the model used in this paper ( figure s4 ) . consequently , we chose the model with the larger training set as the calcium model . although the nonsites in the training set were derived from protein surface points , the model was able to correctly reject most other cationic and anionic sites , displaying a high level of specificity ( table 2 ) . the exceptions to this are the k and mg data sets , for which the model performs poorly in recognizing them as nonsites . it is known that a number of calcium sites can accommodate potassium , sodium , and magnesium ions , and many metal - binding sites are known to bind to multiple metal ions . in particular , magnesium ions have been previously reported to bind to the ef - hand motif , which underlies many calcium - binding sites . in fact , crystallographers often take advantage of the high affinity of calcium sites for magnesium ions by using magnesium ions during the crystallization process . the difficulty in discriminating calcium and magnesium sites has also been documented by other groups . in a recent attempt by wei et al . to discriminate different metal - binding sites computationally , a low auc score of close to 0.5 was obtained when discriminating the two different binding sites . nonetheless , we show that when a higher precision threshold for the model was used the specificity of our model increases across the alternative ion data sets and significantly improves the specificity in the k data set from 58% to a respectable 95% . although sodium and calcium ions have very similar ionic radii , the coordination spheres of both ions are quite different given that the former is an alkaline earth metal , whereas the latter is an alkali metal . the similarity in electron density of sodium ions and water molecules often result in sodium ions being misassigned as water molecules in crystal structures . additionally , rubidium ions are also often used for protein phasing in place of sodium , making it difficult to find well - documented sodium sites in the pdb repository . we attempted to construct a sodium ion test set using the same criteria for the other ions but found that the test set was too small ( only six distinct sites ) to make any meaningful discussion on the absolute specificity . however , we did observe that the specificity improved from 16.7% to 66.7% when the higher precision threshold for the model was used , consistent with the trend reported on the other ions . we also report an algorithm to predict calcium sites in a query protein structure using our feature - based calcium model . although previous work by our group had also shown good performance in classifying points as sites and nonsites , direct application of the model to actual site prediction had not been attempted on a large scale . this is because of the difficulty in transforming high - scoring regions reliably to exact site predictions , given that a significant region around each site may all be classified as sites by the feature model . to reduce the difficulty of the problem , we first ensured that the distribution of feature scores decayed with increasing distance from the calcium site . in our evaluation , we found that the feature scores obtained by our model do indeed show a complete decorrelation with the calcium site when separated by more than 5 and that the greatest drop in signal occurred at 1 to 3 from the calcium site . we showed that the relationship is similar to a logistic curve and that points sampled within 1 of a calcium site would almost always be classified as a true positive ( figure 3 ) . this makes our calcium model well - suited for subsequent incorporation into exact site prediction algorithms . using the curve obtained in figure 3 , we also determined that the maximum grid spacing required for the 95% and 99% precision thresholds were 0.94 and 0.48 , respectively . although the 95% precision threshold allowed for the larger grid spacing and was thus less computationally expensive to scan across a protein , we found that this resulted in a slightly lower site recall compared to that obtained using the finer grid size of 0.48 . in particular , we found that the finer grid size combined with the lower score cutoff corresponding to the 95% precision threshold resulted in the highest site recall but also with the most number of predictions per structure . many of these predictions are likely false positives , given their proximity to each other . the combination of finer grid size and higher score cutoff corresponding to the 99% precision threshold provided a balance , reducing the site recall by around 58% while also reducing the number of predictions made per structure . in this paper , we report the results using both score cutoffs on only the finer grid size . deng et al . employed a merging algorithm that builds on properties of their geometry - based approach to calcium site prediction . nayal and di cera proposed a more general , iterative method by ranking the predictions by valences and taking only the top rank prediction . each time a prediction was accepted , all predictions within 3.5 were removed , and the process was continued until no predictions remained . we adapted this algorithm by refining the predicted site position at each step using the weighted means of all predictions within 3.5 of the top rank prediction . we found that the exponential of the feature scores as weights reduced the noise in the data and led to an overall refinement in the predicted site location . our algorithm outputs a reduced list of predictions that were naturally ranked by the highest feature score within each prediction s cluster . we demonstrated that this algorithm can be used to accurately predict calcium sites with root - mean squared distances ( rmsd ) of less than 1 . in particular , we observed a high site recall ( using a distance cutoff of 3.5 ) of more than 90% across all the data sets with our algorithm and with nearly four - fifths of the highest ranked tpred of each structure corresponding to an actual calcium site . in at least eight of the structures with two or more calcium sites ( pdb ids : 1tf4 , 2amg , 3cln , 2taa , 2tep , 1js4 , 1clx and 1sac ) , we observed that not only were all sites predicted by our algorithm , but the relative rank was also 1 for all the predictions . notably , the median relative rank is 1 for all the data sets , and the prediction redundancy was less than 2 , suggesting that if a priori knowledge of the number of calcium sites in a structure ( e.g. , via stoichiometry ) was used to determine the number of top predictions to retain , the precision of our site prediction algorithm would be close to 100% . we also found that our predictions were extremely close to the sites themselves , with the median distance being less than 0.5 . it is important to note that these results were obtained using a much stricter criterion for predicted sites ( ps ) compared to previous work from our group . in particular , we now define a site as ps if and only if there is a prediction within 3.5 , compared to the 6 used by liang et al . and 7 used by wei and altman . if we had used the 7 criteria in this work , our site recall would increase by 1.53% across the data sets . by considering a rich variety of physiochemical properties , the calcium model also shows better sensitivity than some of the previously published models used for calcium site prediction . the gg algorithm by deng et al . reported a site recall of 91% and 87% for data sets ii and iii , respectively ( using a distance cutoff of 3.5 ) , while our algorithm reports a site recall of 90.2% and 90.4% for those two data sets , respectively , at the precision threshold of 99% . when the lower precision threshold of 95% was used , our model reports a site recall of 97.8% and 96.8% , respectively . in either case , our algorithm shows comparable , if not better , recall than the gg algorithm on those data sets . noted that the gg algorithm had difficulty identifying calcium sites with abnormal ca o distances , and identified six structures ( pdb ids : 1ova , 1cel , 1djx , 1scm , 1bjr and 1ag9 ) in which the gg algorithm failed to detect the presence of sites . our algorithm was not able to identify sites for 1ova as well but performed with varying levels of success for the rest . for 1cel , a prediction for the calcium site was made within 1.29 but had a large relative rank of 18 . for 1djx , all four calcium sites were predicted with rmsd of less than 1 and with relative ranks 3 , 4 , 4 , and 8 . our algorithm performed excellently on 1scm , with predictions for both sites having relative rank 1 and a low rmsd of around 1 . the two calcium sites in 1bjr were predicted by our algorithm at the 95% precision threshold but not at the 99% precision threshold due to the low feature scores of the sites themselves . the calcium site of 1ag9 ( ca350 ) that deng et al . failed to predict was also not predicted by our algorithm due to the ion being chelated by nonprotein oxygen atoms from the cofactor btb ( bis-2-hydroxy - imino - tris - hydroxymethylmethane ) . it is worth pointing out here that the feature software ignores all heteroatoms in structures , making this a potential weakness in detecting sites that depend heavily on nonprotein ligands . although the gg algorithm also ignores oxygen from water molecules , it retains information from other chelating ligands . in the particular case of 1ag9 , however , both algorithms were unable to recover the calcium site . we also report a slightly higher site recall ( 95.4% and 98.4% ) on data set i as compared to the 95% reported by the gg algorithm and the 93% reported by nayal and di cera s method . in a few cases , the ranks of our predictions were also higher than those from nayal s algorithm . for instance , the documented calcium ion in con a ( pdb i d : 3cna , estimated valence 1.41 ) was detected by nayal s method as the 67th rank prediction , with an rmsd of 2 . our method returned a prediction within 0.44 of the site as our first prediction for the structure ( using the 99% precision cutoff ) . similarly , the calcium ion in taka amylase a ( pdb i d : 2taa , estimated valence 1.40 ) was detected by nayal s method in the 61st ranked prediction , with an rmsd of 3.2 . our method returned a prediction within 1.95 of the site as our sixth prediction for the structure ( using the 99% precision cutoff ) . in general , we observed that the performance of nayal s method diminishes when the estimated valence of the calcium site is close to or less than the threshold of 1.4 . nonetheless , our site prediction algorithm suffers the same pitfalls as all scoring - function based models do ; if the calcium site itself does not score above the calcium model s score cutoff , the algorithm typically fails to correctly predict the presence of the site . for instance , the calcium site in tobacco mosaic virus protein ( pdb i d : 2tmv , estimated valence 1.70 ) was recovered by nayal s method , but not our model , which had assigned the site a low feature score of 3.90 . analysis of the calcium site in 2tmv reveals very different feature properties from the other calcium - binding sites in the combined data set ( figure s5 ) . in particular , the site in 2tmv was lacking in some features that were enriched in the calcium model for calcium sites and was instead enriched for other features that were expected to be diminished . additionally , the binding of calcium to the viral protein is enabled by the chelation of rna bases , which are not incorporated into the default feature properties as previously noted . similarly , the second calcium site in flavodoxin ( pdb i d : 1ag9 , a350 ) from data set iii is chelated by btb ( 2-[bis(2-hydroxyl - ethyl)-amino]-2-hydoxymethylpropane-1,3-diol ) and at least three water molecules , all of which were not used in the feature vector . as a result , we note , however , that the generally high recall of our calcium model suggests that such occurrences are not a major problem . while many of the methods currently available for calcium site prediction are often rule - based and dependent on properties such as the presence of glutamate or aspartate or sequence similarities , these methods immediately preclude the possibility of discovering novel calcium sites that do not resemble known calcium sites . a statistical - based method like the one used in our model relaxes the conditions for classification and may be more suitable for evaluating engineered or newly discovered proteins .

metal - binding proteins are ubiquitous in biological systems ranging from enzymes to cell surface receptors . among the various biologically active metal ions , calcium plays a large role in regulating cellular and physiological changes . with the increasing number of high - quality crystal structures of proteins associated with their metal ion ligands , many groups have built models to identify ca2 + sites in proteins , utilizing information such as structure , geometry , or homology to do the inference . we present a feature - based approach in building such a model and show that our model is able to discriminate between nonsites and calcium - binding sites with a very high precision of more than 98% . we demonstrate the high specificity of our model by applying it to test sets constructed from other ions . we also introduce an algorithm to convert high scoring regions into specific site predictions and demonstrate the usage by scanning a test set of 91 calcium - binding protein structures ( 190 calcium sites ) . the algorithm has a recall of more than 93% on the test set with predictions found within 3 of the actual sites .

Introduction Methods Results Discussion

calcium ions ( ca ) participate in a diverse range of biological activities ranging from ventricular contractions to cell motility . as a second messenger in signaling pathways , ca activates regulatory proteins such as calmodulin , which then act on other proteins to cause large - scale physiological changes . ca is also directly involved in the activities of many enzymes , where the metal ion functions as a cofactor ( prothrombinase ) or plays a role in thermostability ( mammalian trypsin ) . the involvement of ca in a range of biological activities has led to interest in identifying calcium - binding proteins and also in designing proteins that can bind to calcium . although the ef - hand motif is common in many calcium - binding proteins , there exist a significant number of calcium binding sites with differing coordinating residues and structures . used hydrophobicity to identify metal - binding sites and noted that long - range properties of the protein , such as electrostatics , did not work as well . nayal and di cera subsequently presented a method to predict calcium sites with higher accuracy using the estimated valence of a point as a predictor of local presence of calcium . wei and altman suggested the use of protein microenvironments as statistical predictors of calcium sites ( feature ) and showed that a nave bayes model trained on just 16 sites and 100 nonsites had a high recall on a small test set of 33 sites . developing further along the idea of a feature - based model , liang et al . also demonstrated the use of graph theory in a geometry - based approach to the site - prediction problem . although the prediction of site regions may be sufficient to infer protein function , prediction of specific site locations is often desirable when designing peptides or re - engineering calcium - binding sites . the difficulty in doing so is that unless a perfect scoring function ( where a high score is given to the exact site location and nowhere else nearby ) is used , significant regions around each site would also have relatively high scores , leading to a large number of false positives when aggregation is performed . while varying levels of success have been reported with earlier methods , many of them do not take full advantage of the large number of calcium sites available in current structure repositories . the exponential growth in the number of crystal structures deposited in the protein data bank ( pdb ) repository over the past two decades has provided an opportunity to build models with better predictive power . in this paper , we revisit the feature - based calcium model first introduced by wei and altman , using a 20-fold increase in training size and performing a more rigorous analysis of the model . we evaluated our model s performance against a range of data sets used in other papers and also on curated alternative ion data sets and show that our current model performs significantly better at site classification than the earlier feature - based calcium model . we also studied our model s sensitivity as a function of distance of a query point from the location of the known calcium sites and showed that the relationship to distance is similar to a logistic function . we applied the algorithm on a set of 91 pdb structures with 190 sites and were able to recover more than 90% of the sites , with more than half of the predictions made within 0.5 from actual sites . our training set for the calcium model consisted of 314 calcium sites and 2735 nonsites from 312 protein structures taken from the rcsb protein data bank ( pdb ) , and the full list of calcium sites is provided in table s1 of the supporting information . the pdb structures with the best resolution in each cluster were chosen , and structures with a resolution of more than 3.5 were rejected . pdb s blastclust results do not cluster proteins with significantly different lengths together and may therefore over - represent the actual number of clusters . in the case where the associated calcium sites in both chains were in different relative positions , we characterized two physical properties of the sites in the training set , specifically the number of protein atoms within 1.25 and 5 of a site , in order to generate matched nonsites . we consider points in protein regions more than 10 from metal ions as potential candidates for nonsites . the nonsites were obtained by first generating protein surface points with pymol from the 312 calcium - binding protein structures , excluding points that were within 10 from any calcium , zinc , sodium , and magnesium ions . the perturbed points were retained if the number of protein atoms found within 1.25 and 5 matched that of the training sites . to avoid any possible sequence - based bias in the nonsites , no more than 10 out of the 1000 points generated were accepted for each protein structure . consist of 14 noncalcium - binding proteins and 92 calcium sites from 40 calcium - binding proteins . four of the calcium - binding structures were repeated in data set i ( pdb ids : 1ova , 1snc , 2por , 4sbv ) , containing a total of eight calcium sites . twelve of the calcium - binding protein structures were also used in our training set ( pdb ids : 1axn , 1clc , 1kit , 1kuh , 1mhl , 1poc , 1scm , 1sra , 1tco , 2aaa , 2scp , 3dni ) , containing a total of 33 calcium sites . six of the protein structures ( pdb ids : 1kit , 2fib , 1mmq , 1bjr , 1sra , 1bf2 ) were also used in our training set , containing a total of nine calcium sites . four of the calcium - binding structures in data set iii also appeared in data set ii ( pdb ids : 1cel , 1esl , 1kit , 1sra ) . the three data sets yielded a total of 154 unique calcium sites from 91 calcium - binding protein structures , excluding all duplicated structures ( and their sites ) from our training set . in reporting the performance metrics , we refer to this set of 154 calcium sites as the a total of 788 nonsites were generated from the 91 calcium - binding protein structures in the combined data set , using the same method that generated the nonsites for the training set . additionally , alternative ( noncalcium ) ion test sets were similarly obtained by querying uniprot for desired ion - binding sites and then clustering the results using blastclust . using a threshold of 70% sequence similarity , we obtained 651 zinc sites , 349 magnesium sites , 59 copper sites , 14 chloride sites , and 19 potassium sites for the test sets . briefly , the microenvironment of the point is partitioned into six 1.25 - thick concentric shell ; 80 physiochemical properties such as hydrophobicity , electrostatic and the number of protein atoms are evaluated within each of those six shells , ultimately returning a combined numeric vector of length 480 for the given point ( 6 shells 80 properties / shell ) . all sites and nonsites a nave bayesian classifier was built using the feature software , with the feature vectors corresponding to the calcium sites and nonsites used as the training data . for each property p , the conditional probability of a value vp falling in bin bp , given that the query was a site , is simply : we then used bayes rule to calculate the posterior probability that vp was drawn from the distribution of the site values rather than the nonsite values : the reported feature score of a model is the summation of the log - odds ratio across all 480 properties and is an indication of the likelihood that a given query is a site . the full set of training data ( 314 calcium sites and 2735 nonsites ) was used to build the calcium model . to determine the score cutoffs corresponding to the desired precision thresholds ( 95% and 99% ) for the calcium model briefly , the full training data set was partitioned into 10 nonoverlapping , equal - sized subsets , and in each fold , a model was trained on nine subsets and tested on the left - out subset . in order to determine the sensitivity of the model s scoring function to distance from actual sites , we studied the distribution of scores at various distances from the sites . the set of all grid points from the various calcium sites were scored using the calcium model and then grouped together based on distance from the calcium sites . the precision and recall of the calcium model were calculated as for the alternative ion test sets , a true negative ( tnion ) was defined as a point that scored below the score cutoff , and a false positive ( fpion ) was defined as a point that scored above the score cutoff . the specificity of the calcium model in each ion test set was calculated as as a simulation of actual use scenario , we scanned across the structures from data set i iii and scored the points in order to predict the location of calcium sites . a cubic grid with a spacing of 0.48 was constructed for each pdb structure , extending by 1 beyond the extreme cartesian coordinates of the structure . the following was then done recursively to obtain aggregate numerous high scoring grid points into a more compact set of site predictions : while ( number of remaining points > 0):(i)take the top scoring point as an initial guess of the site s position. (ii)weigh all points within 3.5 of the initial guess by the exponential of their feature score , and refine the site position by taking the weighted mean of those points. (iii)return the refined position as a predicted site , and remove all points within 3.5 of the refined predicted site . weigh all points within 3.5 of the initial guess by the exponential of their feature score , and refine the site position by taking the weighted mean of those points . return the refined position as a predicted site , and remove all points within 3.5 of the refined predicted site . note that for site predictions , all annotated calcium sites in a structure are retained as actual sites since the scanning was done across the entire structure . the performance of the site prediction algorithm was determined by the site recall ( sr ) , redundancy ( re ) , median relative rank ( mrr ) , first rank percentage ( frp ) , and the median distances between tpred and the actual calcium sites ( md ) . the redundancy is the average number of predictions found within 3.5 in the ps . due to the design of the algorithm , each prediction is associated with a cluster of points within 3.5 , and in nearly all cases , the predictions are ordered by the magnitude of the highest score found within each respective cluster . if this pathological example was sufficiently common , the mrr would be 1 even though a large number of false positives would be made before the first tpred is encountered . the number of protein atoms within 5 of a site ranged from 10 to 50 , with a median of around 30 protein atoms . the number of water molecules found in a resolved crystal structure depended on the crystallization techniques employed and was thus not a good physical property to generate matched nonsites . we initially trained a model using only 70% of the full training set ( 220 sites and 1914 nonsites ) , leaving the remaining 94 sites and 821 nonsites as the test set . the full model ( hereafter referred to as the calcium model ) , which was trained on all 314 sites and 2735 nonsites , was used to perform the site predictions and also scoring of the validation data sets . the training calcium sites showed significantly lower hydrophobicity between shells 2 to 6 ( 1.257.5 from sites ) than the nonsites , which concurs with the observations made by yamashita et al . a generally high recall was obtained across the data sets i iii and the combined data set , with a recall of 94% in the latter when the low score cutoff was used ( table 1 ) . the model also displayed generally high specificity when applied to the alternative ion test sets , with the exception of mg and k ( table 2 ) . ( right ) precision - recall curves obtained by using a range of score cutoffs in each of the 10-fold cv test set . after establishing the performance of the calcium model in classifying sites and nonsites , we evaluated the distribution of feature scores with respect to distance from documented calcium sites . as shown in figure 3 , the proportion of points classified as a site decreases sharply with distance from the actual calcium sites , exhibiting a logistic behavior . our site prediction algorithm reports a very high site recall of more than 90% , even when the high score cutoff ( model s 99% precision threshold ) was used for the calcium model ( table 3 ) . the algorithm has a slightly better site recall when the precision threshold is set lower at 95% . n here is the total number of calcium ions annotated in the crystal structures from the data set . a sectional - plane through three calcium sites in a sarcoplasmic calcium - binding protein from nereis diversicolor ( pdb i d : 2scp ) . ( c ) feature scores for the corresponding grid points when our model is applied . sectional - plane through three calcium sites in calmodulin from rattus rattus ( pdb i d : 3cln ) . in this work , we built a feature - based calcium model for scoring points in 3d structures and also developed an algorithm that utilizes the model for automated calcium site prediction in protein structures . we evaluated the feature - based calcium model on various test sets and showed that the model exhibited high recall for calcium ions across all data sets i iii , with a recall of 94% and 84% on the combined data set when the low and high score cutoff were used , respectively . it is known that a number of calcium sites can accommodate potassium , sodium , and magnesium ions , and many metal - binding sites are known to bind to multiple metal ions . in particular , magnesium ions have been previously reported to bind to the ef - hand motif , which underlies many calcium - binding sites . in fact , crystallographers often take advantage of the high affinity of calcium sites for magnesium ions by using magnesium ions during the crystallization process . to discriminate different metal - binding sites computationally , a low auc score of close to 0.5 was obtained when discriminating the two different binding sites . nonetheless , we show that when a higher precision threshold for the model was used the specificity of our model increases across the alternative ion data sets and significantly improves the specificity in the k data set from 58% to a respectable 95% . we attempted to construct a sodium ion test set using the same criteria for the other ions but found that the test set was too small ( only six distinct sites ) to make any meaningful discussion on the absolute specificity . however , we did observe that the specificity improved from 16.7% to 66.7% when the higher precision threshold for the model was used , consistent with the trend reported on the other ions . we also report an algorithm to predict calcium sites in a query protein structure using our feature - based calcium model . this is because of the difficulty in transforming high - scoring regions reliably to exact site predictions , given that a significant region around each site may all be classified as sites by the feature model . in our evaluation , we found that the feature scores obtained by our model do indeed show a complete decorrelation with the calcium site when separated by more than 5 and that the greatest drop in signal occurred at 1 to 3 from the calcium site . employed a merging algorithm that builds on properties of their geometry - based approach to calcium site prediction . we adapted this algorithm by refining the predicted site position at each step using the weighted means of all predictions within 3.5 of the top rank prediction . in particular , we observed a high site recall ( using a distance cutoff of 3.5 ) of more than 90% across all the data sets with our algorithm and with nearly four - fifths of the highest ranked tpred of each structure corresponding to an actual calcium site . notably , the median relative rank is 1 for all the data sets , and the prediction redundancy was less than 2 , suggesting that if a priori knowledge of the number of calcium sites in a structure ( e.g. , via stoichiometry ) was used to determine the number of top predictions to retain , the precision of our site prediction algorithm would be close to 100% . we also found that our predictions were extremely close to the sites themselves , with the median distance being less than 0.5 . noted that the gg algorithm had difficulty identifying calcium sites with abnormal ca o distances , and identified six structures ( pdb ids : 1ova , 1cel , 1djx , 1scm , 1bjr and 1ag9 ) in which the gg algorithm failed to detect the presence of sites . our algorithm was not able to identify sites for 1ova as well but performed with varying levels of success for the rest . the two calcium sites in 1bjr were predicted by our algorithm at the 95% precision threshold but not at the 99% precision threshold due to the low feature scores of the sites themselves . analysis of the calcium site in 2tmv reveals very different feature properties from the other calcium - binding sites in the combined data set ( figure s5 ) . as a result , we note , however , that the generally high recall of our calcium model suggests that such occurrences are not a major problem . while many of the methods currently available for calcium site prediction are often rule - based and dependent on properties such as the presence of glutamate or aspartate or sequence similarities , these methods immediately preclude the possibility of discovering novel calcium sites that do not resemble known calcium sites . a statistical - based method like the one used in our model relaxes the conditions for classification and may be more suitable for evaluating engineered or newly discovered proteins .

every year in the united states , over 22,000 women are diagnosed with ovarian cancer ( oc ) and more than 15,000 die from the disease.1 the poor prognosis for women diagnosed with oc is largely because of the presence of local and distant metastases in 81% of patients at diagnosis , leading to 5-year survival rates of 69% and 30% , respectively.1 these numbers suggest that both early detection and treatment of metastatic disease are crucial in managing oc . these facts also highlight the need to better understand mechanisms driving progression from primary tumor to metastatic disease . ovarian tumors can arise in the ovary from germ cells , stromal cells , or epithelial cells . they are high - grade serous ( hgs ) and low - grade serous ovarian , mucinous , endometrioid , clear cell , and transitional or undifferentiated.2 hgs is the most common form of oc . the high mortality associated with oc is a direct result of the process of tumor metastasis . oc invasion is a complex process that differs from metastatic programs of other solid tumors , since oc tumor cells shed from the primary site and spread throughout the peritoneal cavity without first entering the blood or lymphatic system.3 oc was originally thought to be derived from the ovarian surface epithelium.4 however , epidemiological , pathological , and molecular studies have made scientists question the origin of oc and hypothesize that different types of ovarian tumors may arise from different precursor cells , including those from the fallopian tube , endometrium , cervix , and gastrointestinal ( gi ) tract.2,57 in addition to the oc subtypes being histologically different , they are also genetically distinct . ( 1 ) type 1 cancers progress from tumors with lower malignant activity , are usually low grade , and contain ras pathway mutations ; these include clear cell , mucinous , endometrioid , and low - grade serous tumors . ( 2 ) type 2 tumors arise rapidly de novo and contain tp53 mutations ; these include hgs , undifferentiated , and carcinosarcomas.8 recent work suggests that some hgs tumors may be initiated in the fimbria of the fallopian tube.8,9 identifying the cell type of origin for oc will change how animal models are generated and evaluated . frequently , as oc progresses , tumor cells accumulate as single - cell and multi - cell aggregates in the ascites fluid.10 the accumulation of ascites fluid in late stage cancer correlates with poor outcome.11 these shed tumor cells metastasize locally . preferred sites of metastasis include the omentum , peritoneal wall , the diaphragm , and small bowel mesentery.3 therefore , design and utilization of mouse models of oc that recapitulate the steps of metastasis are critical . here , we will review many of the common ways oc is studied in mice . we will first discuss xenografts derived from cell lines and patient samples , followed by genetic models of oc . xenograft models are a common way of investigating oc growth , metastasis , and treatment response in a live animal . the process of injecting ovarian tumor cells into mice and monitoring tumor growth has been conducted since at least the early 1980s . in these early studies , the thymus of the mice was removed , mice were treated with cytosine arabinoside , given whole - body irradiation , and then engrafted subcutaneously ( sc ) with either ovarian ascites ( n = 5 ) or a solid peritoneal metastasis from a papillary cystadenocarcinoma ovarian tumor biopsy ( n = 8).12 four of the peritoneal metastases established tumor growth.12 xenograft studies have been used extensively since the 1980s and offer many advantages . xenografts are effective at measuring tumor growth in response to mutations or exogenous gene expression . , many xenograft mouse models are conducted using immunocompromised mice , which will lack the contribution of the full complement of immune cells to the tumor growth process . the use of xenograft models enables researchers to test factors that influence tumor growth , spread , and drug response in a live animal . these elements can not be entirely recapitulated in tissue culture . to study tumor growth in mice , murine or human oc cells cells from different genetic backgrounds are injected into immunocompromised mice such as nude ( foxn1 , nu / nu ) , scid , nod / scid , or nod - scid il2rcnull ( nsg ) to enable the cells to engraft without being eliminated by the immune system . cells are injected sc , intraperitoneally ( ip ) , or intrabursally [ ( ib ) into the bursa that surrounds the mouse ovary ] . the sc model is not well suited for ovarian metastasis studies as the tumors do not typically metastasize , and the tumor is not positioned in the right anatomic location or microenvironment . one advantage of the sc model is that it is well suited for investigation with imaging modalities such as two - photon microscopy ( fig . 1 ) . in two - photon microscopy , tumor vasculature ( with fluorescent dextrans ) , collagens ( with second harmonic imaging ) , and fluorescently labeled tumor cells can be measured simultaneously in a live animal . this can not be done with ip or ib injections because of the depth of the tumors . however , ip and ib tumors can be imaged in live animals with optical imaging approaches using fluorescence or luminescence . in contrast to sc tumors , ip and ib injections of tumor cells into mice can mimic aspects of tumor metastasis , particularly metastatic dissemination . while ip injection can not mimic the initial steps in metastasis , ip - injected tumor cells such as skov3 metastasize to the ovary , peritoneal wall , diaphragm , and form ascites fluid similar to human disease.13 the skov3ip cell line is derived from ascites cells that developed in a mouse ip cavity injected with skov3 cells.13 compared to the parental skov3 cells , the skov3ip cells grow faster , disseminate more , and exhibit overexpressed erbb2.13 many cells ( including the skov3 lines ) are frequently transfected or transduced with fluorescence or luminescence - expressing vectors to monitor tumor growth in vivo.14 similarly , ovcar3 cells metastasize to the gi tract , omentum , pancreas , kidney , and liver ( unpublished data ) when injected ip . in some ways , the ib injection mimics the initial steps in metastasis , as the tumor cells exit the bursa to spread throughout the peritoneal cavity . many cell lines metastasize following ib injection , including a2780 , skov3 , and hey cells.15,16 sites of metastasis include diaphragm , mesentery , bowel , and liver.16 in particular , the a2780 cells have been used to study not only metastasis but also chemoresistance . a2780 that are sensitive to cisplatin and a subline of a2780 cells that are resistant to cisplatin cells are also commonly used in xenograft studies of chemoresistance in vivo.17 many other cell lines not discussed also demonstrate growth in xenograft models.18 compared to genetic models , xenograft models may require less time to form tumors , from a few weeks to a few months , depending on the cell line and the number of injected tumor cells . tumor cells can be genetically manipulated in culture , and then implanted in mice ( fig . 2 ) . this allows for the study of how individual genes impact tumor development or metastasis . however , by using xenografts in immunocom - promised mice to study oc , the response of the immune system is ignored.19 a thematically related model of oc is patient - derived xenografts ( pdxs ) . to derive pdxs , ovarian tumors are isolated from patients , and then surgically grafted or injected into mice . tumors from diverse genetic backgrounds can be grafted , and the tumor growth can be monitored . furthermore , the pdxs recapitulate aspects of oc like metastasis and ascites formation . typically , small fragments of a tumor are isolated and injected sc or orthotopically into immune compromised mice . tumor engraftment percentages and time to develop tumors vary widely,19 and some xenograft studies result in less than 50% of the mice developing tumors.20 after the tumor reaches a critical size , it can be excised and reimplanted serially into subsequent mice . the original tumor and the serial pdx tumors are characterized for gene expression and mutation profile to assess if the pdxs retain the genetics and behaviors of the original tumor . pdx models are used to examine tumor progression and metastasis when the pdxs are implanted into the peritoneal cavity . in one model , human oc biopsies were dissociated and injected in nsg mice.21 the tumor aggregates that were injected contained tumor - associated cells , including lymphocytes and fibroblasts.21 these tumors metastasized to the omentum , ovary , pancreas , uterus , spleen , and liver . twenty - six percent of mice were found to have lung micrometastases as commonly found in stage iv disease.21 furthermore , a large study of 241 tumors demonstrated that the pdxs retain the genetics and metastatic spreading pattern of the original tumors.22 minced tumors were injected ip into female scid mice , and 74% ( 168 ) of these tumors engrafted , mostly in the pelvis.22 almost 50% of the mice developed metastases in the intestines , mesentery , liver , spleen , diaphragm , and/or omentum , and 17 of these mice developed ascites.22 one intriguing study found that pdxs with ascites or plural effusions were more likely to generate xenograft tumors than solid tumors.23 this raises questions about the metastatic unit in oc . oc stem cells that have been isolated from serous adenocarcinomas based on surface expression of cd177 and cd44 were demonstrated to have increased tumorigenic capacity in xenografts.24 a more recent study used a pdx model of oc to demonstrate that high expression type i receptor tyrosine kinase - like orphan receptor in oc stem cells results in higher tumorigenesis.21,25 more studies on the link between cancer stem cells and metastasis should be conducted using models like the pdxs to discover novel strategies to limit metastatic spread and appropriately target cells with metastatic potential . one of the major advantages of the pdx models is the ability to investigate the response of tumors to therapeutic agents . treating pdx tumors with il-12 , flt-3 , and cd40 decreased tumor growth.2628 similarly , pdx tumors have been studied for their ability to predict patient response to drug therapy ( previously reviewed by scott et al).19 in one study , hgs tumors were engrafted into ngs mice with an 83% success rate.29 after treatment with cisplatin , it was found that 40% of the mice were platinum sensitive , 30% were refractory , and 30% were platinum resistant.29 these responses correlated with patient outcome,29 which suggests that pdxs retain the genetic / epigenetic program of the original tumor and that the response to standard chemotherapy present in the original tumor is retained . additionally , access to patient samples is an impediment to using pdxs as a model system . murine ovarian surface epithelial cells from c57bl/6 mice were transformed ( id8 cells ) , and then injected back into c57bl/6 mice so that tumor metastasis or growth can be monitored using mice with intact immune systems.30 more recent syngeneic mouse models use cells with spontaneously generated increased aggressiveness to study resistance to anoikis.31 the tumors generated in this syngeneic model develop extensive metastasis in the peritoneal cavity reminiscent of advanced oc . mccloskey et al also described a strategy for creating spontaneously transformed ovarian surface epithelial cell lines by maintaining cells for many passages . these transformed cells exhibited aberrant wnt and nfkb signaling and resulted in tumors resembling hgs cancer when injected into the ip cavity of fvb / n mice.32 the syngeneic experimental design more accurately reflects the response of the immune system during tumor progression than immunocompromised models . however , the cells are of murine origin , and thus may not resemble human cancer as well as models using human cells . genetically engineered mouse models ( gemms ) have yielded many important advances in understanding cancer . mouse models of leukemia , breast cancer , and colon cancer can faithfully recapitulate many aspects of human disease . the oc genetic models have been more complicated to generate , partially because of the lack of understanding of oc biology and the heterogeneity of oc . the controversy about the cell type of origin for oc and identification of transgenic promoters that reflect the proper cell type of origin are further complications . gemms are especially important for studying the beginning stages of oc that can not be mimicked in xenografts . some ocs may originate in the fallopian tube , while others may arise from ovarian epithelium.5 different gemms allow us to evaluate how tumors arising from mutations at different anatomic locations compare to human disease . thus , gemms provide an invaluable tool for studying the development of oc with mutations observed in patients . for example , women with familial brca1 mutations have increased the risk of developing oc and develop cancer at a younger age than those with sporadic disease.33 thus , mouse models with alterations to brca1 and other genes commonly mutated in oc , such as tp53 , c - myc , kras , akt , and brca2,3436 are important to increasing our understanding regarding how genetic predisposition leads to oc ( summarized in table 1 ) . the most common mutation in hgs oc occurs in tp53,37 which is mutated in about 48% of sporadic ocs and 96% of hgs cancers.37,38 in addition , rb1 is dysregulated in about 67% of hgs ocs.38 gemms can be used to study these tumor suppressors as well as oncogenes such as c - myc , kras , and akt1 . an early method for inhibiting both of tp53 and rb1 is to express simian virus 40 ( sv40 ) large t antigen that binds to and inactivates both p53 and prb.39 transgenic mice expressing sv40 under control of the promoter for mullerian inhibiting substance type ii receptor ( misiir ) develop poorly differentiated ovarian carcinomas.40 misiir is expressed in 92% of oc , but most nongynecological tissues do not express this receptor.41 these carcinomas express cyto - keratins 8 and 19 and lack alpha - inhibin , suggesting that they are derived from epithelium and not derived from granulosa cells.40 these tumors result in ascites formation and metastatic spread to omentum.40 one mouse demonstrated metastasis to the kidney and one had metastasis to the uterus , but metastasis to other organs was not observed.40 this was the first published model of a transgenic model of epithelial oc . a major disadvantage to this approach is that the mice were infertile because of the ovarian tumors . therefore , other approaches to inactivate tumor suppressors that may be more indicative of human cancer needed to be developed . conditional knockouts of tp53 and rb1 have also been used to generate mouse models of oc . as reported by flesken - nikitin , mice with tp53 and rb1 flanked by loxp sites ( floxed ) were injected into the bursa ovary with an adenovirus expressing cre recombinase under control of a cmv promoter.34 this double knockout resulted in a metastatic oc resembling human ovarian epithelial cancer , which spread to the lung , liver , and noninjected ovary.34 a total of 97% of the mice with knockout of tp53 and rb1 in the ovary died within eight months.34 single knockouts of either tp53 or rb1 were much less efficient at inducing metastatic cancer : 13% of tp53 knockout mice and 3% of rb1 knockout mice developed tumors.34 importantly , these studies reveal that tp53 mutations alone are sufficient for ovarian tumor initiation , but mutation in both tp53 and rb1 lead to metastatic oc.34 in a more recent study , combinations of tp53 mutation or loss , loss of pocket protein function , and loss of brca1/2 were generated to thoroughly examine the contributions of these genes to oc . expression of t121 ( a domain of the sv40 large t antigen ) in the surface epithelium is induced by injecting cre - expressing adenovirus ( adeno - cre ) . t121 is in a cre - conditional loxp - gfp - stop - loxp ( lsl ) cassette driven by the cytokeratin 18 promoter . injection of adeno - cre resulted in inhibition of rb1 , p107 , and p130 , also known as the pocket proteins . in this study , antigen t121 is used in concert with floxed alleles for tp53 , a point mutation in tp53 ( p53 ) , and inactivation of either brca1 or brca2.35 inactivation of the pocket proteins was sufficient for tumorigenesis , resulting in stage i serous epithelial oc.35 however , loss of prb , p53 , and brca1 or brca2 in mice more closely mimics serous oc.35 when brca1 or brca2 were deleted in this model ( in addition to inhibition of tp53 and the rb pathway ) , the tumors metastasized to the liver and demonstrated a gene expression profile resembling serous oc.35 a later version of this gemm was used to study drug response in brca1-deficient mice , showing that tumors in these mice respond to a combination chemotherapy of cisplatin and olaparib.42 this provides an example of how gemms can be used to test effective therapies . through these varying combinations of genetic alterations , different stages of oc progression were modeled , from early stage i epithelial oc ( rb pathway disruption alone ) to an advanced metastatic cancer ( rb loss / tp53/brca1/brca2 alterations ) . in addition to disrupting tumor suppressor genes , introduction of oncogenes has also been employed to generate mouse models of oc . transgenic mice expressing the avian retroviral receptor tva ( with a - actin or keratin 5 promoter ) were generated.36 these mice were then crossed with p53 null mice.36 ovarian surface epithelial cells were isolated from the mice and transduced in vitro with replication - competent avian - leukemia - virus splice - acceptor ( rcas)43 viral vectors . these viral vectors expressed common oc oncogenes : c - myc , kras , or akt1 . subcutaneous tumor formation resulted when any two of these oncogenes were expressed in ovarian cells that were injected sc into nude mice.36 ib injection of the -actin - tva / p53 cells with the rcas viruses containing c - myc or kras oncogenes resulted in metastastic oc.36 metastasis to the liver , spleen , intestine , kidney , and malignant ascites was observed.36 this result mirrors that of later mouse models from the same laboratory , showing that tva / rcas - induced expression of c - myc is sufficient to induce transformation in ovarian cells from mice with floxed tp53 and brca1.44 these transformed cells displayed chromosomal defects and an unusually high centromere count ; injection of these modified cells into the ovary resulted in metastatic tumors that resemble serous ovarian carcinomas and spread to the peritoneum , pancreas , and intestines.44 collectively , these models demonstrate that tp53 mutation in concert with oncogene expression or loss of other tumor suppressors results in oc generation and metastasis . as our understanding of the genetics and epigenetics of oc evolves , so must the ways of modeling the disease . it has been hypothesized that different cell types of origin and different genetic mutations are responsible for the distinct histological subtypes of oc . therefore , establishing models of fallopian tube and uterine - derived oc is essential . to determine if high - grade serous ovarian cancer ( hgsoc ) can originate from fallopian tube , pax8-driven cre recombinase was used to excise brca1 , brca2 , tp53 , and/or pten.45 loss of brca1/2 , tp53 mutation , and pten loss resulted in fallopian tube serous tubal intraepithelial carcinomas ( stics ) , ovarian metastases , and peritoneal metastases.45 the histology , metastatic profile , and transcriptome of these tumors closely mimics hgs oc.45 another method to transform the fallopian tube used sv40 large t antigen expressed under control of the mullerian ovgp-1 promoter.46 this model results in stics and invasive adenocarcinomas that spread to the ovaries.46 therefore , this model is capable of recapitulating early development of oc46 in the fallopian tube . one additional advantage of this model is that the ovgp-1 promoter ensures tissue - specific expression of sv40 in mice that have reached sexual maturity . since the ovary , uterus , and fallopian tube may be sites of origin for different types of oc , a comparison of transformation at each of these locations was conducted . to compare tumorigenesis in the ovary , oviduct , and endometrium using the same genetic backgrounds , a triple transgenic mouse model was created.47 in these mice , kras ( using a lsl cassette ) is induced , and floxed pten is deleted using adeno - cre in different locations of the female reproductive tract.48 additionally , these transgenic mice also express the human glycoprotein muc1 that is often overexpressed in oc.48 injection of adeno - cre into the ovary , oviduct , or uterus resulted in the formation of epithelial tumors capable of metastasis , spreading to the pancreas and diaphragm.47 however , the nuclear grade of the tumors and survival of the mice varied depending on the anatomical site of injection.47 tumors arising from ovaries , the oviduct , and uterus generated endometrioid histology and metastasis to spleen , diaphragm , and liver were detected.47 the oviduct tumors developed into poorly differentiated high - grade tumors , while ovarian and uterine tumors were more differentiated.47 mice with ovarian and oviduct tumors exhibited the shortest mean survival times compared to mice with uterine tumors ( 89 , 82 , and 132.5 days , respectively ) . a key feature of this model is that overexpression of muc1 results in a similar immune response as that seen in human patients.47 this enables testing of immunotherapy treatments that target oc cells over - expressing muc1.47 while many of the recent gemms are aimed at understanding the origin of oc or recapitulating hgsoc , mouse models of other oc histotypes have also emerged . ovarian endometrioid adenocarcinoma is a subtype of oc originating from malignant uterine cells created during endometriosis.6 some models of endometrioid oc resulted from oncogenic kras and a conditional pten deletion.47,49 however , kras mutations are uncommon in human endometrioid cancer . to address this issue , a mouse model of endometrioid oc was created in which apc and pten are deleted , since perturbations in wnt and phosphoinositide 3-kinase ( pi3k ) signaling are linked in some endometrioid oc.50 mice with floxed apc and pten were ib injected with adeno - cre.50 resultant adenocarcinomas were similar to metastatic human ovarian endome - trioid adenocarcinoma and50 were generally low grade , unlike those resulting from tp53 mutations.50 the tumors resulting from pten / apc knockout showed increased nuclear -catenin and overactive akt signaling.50 these gemms have been used to test therapeutics against endometrioid cancer ; a combination of cisplatin and paclitaxel together with inhibitors of mtor and akt signaling has proven effective in mouse models.51 this work highlights the possibility of using treatments that target the wnt and pi3k pathways to combat endometrioid cancer . the survival rates and treatment for oc have remained relatively unchanged for the past two decades . recent advances in understanding the genetic landscape of the histological subtypes of oc generate hope that targeted therapies can be devised that will increase patient survival . in order to see these targeted therapies come into practice , preclinical mouse models that reflect the genetics , metastatic potential , and different responses to therapy need to be used . in the last 30 years , mouse models have progressed from early xenografts to xenografts with immunocompetant mice , and pdxs that reflect individual patient s genetics and therapeutic response . moreover , genetic models of cancer have increased our understanding of how oc originates and what mutations or combinations of mutations may lead to tumor establishment . a recent mouse model with mutations in c - kit further allows the study of ovarian tumors in the context of a postmenopausal environment , potentially allowing for the above - discussed genetic mutations to be further improved by incorporating hormone responsiveness seen in the older population most likely to be affected by oc.52 now that we have a variety of mouse models ( summarized in table 2 ) ( as well as other animal models , such as the laying hen)53 at our disposal that reflect a wide range of steps of oc progression , we can develop diagnostic , prognostic , and therapeutic approaches that will increase oc survival .

ovarian cancer ( oc ) is the leading cause of death from a gynecological malignancy in the united states . by the time a woman is diagnosed with oc , the tumor has usually metastasized . mouse models that are used to recapitulate different aspects of human oc have been evolving for nearly 40 years . xenograft studies in immunocompromised and immunocompetent mice have enhanced our knowledge of metastasis and immune cell involvement in cancer . patient - derived xenografts ( pdxs ) can accurately reflect metastasis , response to therapy , and diverse genetics found in patients . additionally , multiple genetically engineered mouse models have increased our understanding of possible tissues of origin for oc and what role individual mutations play in establishing ovarian tumors . many of these models are used to test novel therapeutics . as no single model perfectly copies the human disease , we can use a variety of oc animal models in hypothesis testing that will lead to novel treatment options . the goal of this review is to provide an overview of the utility of different mouse models in the study of oc and their suitability for cancer research .

Introduction Xenograft Models of OC Genetically Engineered Mouse Models of OC Summary

every year in the united states , over 22,000 women are diagnosed with ovarian cancer ( oc ) and more than 15,000 die from the disease.1 the poor prognosis for women diagnosed with oc is largely because of the presence of local and distant metastases in 81% of patients at diagnosis , leading to 5-year survival rates of 69% and 30% , respectively.1 these numbers suggest that both early detection and treatment of metastatic disease are crucial in managing oc . they are high - grade serous ( hgs ) and low - grade serous ovarian , mucinous , endometrioid , clear cell , and transitional or undifferentiated.2 hgs is the most common form of oc . oc invasion is a complex process that differs from metastatic programs of other solid tumors , since oc tumor cells shed from the primary site and spread throughout the peritoneal cavity without first entering the blood or lymphatic system.3 oc was originally thought to be derived from the ovarian surface epithelium.4 however , epidemiological , pathological , and molecular studies have made scientists question the origin of oc and hypothesize that different types of ovarian tumors may arise from different precursor cells , including those from the fallopian tube , endometrium , cervix , and gastrointestinal ( gi ) tract.2,57 in addition to the oc subtypes being histologically different , they are also genetically distinct . ( 2 ) type 2 tumors arise rapidly de novo and contain tp53 mutations ; these include hgs , undifferentiated , and carcinosarcomas.8 recent work suggests that some hgs tumors may be initiated in the fimbria of the fallopian tube.8,9 identifying the cell type of origin for oc will change how animal models are generated and evaluated . preferred sites of metastasis include the omentum , peritoneal wall , the diaphragm , and small bowel mesentery.3 therefore , design and utilization of mouse models of oc that recapitulate the steps of metastasis are critical . here , we will review many of the common ways oc is studied in mice . xenograft models are a common way of investigating oc growth , metastasis , and treatment response in a live animal . in these early studies , the thymus of the mice was removed , mice were treated with cytosine arabinoside , given whole - body irradiation , and then engrafted subcutaneously ( sc ) with either ovarian ascites ( n = 5 ) or a solid peritoneal metastasis from a papillary cystadenocarcinoma ovarian tumor biopsy ( n = 8).12 four of the peritoneal metastases established tumor growth.12 xenograft studies have been used extensively since the 1980s and offer many advantages . , many xenograft mouse models are conducted using immunocompromised mice , which will lack the contribution of the full complement of immune cells to the tumor growth process . to study tumor growth in mice , murine or human oc cells cells from different genetic backgrounds are injected into immunocompromised mice such as nude ( foxn1 , nu / nu ) , scid , nod / scid , or nod - scid il2rcnull ( nsg ) to enable the cells to engraft without being eliminated by the immune system . the sc model is not well suited for ovarian metastasis studies as the tumors do not typically metastasize , and the tumor is not positioned in the right anatomic location or microenvironment . while ip injection can not mimic the initial steps in metastasis , ip - injected tumor cells such as skov3 metastasize to the ovary , peritoneal wall , diaphragm , and form ascites fluid similar to human disease.13 the skov3ip cell line is derived from ascites cells that developed in a mouse ip cavity injected with skov3 cells.13 compared to the parental skov3 cells , the skov3ip cells grow faster , disseminate more , and exhibit overexpressed erbb2.13 many cells ( including the skov3 lines ) are frequently transfected or transduced with fluorescence or luminescence - expressing vectors to monitor tumor growth in vivo.14 similarly , ovcar3 cells metastasize to the gi tract , omentum , pancreas , kidney , and liver ( unpublished data ) when injected ip . in some ways , the ib injection mimics the initial steps in metastasis , as the tumor cells exit the bursa to spread throughout the peritoneal cavity . many cell lines metastasize following ib injection , including a2780 , skov3 , and hey cells.15,16 sites of metastasis include diaphragm , mesentery , bowel , and liver.16 in particular , the a2780 cells have been used to study not only metastasis but also chemoresistance . a2780 that are sensitive to cisplatin and a subline of a2780 cells that are resistant to cisplatin cells are also commonly used in xenograft studies of chemoresistance in vivo.17 many other cell lines not discussed also demonstrate growth in xenograft models.18 compared to genetic models , xenograft models may require less time to form tumors , from a few weeks to a few months , depending on the cell line and the number of injected tumor cells . however , by using xenografts in immunocom - promised mice to study oc , the response of the immune system is ignored.19 a thematically related model of oc is patient - derived xenografts ( pdxs ) . furthermore , the pdxs recapitulate aspects of oc like metastasis and ascites formation . tumor engraftment percentages and time to develop tumors vary widely,19 and some xenograft studies result in less than 50% of the mice developing tumors.20 after the tumor reaches a critical size , it can be excised and reimplanted serially into subsequent mice . pdx models are used to examine tumor progression and metastasis when the pdxs are implanted into the peritoneal cavity . in one model , human oc biopsies were dissociated and injected in nsg mice.21 the tumor aggregates that were injected contained tumor - associated cells , including lymphocytes and fibroblasts.21 these tumors metastasized to the omentum , ovary , pancreas , uterus , spleen , and liver . twenty - six percent of mice were found to have lung micrometastases as commonly found in stage iv disease.21 furthermore , a large study of 241 tumors demonstrated that the pdxs retain the genetics and metastatic spreading pattern of the original tumors.22 minced tumors were injected ip into female scid mice , and 74% ( 168 ) of these tumors engrafted , mostly in the pelvis.22 almost 50% of the mice developed metastases in the intestines , mesentery , liver , spleen , diaphragm , and/or omentum , and 17 of these mice developed ascites.22 one intriguing study found that pdxs with ascites or plural effusions were more likely to generate xenograft tumors than solid tumors.23 this raises questions about the metastatic unit in oc . oc stem cells that have been isolated from serous adenocarcinomas based on surface expression of cd177 and cd44 were demonstrated to have increased tumorigenic capacity in xenografts.24 a more recent study used a pdx model of oc to demonstrate that high expression type i receptor tyrosine kinase - like orphan receptor in oc stem cells results in higher tumorigenesis.21,25 more studies on the link between cancer stem cells and metastasis should be conducted using models like the pdxs to discover novel strategies to limit metastatic spread and appropriately target cells with metastatic potential . treating pdx tumors with il-12 , flt-3 , and cd40 decreased tumor growth.2628 similarly , pdx tumors have been studied for their ability to predict patient response to drug therapy ( previously reviewed by scott et al).19 in one study , hgs tumors were engrafted into ngs mice with an 83% success rate.29 after treatment with cisplatin , it was found that 40% of the mice were platinum sensitive , 30% were refractory , and 30% were platinum resistant.29 these responses correlated with patient outcome,29 which suggests that pdxs retain the genetic / epigenetic program of the original tumor and that the response to standard chemotherapy present in the original tumor is retained . murine ovarian surface epithelial cells from c57bl/6 mice were transformed ( id8 cells ) , and then injected back into c57bl/6 mice so that tumor metastasis or growth can be monitored using mice with intact immune systems.30 more recent syngeneic mouse models use cells with spontaneously generated increased aggressiveness to study resistance to anoikis.31 the tumors generated in this syngeneic model develop extensive metastasis in the peritoneal cavity reminiscent of advanced oc . genetically engineered mouse models ( gemms ) have yielded many important advances in understanding cancer . mouse models of leukemia , breast cancer , and colon cancer can faithfully recapitulate many aspects of human disease . the oc genetic models have been more complicated to generate , partially because of the lack of understanding of oc biology and the heterogeneity of oc . the controversy about the cell type of origin for oc and identification of transgenic promoters that reflect the proper cell type of origin are further complications . thus , gemms provide an invaluable tool for studying the development of oc with mutations observed in patients . for example , women with familial brca1 mutations have increased the risk of developing oc and develop cancer at a younger age than those with sporadic disease.33 thus , mouse models with alterations to brca1 and other genes commonly mutated in oc , such as tp53 , c - myc , kras , akt , and brca2,3436 are important to increasing our understanding regarding how genetic predisposition leads to oc ( summarized in table 1 ) . an early method for inhibiting both of tp53 and rb1 is to express simian virus 40 ( sv40 ) large t antigen that binds to and inactivates both p53 and prb.39 transgenic mice expressing sv40 under control of the promoter for mullerian inhibiting substance type ii receptor ( misiir ) develop poorly differentiated ovarian carcinomas.40 misiir is expressed in 92% of oc , but most nongynecological tissues do not express this receptor.41 these carcinomas express cyto - keratins 8 and 19 and lack alpha - inhibin , suggesting that they are derived from epithelium and not derived from granulosa cells.40 these tumors result in ascites formation and metastatic spread to omentum.40 one mouse demonstrated metastasis to the kidney and one had metastasis to the uterus , but metastasis to other organs was not observed.40 this was the first published model of a transgenic model of epithelial oc . conditional knockouts of tp53 and rb1 have also been used to generate mouse models of oc . as reported by flesken - nikitin , mice with tp53 and rb1 flanked by loxp sites ( floxed ) were injected into the bursa ovary with an adenovirus expressing cre recombinase under control of a cmv promoter.34 this double knockout resulted in a metastatic oc resembling human ovarian epithelial cancer , which spread to the lung , liver , and noninjected ovary.34 a total of 97% of the mice with knockout of tp53 and rb1 in the ovary died within eight months.34 single knockouts of either tp53 or rb1 were much less efficient at inducing metastatic cancer : 13% of tp53 knockout mice and 3% of rb1 knockout mice developed tumors.34 importantly , these studies reveal that tp53 mutations alone are sufficient for ovarian tumor initiation , but mutation in both tp53 and rb1 lead to metastatic oc.34 in a more recent study , combinations of tp53 mutation or loss , loss of pocket protein function , and loss of brca1/2 were generated to thoroughly examine the contributions of these genes to oc . in this study , antigen t121 is used in concert with floxed alleles for tp53 , a point mutation in tp53 ( p53 ) , and inactivation of either brca1 or brca2.35 inactivation of the pocket proteins was sufficient for tumorigenesis , resulting in stage i serous epithelial oc.35 however , loss of prb , p53 , and brca1 or brca2 in mice more closely mimics serous oc.35 when brca1 or brca2 were deleted in this model ( in addition to inhibition of tp53 and the rb pathway ) , the tumors metastasized to the liver and demonstrated a gene expression profile resembling serous oc.35 a later version of this gemm was used to study drug response in brca1-deficient mice , showing that tumors in these mice respond to a combination chemotherapy of cisplatin and olaparib.42 this provides an example of how gemms can be used to test effective therapies . subcutaneous tumor formation resulted when any two of these oncogenes were expressed in ovarian cells that were injected sc into nude mice.36 ib injection of the -actin - tva / p53 cells with the rcas viruses containing c - myc or kras oncogenes resulted in metastastic oc.36 metastasis to the liver , spleen , intestine , kidney , and malignant ascites was observed.36 this result mirrors that of later mouse models from the same laboratory , showing that tva / rcas - induced expression of c - myc is sufficient to induce transformation in ovarian cells from mice with floxed tp53 and brca1.44 these transformed cells displayed chromosomal defects and an unusually high centromere count ; injection of these modified cells into the ovary resulted in metastatic tumors that resemble serous ovarian carcinomas and spread to the peritoneum , pancreas , and intestines.44 collectively , these models demonstrate that tp53 mutation in concert with oncogene expression or loss of other tumor suppressors results in oc generation and metastasis . as our understanding of the genetics and epigenetics of oc evolves , so must the ways of modeling the disease . to determine if high - grade serous ovarian cancer ( hgsoc ) can originate from fallopian tube , pax8-driven cre recombinase was used to excise brca1 , brca2 , tp53 , and/or pten.45 loss of brca1/2 , tp53 mutation , and pten loss resulted in fallopian tube serous tubal intraepithelial carcinomas ( stics ) , ovarian metastases , and peritoneal metastases.45 the histology , metastatic profile , and transcriptome of these tumors closely mimics hgs oc.45 another method to transform the fallopian tube used sv40 large t antigen expressed under control of the mullerian ovgp-1 promoter.46 this model results in stics and invasive adenocarcinomas that spread to the ovaries.46 therefore , this model is capable of recapitulating early development of oc46 in the fallopian tube . since the ovary , uterus , and fallopian tube may be sites of origin for different types of oc , a comparison of transformation at each of these locations was conducted . to compare tumorigenesis in the ovary , oviduct , and endometrium using the same genetic backgrounds , a triple transgenic mouse model was created.47 in these mice , kras ( using a lsl cassette ) is induced , and floxed pten is deleted using adeno - cre in different locations of the female reproductive tract.48 additionally , these transgenic mice also express the human glycoprotein muc1 that is often overexpressed in oc.48 injection of adeno - cre into the ovary , oviduct , or uterus resulted in the formation of epithelial tumors capable of metastasis , spreading to the pancreas and diaphragm.47 however , the nuclear grade of the tumors and survival of the mice varied depending on the anatomical site of injection.47 tumors arising from ovaries , the oviduct , and uterus generated endometrioid histology and metastasis to spleen , diaphragm , and liver were detected.47 the oviduct tumors developed into poorly differentiated high - grade tumors , while ovarian and uterine tumors were more differentiated.47 mice with ovarian and oviduct tumors exhibited the shortest mean survival times compared to mice with uterine tumors ( 89 , 82 , and 132.5 days , respectively ) . a key feature of this model is that overexpression of muc1 results in a similar immune response as that seen in human patients.47 this enables testing of immunotherapy treatments that target oc cells over - expressing muc1.47 while many of the recent gemms are aimed at understanding the origin of oc or recapitulating hgsoc , mouse models of other oc histotypes have also emerged . to address this issue , a mouse model of endometrioid oc was created in which apc and pten are deleted , since perturbations in wnt and phosphoinositide 3-kinase ( pi3k ) signaling are linked in some endometrioid oc.50 mice with floxed apc and pten were ib injected with adeno - cre.50 resultant adenocarcinomas were similar to metastatic human ovarian endome - trioid adenocarcinoma and50 were generally low grade , unlike those resulting from tp53 mutations.50 the tumors resulting from pten / apc knockout showed increased nuclear -catenin and overactive akt signaling.50 these gemms have been used to test therapeutics against endometrioid cancer ; a combination of cisplatin and paclitaxel together with inhibitors of mtor and akt signaling has proven effective in mouse models.51 this work highlights the possibility of using treatments that target the wnt and pi3k pathways to combat endometrioid cancer . recent advances in understanding the genetic landscape of the histological subtypes of oc generate hope that targeted therapies can be devised that will increase patient survival . in order to see these targeted therapies come into practice , preclinical mouse models that reflect the genetics , metastatic potential , and different responses to therapy need to be used . in the last 30 years , mouse models have progressed from early xenografts to xenografts with immunocompetant mice , and pdxs that reflect individual patient s genetics and therapeutic response . moreover , genetic models of cancer have increased our understanding of how oc originates and what mutations or combinations of mutations may lead to tumor establishment . a recent mouse model with mutations in c - kit further allows the study of ovarian tumors in the context of a postmenopausal environment , potentially allowing for the above - discussed genetic mutations to be further improved by incorporating hormone responsiveness seen in the older population most likely to be affected by oc.52 now that we have a variety of mouse models ( summarized in table 2 ) ( as well as other animal models , such as the laying hen)53 at our disposal that reflect a wide range of steps of oc progression , we can develop diagnostic , prognostic , and therapeutic approaches that will increase oc survival .

existing theories of public involvement embody certain assumptions and hypotheses about what public involvement interventions are made of ( their principal components ) and how they are expected to work ( their internal dynamic explaining outcomes).3640 three main theoretical constructs have been proposed to explain the public 's influence on collective health care decisions : ( 1 ) public members credibility and ability to contribute knowledge that is considered valid and relevant to inform collective health care decisions , ( 2 ) their legitimacy to speak on behalf of people affected directly or indirectly by health care services and policies , and ( 3 ) their power and ability to influence collective health care choices.21 technocratic theories of public involvement stress the importance of the public 's ability to demonstrate credible expertise . rowe and frewer contend that information sharing is the core process underlying public involvement and classify interventions according to the flow of information between professionals and the public : communication methods ( in which information is communicated to the public ) , consultation ( information is collected from the public ) , and participation ( information is exchanged between professionals and the public).41 such an information flow perspective is particularly well developed in deliberative theories , which anchor public involvement around the careful weighing of reasons for and against collective action propositions.42 the core hypothesis of deliberative theories is that the exchange of reasonable and credible arguments should result in mutual learning and in the generation of solutions that can be rationally justified to those affected by it.4346 from this perspective , access to the best available evidence through training and preparation is a critical component of effective public involvement interventions , ensuring that valid and relevant information is collected and exchanged among competent participants.35,41 collins and evans contend that members of the general public are experience - based experts whose knowledge is based mainly on their personal experiences rather than on their degrees and professional qualifications.47 these authors further distinguish among 3 levels of public expertise . at the most basic level , public members have no expertise at all in a specific domain and are unable to understand professionals . at the highest level , some public members have developed contributory expertise in a specific domain , meaning that they can contribute new knowledge to a specific aspect of collective health care decisions ( eg , as a chronic disease patient with long - term experience of a given health condition ) . at the intermediate level of interactional expertise , public members have developed knowledge that allows them to interact meaningfully with professionals . interactional expertise is assumed to be particularly important when professionals and the public deliberate , enabling both parties to understand how their respective expertise can contribute to collective health care decisions . this distinction between interactional and contributory expertise challenges sponsors of public involvement to clarify what specific knowledge and experience public members are expected to contribute to collective decisions and what forms of expertise professionals and the public must have in order to interact meaningfully with each other . democratic theories emphasize the importance of the public 's legitimacy to speak on behalf of others ; indeed , it is seen as a critical component of effective public involvement.27,45,4851 judging the public members legitimacy requires paying close attention to the social practice of public involvement and the political and organizational context in which divergent notions of representativeness are deployed in pursuit of differing roles for public participation.48 observations of real - life involvement interventions suggest that professionals use different criteria to support ( or question ) the public 's legitimacy , such as the participants statistical representativeness , the use of formal public representatives selection and nomination procedures , and the participants accountability to the community members they represent.48,49,51 finally , a number of authors have highlighted the importance of power as a core ingredient for explaining the public 's relative influence on collective decision making . power may be exercised over who is allowed or not allowed to participate in collective decision making , as well as what kinds of arguments and propositions are regarded as receivable or not.52 deliberation theory emphasizes the importance of balancing the participants power differences in order to agree on the best rational arguments , open to all competent speakers and free of coercion or manipulation.44,53 but observations of actual deliberation processes suggest that these egalitarian ideals may not always operate in practice . for example , research on small - group decision making suggests that collective decisions often tend to move toward the majority 's view.53,54 in the context of collective health care improvement and policy decisions , power may also be determined by professional and hierarchical status.27,48 in its more subtle and discursive forms , power may be exerted through the types of assumptions that are taken for granted and accepted by the group.45 finally , power struggles can be woven into the fabric of a public involvement intervention itself , shaping the selected method of involvement , who is asked to participate , and how its sponsors define effectiveness.55 while power is closely connected to and contingent on the public 's credibility and legitimacy , it also points to other important components of public involvement interventions , including control over agenda and procedures , and decisions about how public recommendations will be incorporated in actual policy decisions.56 in summary , the theoretical literature points to the need to look closely at how public involvement interventions frame and foster public members ' legitimacy , credibility , and power , in order to explain their ability to influence collective decisions . the literature suggests that process attributes of involvement interventions ( eg , their principal components and internal dynamics ) are likely to affect outcomes differentially . this requires clarifying for whom public representatives are speaking and the specific expertise they are expected to contribute to collective decision making . by looking at what happens when members of the public interact with professionals , and by mobilizing a theory - informed understanding of the key processes at play , this article seeks to understand the involvement process informing the development of more effective interventions . we conducted a qualitative process evaluation of a public involvement trial ( see box 1 ) . details of the development and testing of the public involvement intervention have been published elsewhere.33,57 in a nutshell , we randomized health and social services centers in a canadian health region in intervention and control sites . individual participants included members of the public ( chronic disease patients , caregivers , and healthy adults ) and professionals ( clinicians and managers ) . we asked the participants to select health care improvement priorities from a list of 37 validated quality indicators for chronic disease prevention and management in primary care . in the intervention sites , the public members ( 1 ) attended a 1-day preparation meeting , ( 2 ) were consulted by vote on their local priorities for improvement , and ( 3 ) participated in a 2-day meeting to deliberate with professionals and reach agreement with them on local health care improvement priorities . the 2-day meeting between public members and professionals included small - group deliberation , feedback of the public consultation results , and voting . in the control sites , the professionals among themselves prioritized quality indicators without public involvement ( no participation of public members or feedback about the public consultation ) . design : cluster randomized trial.context : health and social services centers in a canadian region ( n = 6 ) were required to select health care improvement priorities to be incorporated in their financial accountability contract with the regional health authority . health and social services centers could choose from a list of 37 validated quality indicators for chronic disease prevention and management in primary care ( including indicators on access , integration of services , technical quality of care , quality of interpersonal relationships , and health outcomes).baseline public priorities : public members ( n = 83 ) , including chronic disease patients , caregivers , and healthy adults , were recruited from all participating sites . all public members participated in a 1-day preparation meeting and were consulted by vote on their local priorities for improvement.randomization : health and social services centers were randomized in intervention ( n = 3 ) and control sites ( n = 3 ) to compare health care improvement priority setting with and without public involvement.intervention : health care improvement priorities were selected by both professionals and public members . professionals received feedback about the public consultation and deliberated with public representatives in a face - to - face meeting.control : health care improvement priorities were selected by professionals only , without public involvement . professionals did not receive feedback about the public consultation and deliberated among themselves , without public representatives.participants : in all , 172 individuals from 6 health and social services centers participated in the study . public representatives ( n = 83 ) included chronic disease patients ( 81% ) , caregivers , and healthy adults . forty - seven percent had a primary or high - school education ; 56% had an annual household income of less than us$40,000 ; and 23% had been hospitalized during the previous year . professionals ( n = 89 ) included managers ( 35% ) , physicians ( 13% ) , nurses ( 24% ) , and allied health professionals ( 28%).results : priorities selected in intervention sites placed more emphasis on access to primary care , self - care support , patients participation in clinical decisions , and partnership with community organizations ( p < 0.01).agreement between public representatives ' and professionals improvement priorities increased by 41% in favor of intervention sites ( 95% ci + 12 , + 58% , p < 0.01 ) . professionals choices moved toward indicators prioritized by the public ( eg , access ) , and public representatives choices moved toward indicators prioritized by professionals ( eg , self - care support).participants ' perception of the quality of the deliberation process scored high in all domains ( quality of the information received , procedures and moderation , interaction among participants , and overall satisfaction).there was no significant difference in professionals intentions to use the selected quality indicators for health care improvement , which scored high in both intervention and control groups . context : health and social services centers in a canadian region ( n = 6 ) were required to select health care improvement priorities to be incorporated in their financial accountability contract with the regional health authority . health and social services centers could choose from a list of 37 validated quality indicators for chronic disease prevention and management in primary care ( including indicators on access , integration of services , technical quality of care , quality of interpersonal relationships , and health outcomes ) . baseline public priorities : public members ( n = 83 ) , including chronic disease patients , caregivers , and healthy adults , were recruited from all participating sites . all public members participated in a 1-day preparation meeting and were consulted by vote on their local priorities for improvement . randomization : health and social services centers were randomized in intervention ( n = 3 ) and control sites ( n = 3 ) to compare health care improvement priority setting with and without public involvement . intervention : health care improvement priorities were selected by both professionals and public members . professionals received feedback about the public consultation and deliberated with public representatives in a face - to - face meeting . control : health care improvement priorities were selected by professionals only , without public involvement . professionals did not receive feedback about the public consultation and deliberated among themselves , without public representatives . participants : in all , 172 individuals from 6 health and social services centers participated in the study . public representatives ( n = 83 ) included chronic disease patients ( 81% ) , caregivers , and healthy adults . forty - seven percent had a primary or high - school education ; 56% had an annual household income of less than us$40,000 ; and 23% had been hospitalized during the previous year . professionals ( n = 89 ) included managers ( 35% ) , physicians ( 13% ) , nurses ( 24% ) , and allied health professionals ( 28% ) . results : priorities selected in intervention sites placed more emphasis on access to primary care , self - care support , patients participation in clinical decisions , and partnership with community organizations ( p < 0.01).agreement between public representatives ' and professionals improvement priorities increased by 41% in favor of intervention sites ( 95% ci + 12 , + 58% , p < 0.01 ) . professionals choices moved toward indicators prioritized by the public ( eg , access ) , and public representatives choices moved toward indicators prioritized by professionals ( eg , self - care support).participants ' perception of the quality of the deliberation process scored high in all domains ( quality of the information received , procedures and moderation , interaction among participants , and overall satisfaction).there was no significant difference in professionals intentions to use the selected quality indicators for health care improvement , which scored high in both intervention and control groups . priorities selected in intervention sites placed more emphasis on access to primary care , self - care support , patients participation in clinical decisions , and partnership with community organizations ( p < 0.01 ) . agreement between public representatives ' and professionals improvement priorities increased by 41% in favor of intervention sites ( 95% ci + 12 , + 58% , p < 0.01 ) . professionals choices moved toward indicators prioritized by the public ( eg , access ) , and public representatives choices moved toward indicators prioritized by professionals ( eg , self - care support ) . participants ' perception of the quality of the deliberation process scored high in all domains ( quality of the information received , procedures and moderation , interaction among participants , and overall satisfaction ) . there was no significant difference in professionals intentions to use the selected quality indicators for health care improvement , which scored high in both intervention and control groups . at the end of the trial , the priorities established with public involvement were significantly different from those selected by the professionals alone . we observed variations in the effects of public involvement across intervention sites , topic areas , and after the introduction of different components of the intervention . for example , the public 's ability to influence the professionals was more pronounced for some dimensions of quality , like access to primary care ( see table 1 ) . the public 's influence also was greater when public participation in small - group deliberation was combined with feedback from public consultation ( see table 2 ) . changes in professionals priorities during the involvement intervention changes in professionals ' priorities during the involvement intervention , in relation to public members top 3 priorities ( rank 1 = priority selected by the highest proportion of participants ) . agreement between professionals and the public at different stages of the intervention agreement between public representatives and professionals priorities at different stages of the intervention , calculated using spearman 's correlation coefficients ( 0 = no agreement , 1 = perfect agreement ) . the trial 's process evaluation sought to elucidate these different results by drawing from the qualitative data ( direct observation and videorecording ) collected during the trial . davies and colleagues showed the value of combining videorecording and direct observations to explore both the content of the deliberations and the participants social interactions during a public involvement intervention.45 we videorecorded 14 one - day meetings , and 2 independent , nonparticipant observers used structured observation charts to describe the deliberation content , the types of arguments used , and the participants interactions . during all phases of the project , we took field notes to record informal interactions that were not captured on video . structured debriefing sessions with the observers and moderators were held immediately after each meeting , during which key interactive moments were flagged and all observations were linked to the videotranscript using time codes to allow later validation against the original videorecording . our primary empirical materials were our observations and field notes , as well as a full verbatim transcription of videorecording from a sample meeting in each intervention site . the key moments flagged from all our meetings also were transcribed from the original recording . that is , we tried to identify varying degrees of public influence among the different sites , over time , and across topic areas to identify the components of the interventions that could help explain these variations . given our applied policy research focus , we used framework analysis58 to chart all transcribed material and notes and to graphically map the main aspects of the public involvement process and their relationships to outcomes . the principal investigator ( antoine boivin ) attended all the meetings with a research assistant , transcribed the recordings , and conducted the initial analysis . the coprincipal investigator ( pascale lehoux ) attended some of the meetings and validated the initial analysis against the original transcript . our analysis was structured around the main public involvement intervention components , including ( 1 ) building a policy coalition supportive of public involvement , ( 2 ) public recruitment and training , ( 3 ) public participation in face - to - face deliberation with professionals , ( 4 ) group moderation , and ( 5 ) feedback from a wider public consultation . while some of these components were identified from our review of public involvement theory , others emerged inductively . design : cluster randomized trial.context : health and social services centers in a canadian region ( n = 6 ) were required to select health care improvement priorities to be incorporated in their financial accountability contract with the regional health authority . health and social services centers could choose from a list of 37 validated quality indicators for chronic disease prevention and management in primary care ( including indicators on access , integration of services , technical quality of care , quality of interpersonal relationships , and health outcomes).baseline public priorities : public members ( n = 83 ) , including chronic disease patients , caregivers , and healthy adults , were recruited from all participating sites . all public members participated in a 1-day preparation meeting and were consulted by vote on their local priorities for improvement.randomization : health and social services centers were randomized in intervention ( n = 3 ) and control sites ( n = 3 ) to compare health care improvement priority setting with and without public involvement.intervention : health care improvement priorities were selected by both professionals and public members . professionals received feedback about the public consultation and deliberated with public representatives in a face - to - face meeting.control : health care improvement priorities were selected by professionals only , without public involvement . professionals did not receive feedback about the public consultation and deliberated among themselves , without public representatives.participants : in all , 172 individuals from 6 health and social services centers participated in the study . public representatives ( n = 83 ) included chronic disease patients ( 81% ) , caregivers , and healthy adults . forty - seven percent had a primary or high - school education ; 56% had an annual household income of less than us$40,000 ; and 23% had been hospitalized during the previous year . professionals ( n = 89 ) included managers ( 35% ) , physicians ( 13% ) , nurses ( 24% ) , and allied health professionals ( 28%).results : priorities selected in intervention sites placed more emphasis on access to primary care , self - care support , patients participation in clinical decisions , and partnership with community organizations ( p < 0.01).agreement between public representatives ' and professionals improvement priorities increased by 41% in favor of intervention sites ( 95% ci + 12 , + 58% , p < 0.01 ) . professionals choices moved toward indicators prioritized by the public ( eg , access ) , and public representatives choices moved toward indicators prioritized by professionals ( eg , self - care support).participants ' perception of the quality of the deliberation process scored high in all domains ( quality of the information received , procedures and moderation , interaction among participants , and overall satisfaction).there was no significant difference in professionals intentions to use the selected quality indicators for health care improvement , which scored high in both intervention and control groups . context : health and social services centers in a canadian region ( n = 6 ) were required to select health care improvement priorities to be incorporated in their financial accountability contract with the regional health authority . health and social services centers could choose from a list of 37 validated quality indicators for chronic disease prevention and management in primary care ( including indicators on access , integration of services , technical quality of care , quality of interpersonal relationships , and health outcomes ) . baseline public priorities : public members ( n = 83 ) , including chronic disease patients , caregivers , and healthy adults , were recruited from all participating sites . all public members participated in a 1-day preparation meeting and were consulted by vote on their local priorities for improvement . randomization : health and social services centers were randomized in intervention ( n = 3 ) and control sites ( n = 3 ) to compare health care improvement priority setting with and without public involvement . professionals received feedback about the public consultation and deliberated with public representatives in a face - to - face meeting . control : health care improvement priorities were selected by professionals only , without public involvement . professionals did not receive feedback about the public consultation and deliberated among themselves , without public representatives . participants : in all , 172 individuals from 6 health and social services centers participated in the study . public representatives ( n = 83 ) included chronic disease patients ( 81% ) , caregivers , and healthy adults . forty - seven percent had a primary or high - school education ; 56% had an annual household income of less than us$40,000 ; and 23% had been hospitalized during the previous year . professionals ( n = 89 ) included managers ( 35% ) , physicians ( 13% ) , nurses ( 24% ) , and allied health professionals ( 28% ) . results : priorities selected in intervention sites placed more emphasis on access to primary care , self - care support , patients participation in clinical decisions , and partnership with community organizations ( p < 0.01).agreement between public representatives ' and professionals improvement priorities increased by 41% in favor of intervention sites ( 95% ci + 12 , + 58% , p < 0.01 ) . professionals choices moved toward indicators prioritized by the public ( eg , access ) , and public representatives choices moved toward indicators prioritized by professionals ( eg , self - care support).participants ' perception of the quality of the deliberation process scored high in all domains ( quality of the information received , procedures and moderation , interaction among participants , and overall satisfaction).there was no significant difference in professionals intentions to use the selected quality indicators for health care improvement , which scored high in both intervention and control groups . priorities selected in intervention sites placed more emphasis on access to primary care , self - care support , patients participation in clinical decisions , and partnership with community organizations ( p < 0.01 ) . agreement between public representatives ' and professionals improvement priorities increased by 41% in favor of intervention sites ( 95% ci + 12 , + 58% , p < 0.01 ) . professionals choices moved toward indicators prioritized by the public ( eg , access ) , and public representatives choices moved toward indicators prioritized by professionals ( eg , self - care support ) . participants ' perception of the quality of the deliberation process scored high in all domains ( quality of the information received , procedures and moderation , interaction among participants , and overall satisfaction ) . there was no significant difference in professionals intentions to use the selected quality indicators for health care improvement , which scored high in both intervention and control groups . figure 1 illustrates the 6 main components of the public involvement intervention and their relationship to outcomes . in the next section key components of a public involvement intervention during the design and pilot phase of the study , we consulted various stakeholders about the proposed public involvement intervention , including regional and health and social services center directors , medical leaders , as well as public representatives sitting on local and regional health authorities boards . while all stakeholder groups were supportive of public involvement , their endorsement was motivated by different reasons and was shaped by the surrounding organizational context . for example , the regional health authority directors saw public involvement as a lever for influencing the health and social services centers priorities and aligning these with public needs . in contrast , local chief executive officers ( ceos ) saw public involvement as an opportunity to educate the public and legitimize their own organizations ' priorities : ( note that quotations are from the a , b , and c intervention sites and that pseudonyms are used throughout the text . ) clinicians and medical leaders saw the intervention as a way to promote patients responsibility for their own individual care : it is winning to involve people in taking care of themselves , that is why it is important that they be present when big decisions are made public members motivation to support and participate in the trial was either to improve local services or to learn about health and health care : i am curious to see what we will learn ; i want to bring my experience to improve services our tested intervention was therefore supported by a coalition of organizations and individuals with different expectations about the actual goals of public involvement . these competing expectations created tensions as the intervention was implemented in practice and the abstract idea of public involvement became more concrete . these tensions revolved around the degree of legitimacy and credibility of public involvement , as well as the actual power that should be delegated to public representatives in the collective decision process . for example , as the group 's task became clearer , public members questioned how they were expected to contribute to health care priority setting and what rules would be put in place to address power imbalances : [ i 'm wondering , ] not in a pejorative sense , but with the level of the debate , will the population 's comments have a similar weight than those of a chief executive officer ? ( public , site a ) . public members also questioned the credibility of their contribution in certain aspects of their task and negotiated the boundaries of their role to ensure its coherence with their specific expertise . for example , in the pilot phase of the project , public members were asked to rate the feasibility of using each quality indicator in clinical practice , a task they did not feel sufficiently prepared to carry out . accordingly , the definition of the public 's task was restricted to rating the perceived importance of each quality indicator for improved patient care . these negotiations among stakeholders resulted in varying degrees of policy support for public involvement . across the sites , the amount of public influence was related to the levels of support from professionals in positions of power , especially ceos and medical leaders . for example , in intervention sites a and b , ceos and medical leaders proactively sought advice from public members during deliberation meetings and promoted policies important to the public ( eg , access to a family physician and a primary care professional ) . in intervention site c , the ceo sought to adopt a priority that was highly ranked by physicians ( screening for high blood pressure ) , which limited the public impact on group choices . our recruitment strategy was to identify members of the public who would be considered legitimate in the eyes of local professionals and members of the public . we thus delegated the identification of potential candidates to local recruitment teams composed of a senior manager , a physician , and a patient representative sitting on the health and social services centers users committee . we assumed that recruiting patients and caregivers with personal experiences of different health conditions and from various sociodemographic groups would further increase the public 's legitimacy . a member of the research team selected members of the public from local lists of proposed candidates to ensure a balanced representation in age , gender , health , and socioeconomic status . this recruitment strategy proved effective in reaching public members from low socioeconomic groups and building a sample that was representative of both local patients with chronic disease and caregivers ( box 1 ) . interestingly , we observed later in the trial that professionals were more sensitive to the priorities expressed by people from their own local territory ( although these were drawn from small samples of 12 to 15 public members ) than from the pooled regional priorities collected from the total sample of 83 public members involved in the trial . this suggests that professionals judgments about public members legitimacy may be guided by the recruitment of people representing relevant ( local ) perspectives rather than by strict statistical representativeness criteria based on demographic characteristics . we also observed that public members with previous experience of representation and group committee process ( eg , as a board member , as a patient representative on committees , or through professional experience outside health care ) were somewhat more influential than patients without such previous experience . it is worth pointing out that public members with a lower level of education were often equally or more influential than those with more education , possibly because deliberation with professionals involved group talk and the sharing of lived experiences , rather than abstract or written tasks . the public representatives preparation day contributed to the intervention 's impact by building the participants sense of credibility and their ability to contribute specific expertise to the prioritization task . the preparation meeting allowed the public representatives to ask questions of clarification in a nonthreatening environment and to build their confidence . this preparation put them in a favorable position when they later met with professionals : i am part of those who had a warm - up ! ( public , site a ) . public members also felt more competent to contribute to the group 's task as they progressed through the intervention : the preparation day also contributed to the participants sense of legitimacy as public representatives , which was critical to their ability to later influence group decisions . as members of the public discussed their personal experiences as patients and caregivers during the training day , we observed a broadening of the participants perspectives and their growing sense of a collective public representative identity . one participant noted that hearing the experience of others made her more aware of the needs of different people in her community : i have a family physician myself , but i have become more conscious of the difficulties for people who do n't . in the end , i voted to help paul and to help clare [ who do not have a physician ] these observations suggest that the participants progress in understanding their representation role , and also how their legitimacy and credibility evolved over time , was partly framed by how they were recruited and selected , their opportunity to interact with other public members , and their preparation . the tested public involvement intervention included different involvement methods ( public participation in small - group deliberation , public consultation through voting , and public communication of group decisions ) . these components were introduced sequentially , which allowed us to explore their respective impact on group decision making ( figure 1 ) . the participation component of the intervention ( days 1 and 2 ) was important to fostering mutual influence between the public and professionals . the public members influence was linked with ( 1 ) their ability to use rational and credible arguments in areas of deliberation in which they were considered experts , ( 2 ) their adoption of collective speech strategies that supported their representation role , and ( 3 ) through informal interactions favoring the establishment of alliances and coalitions among public members and professionals . our observation of small - group deliberation content revealed specific areas in which professionals saw public members as credible experts , which allowed them to contribute arguments perceived as rational . this included issues such as their personal experience of care , their expectations and needs , their perception of the public acceptability of health care innovations , as well as their knowledge of existing community organizations and services . professionals actively sought validation from public members when they discussed issues like access to care or the quality of interpersonal relationships : our final group priority is patient participation in clinical decision - making, where one of our health care users said a key sentence : working in team with the patient. do user representatives around the table agree with all that ? in other areas of deliberation , such as the psychometric properties of the proposed quality indicators , the clinical value of different treatment options , and the resource implications of the proposed changes , public members increasingly displayed interactional expertise as they became more familiar with the issues , but the professionals essentially still made the authoritative contributions in these areas . the public members increasingly endorsed a representation role as they deliberated with the professionals , not only drawing from their own individual experience but also voicing concerns raised by other participants . their ability to refer back to experiences voiced by other public members in the preparation meeting supported the public representatives ability to legitimately claim that they spoke on behalf of a wider constituency . this collective perspective was also reflected in the way that the public members portrayed their own role . although their ability to use personal stories and anecdotes was important to establish their position as real patients , those public members who brought only their own experience to the table were less effective in influencing professionals . for example , many public members originally identified themselves through their disease ( eg , i am john , and i have diabetes ) but later framed their role and contribution as population , public , or the public members adoption of collective speech strategies and their ability to support their claims of representing a larger constituency through concrete examples drawn from others experiences thus contributed to their influence in the group deliberations . finally , the impact of the public participation component of the intervention also appeared to be mediated by informal social interactions between professionals and public members , which led to greater mutual understanding and helped lessen the power differential . we observed that informal interactions during breaks and lunch time supported this change in perspective : we see that they are like us , fathers and mothers ; when we hear health professionals through their union 's representatives [ on tv ] , it is not very positive and we gain from meeting them ; [ interacting with these ] professionals allowed us to see the other side of the coin because we are on one side , in the waiting room , and they are on the other side , waiting for us ( public , site b ) . as a consequence of this face - to - face interaction , dissent rarely put professionals and the public into a us versus them position but instead led to shifting alliances among the participants . in intervention site a , for example , public representatives allied with managers and nurses to support their claim that access to a family physician was problematic in their community and should be prioritized . similarly , in intervention site b , the ceo and the medical leader actively sought validation and support from public members to support their view that patients participation in clinical decision making should be prioritized . the weight of majority was strongly at play during deliberation and supported this need for strategic alliances . indicators that received only a few votes in the first round of voting tended to be marginalized in group deliberation unless their proponents could rally support from other influential group members . this may partly explain why indicators that generated wide consensus among public members ( eg , access ) were more influential than those identified by a minority ( eg , respect and empathy ) ( table 1 ) . in some cases , alliances of professionals and public members resulted in compromises between both perspectives . in intervention site a , for example , the public participants insistence over access to a family physician ( their top priority at baseline ) was countered by the professionals belief that greater access could realistically be achieved only by investing in interdisciplinary teams ( professionals top priority at baseline ) . deliberation in this site led to the adoption of a compromise indicator on the difficulty of obtaining an appointment with a primary care professional , thereby bridging public members concerns over access and professionals insistence on supporting primary care teams . in sum , direct public participation in deliberation meetings with professionals supported the public members influence on group decisions through their ability to display rational arguments , their use of collective speech strategies , and the establishment of strategic alliances with professionals . these were facilitated by informal social interactions with professionals and built on experience shared among public members during the preparation meeting . we hired an expert in communication as our lead moderator , who was assisted by 2 comoderators with formal training in health care ( employees of the regional health authority ) . we observed that the lead moderator had less content expertise and was more focused on effective group processes , paying close attention to the setting and enforcing ground rules , supporting a relaxed atmosphere conducive to deliberation and compromise , and asking for frequent clarifications when technical language was used . the lead moderator also used a number of strategies to minimize power differences , by actively seeking public members opinions during discussions or by using seating plans ( eg , not letting lead physicians sit with ceos and seating public members in pairs ) . the professionals and the public members expression of different perspectives did cost time and effort in reaching a consensus . we observed that debates were more dynamic and lively in those groups including public representatives but that group deliberation lasted , on average , 10% longer . the moderator therefore needed to keep the participants focused on the task ( eg , when you say that , what indicator are you arguing for ? ) , closing debates when acceptable compromises and consensus were emerging ( eg , ok , it seems that we will definitely keep this indicator ) , and storing away persistent disagreements and unresolved issues ( eg , you clearly will need to discuss your action plan among yourselves ) . the participants tasks and ground rules presented at the beginning of each meeting emphasized the value of expressing different views , even though this was difficult for many participants . lecture public members about proper health - oriented behaviors ( ie , tobacco cessation ) , which made it more difficult to voice concerns raised in the public - only preparation meeting , in which a number of public members criticized tobacco cessation programs . during the deliberations , the moderators played a pivotal role in ensuring that the public members engaged in debates , by actively seeking their dissenting views : physician : i have a lot of difficulty with [ the indicator measured from ] the patients perceptions . i would be interested to hear from someone who has the opposite opinion?manager : i disagree with dr . smith on the uselessness of patients perceptions . if we measure it as a general tendency , it is important.public : i agree with dr . ( site c ) physician : i have a lot of difficulty with [ the indicator measured from ] the patients perceptions . i would be interested to hear from someone who has the opposite opinion ? manager : ( site c ) moderation techniques that actively sought the expression of marginal voices ( eg , i would be interested to hear from someone with the opposite opinion ) encouraged the active involvement of public representatives by offering a safe opportunity to express dissent with powerful participants ( i agree with dr . however , ) . whereas our lead moderator was good at facilitating the group process and the expression of divergent opinions , the 2 health professionals acting as comoderators tended to focus more on discussion content . these content - expert moderators sometimes fell into a participant role , arguing , for example , in favor of the robustness of certain indicators as a strategy to build consensus . as a result , we noticed that our lead moderator was somewhat more effective in supporting public participation , because of her focus on listening to and facilitating the group process rather than being distracted by or trying to contribute to the subject matter . although public participation alone brought some change in the professionals priorities , we observed that it was somewhat ineffective in challenging more entrenched opinions ( table 2 ) . part of the public members continuing difficulty was legitimizing their claim of speaking on behalf of the wider population rather than relying on anecdotal personal experiences . these criticisms of public members representativeness became more pointed when disagreements with professionals could not be resolved . for instance , one manager argued that comments from those people here [ pointing at public members ] gave her little information about what our population wants the public members baseline priorities collected at the beginning of the study were contrasted with those of the professionals , and descriptive statistics were presented for the overall region and for each local intervention site . the professionals saw the public consultation feedback as contributing to a better understanding of their population needs , and it played various roles in subsequent deliberations . first , it made more visible the gap between the professionals and the public members priorities . second , it added weight to individual public members claims of representing a wider group of people and opened the door to exploring differences of opinion : public 1 : i will tell you frankly , i am surprised that there are 3 indicators on which we agreed and the rest for me as a member of the public.public 2 : there are important things that have been left aside . i will tell you frankly , i am surprised that there are 3 indicators on which we agreed and the rest for me as a member of the public . ( site c ) third , because the public representatives who had been consulted at baseline were also present to discuss statistical findings with professionals , they could claim specific expertise in the interpretation of results . the public members participation in the intervention sites shaped in different ways the interpretation of the credibility of statistical summaries . in some instances , the public members kept their distance from the public consultation findings ( self - care support , in reality , i participated in this consultation [ ] maybe we did not understand the question and did not put enough importance on this [ public , site b ] ) , while in other cases , their presence supported the credibility of the findings and ensured that they were not simply tossed aside by the professionals : public : there is an enormous gap between public and professionals priorities.moderator : do you think this is a [ public ] misunderstanding problem?public : not at all . ( site a ) public : there is an enormous gap between public and professionals priorities . moderator : do you think this is a [ public ] misunderstanding problem ? public : not at all . ( site a ) overall , the statistical feedback of the public consultation prompted the participants to negotiate further any differences of opinions . the intervention 's public consultation component also added legitimacy and credibility to the public members role in setting priorities . more precisely , the combination of public consultation and direct public participation acted synergistically and increased the public members influence on the professionals . this synergy was particularly visible in intervention site a , where the introduction of the public consultation feedback altered the balance of power and shifted professionals opinions in favor of public priorities on access to a primary care professional . during the design and pilot phase of the study , we consulted various stakeholders about the proposed public involvement intervention , including regional and health and social services center directors , medical leaders , as well as public representatives sitting on local and regional health authorities boards . while all stakeholder groups were supportive of public involvement , their endorsement was motivated by different reasons and was shaped by the surrounding organizational context . for example , the regional health authority directors saw public involvement as a lever for influencing the health and social services centers priorities and aligning these with public needs . in contrast , local chief executive officers ( ceos ) saw public involvement as an opportunity to educate the public and legitimize their own organizations ' priorities : ( note that quotations are from the a , b , and c intervention sites and that pseudonyms are used throughout the text . ) clinicians and medical leaders saw the intervention as a way to promote patients responsibility for their own individual care : it is winning to involve people in taking care of themselves , that is why it is important that they be present when big decisions are made public members motivation to support and participate in the trial was either to improve local services or to learn about health and health care : i am curious to see what we will learn ; i want to bring my experience to improve services our tested intervention was therefore supported by a coalition of organizations and individuals with different expectations about the actual goals of public involvement . these competing expectations created tensions as the intervention was implemented in practice and the abstract idea of public involvement became more concrete . these tensions revolved around the degree of legitimacy and credibility of public involvement , as well as the actual power that should be delegated to public representatives in the collective decision process . for example , as the group 's task became clearer , public members questioned how they were expected to contribute to health care priority setting and what rules would be put in place to address power imbalances : [ i 'm wondering , ] not in a pejorative sense , but with the level of the debate , will the population 's comments have a similar weight than those of a chief executive officer ? ( public , site a ) . public members also questioned the credibility of their contribution in certain aspects of their task and negotiated the boundaries of their role to ensure its coherence with their specific expertise . for example , in the pilot phase of the project , public members were asked to rate the feasibility of using each quality indicator in clinical practice , a task they did not feel sufficiently prepared to carry out . accordingly , the definition of the public 's task was restricted to rating the perceived importance of each quality indicator for improved patient care . these negotiations among stakeholders resulted in varying degrees of policy support for public involvement . across the sites , the amount of public influence was related to the levels of support from professionals in positions of power , especially ceos and medical leaders . for example , in intervention sites a and b , ceos and medical leaders proactively sought advice from public members during deliberation meetings and promoted policies important to the public ( eg , access to a family physician and a primary care professional ) . in intervention site c , the ceo sought to adopt a priority that was highly ranked by physicians ( screening for high blood pressure ) , which limited the public impact on group choices . our recruitment strategy was to identify members of the public who would be considered legitimate in the eyes of local professionals and members of the public . we thus delegated the identification of potential candidates to local recruitment teams composed of a senior manager , a physician , and a patient representative sitting on the health and social services centers users committee . we assumed that recruiting patients and caregivers with personal experiences of different health conditions and from various sociodemographic groups would further increase the public 's legitimacy . a member of the research team selected members of the public from local lists of proposed candidates to ensure a balanced representation in age , gender , health , and socioeconomic status . this recruitment strategy proved effective in reaching public members from low socioeconomic groups and building a sample that was representative of both local patients with chronic disease and caregivers ( box 1 ) . interestingly , we observed later in the trial that professionals were more sensitive to the priorities expressed by people from their own local territory ( although these were drawn from small samples of 12 to 15 public members ) than from the pooled regional priorities collected from the total sample of 83 public members involved in the trial . this suggests that professionals judgments about public members legitimacy may be guided by the recruitment of people representing relevant ( local ) perspectives rather than by strict statistical representativeness criteria based on demographic characteristics . we also observed that public members with previous experience of representation and group committee process ( eg , as a board member , as a patient representative on committees , or through professional experience outside health care ) were somewhat more influential than patients without such previous experience . it is worth pointing out that public members with a lower level of education were often equally or more influential than those with more education , possibly because deliberation with professionals involved group talk and the sharing of lived experiences , rather than abstract or written tasks . the public representatives preparation day contributed to the intervention 's impact by building the participants sense of credibility and their ability to contribute specific expertise to the prioritization task . the preparation meeting allowed the public representatives to ask questions of clarification in a nonthreatening environment and to build their confidence . this preparation put them in a favorable position when they later met with professionals : i am part of those who had a warm - up ! ( public , site a ) . public members also felt more competent to contribute to the group 's task as they progressed through the intervention : the preparation day also contributed to the participants sense of legitimacy as public representatives , which was critical to their ability to later influence group decisions . as members of the public discussed their personal experiences as patients and caregivers during the training day , we observed a broadening of the participants perspectives and their growing sense of a collective public representative identity . one participant noted that hearing the experience of others made her more aware of the needs of different people in her community : i have a family physician myself , but i have become more conscious of the difficulties for people who do n't . in the end , i voted to help paul and to help clare [ who do not have a physician ] these observations suggest that the participants progress in understanding their representation role , and also how their legitimacy and credibility evolved over time , was partly framed by how they were recruited and selected , their opportunity to interact with other public members , and their preparation . the tested public involvement intervention included different involvement methods ( public participation in small - group deliberation , public consultation through voting , and public communication of group decisions ) . these components were introduced sequentially , which allowed us to explore their respective impact on group decision making ( figure 1 ) . the participation component of the intervention ( days 1 and 2 ) was important to fostering mutual influence between the public and professionals . the public members influence was linked with ( 1 ) their ability to use rational and credible arguments in areas of deliberation in which they were considered experts , ( 2 ) their adoption of collective speech strategies that supported their representation role , and ( 3 ) through informal interactions favoring the establishment of alliances and coalitions among public members and professionals . our observation of small - group deliberation content revealed specific areas in which professionals saw public members as credible experts , which allowed them to contribute arguments perceived as rational . this included issues such as their personal experience of care , their expectations and needs , their perception of the public acceptability of health care innovations , as well as their knowledge of existing community organizations and services . professionals actively sought validation from public members when they discussed issues like access to care or the quality of interpersonal relationships : our final group priority is patient participation in clinical decision - making, where one of our health care users said a key sentence : working in team with the patient. do user representatives around the table agree with all that ? ( manager , site b ) . in other areas of deliberation , such as the psychometric properties of the proposed quality indicators , the clinical value of different treatment options , and the resource implications of the proposed changes , public members increasingly displayed interactional expertise as they became more familiar with the issues , but the professionals essentially still made the authoritative contributions in these areas . the public members increasingly endorsed a representation role as they deliberated with the professionals , not only drawing from their own individual experience but also voicing concerns raised by other participants . their ability to refer back to experiences voiced by other public members in the preparation meeting supported the public representatives ability to legitimately claim that they spoke on behalf of a wider constituency . this collective perspective was also reflected in the way that the public members portrayed their own role . although their ability to use personal stories and anecdotes was important to establish their position as real patients , those public members who brought only their own experience to the table were less effective in influencing professionals . for example , many public members originally identified themselves through their disease ( eg , i am john , and i have diabetes ) but later framed their role and contribution as population , public , or the public members adoption of collective speech strategies and their ability to support their claims of representing a larger constituency through concrete examples drawn from others experiences thus contributed to their influence in the group deliberations . finally , the impact of the public participation component of the intervention also appeared to be mediated by informal social interactions between professionals and public members , which led to greater mutual understanding and helped lessen the power differential . we observed that informal interactions during breaks and lunch time supported this change in perspective : we see that they are like us , fathers and mothers ; when we hear health professionals through their union 's representatives [ on tv ] , it is not very positive and we gain from meeting them ; [ interacting with these ] professionals allowed us to see the other side of the coin because we are on one side , in the waiting room , and they are on the other side , waiting for us ( public , site b ) . as a consequence of this face - to - face interaction , dissent rarely put professionals and the public into a us versus them position but instead led to shifting alliances among the participants . in intervention site a , for example , public representatives allied with managers and nurses to support their claim that access to a family physician was problematic in their community and should be prioritized . similarly , in intervention site b , the ceo and the medical leader actively sought validation and support from public members to support their view that patients participation in clinical decision making should be prioritized . the weight of majority was strongly at play during deliberation and supported this need for strategic alliances . indicators that received only a few votes in the first round of voting tended to be marginalized in group deliberation unless their proponents could rally support from other influential group members . this may partly explain why indicators that generated wide consensus among public members ( eg , access ) were more influential than those identified by a minority ( eg , respect and empathy ) ( table 1 ) . in some cases , alliances of professionals and public members resulted in compromises between both perspectives . in intervention site a , for example , the public participants insistence over access to a family physician ( their top priority at baseline ) was countered by the professionals belief that greater access could realistically be achieved only by investing in interdisciplinary teams ( professionals top priority at baseline ) . deliberation in this site led to the adoption of a compromise indicator on the difficulty of obtaining an appointment with a primary care professional , thereby bridging public members concerns over access and professionals insistence on supporting primary care teams . in sum , direct public participation in deliberation meetings with professionals supported the public members influence on group decisions through their ability to display rational arguments , their use of collective speech strategies , and the establishment of strategic alliances with professionals . these were facilitated by informal social interactions with professionals and built on experience shared among public members during the preparation meeting . we hired an expert in communication as our lead moderator , who was assisted by 2 comoderators with formal training in health care ( employees of the regional health authority ) . we observed that the lead moderator had less content expertise and was more focused on effective group processes , paying close attention to the setting and enforcing ground rules , supporting a relaxed atmosphere conducive to deliberation and compromise , and asking for frequent clarifications when technical language was used . the lead moderator also used a number of strategies to minimize power differences , by actively seeking public members opinions during discussions or by using seating plans ( eg , not letting lead physicians sit with ceos and seating public members in pairs ) . the professionals and the public members expression of different perspectives did cost time and effort in reaching a consensus . we observed that debates were more dynamic and lively in those groups including public representatives but that group deliberation lasted , on average , 10% longer . the moderator therefore needed to keep the participants focused on the task ( eg , when you say that , what indicator are you arguing for ? ) , closing debates when acceptable compromises and consensus were emerging ( eg , ok , it seems that we will definitely keep this indicator ) , and storing away persistent disagreements and unresolved issues ( eg , you clearly will need to discuss your action plan among yourselves ) . the participants tasks and ground rules presented at the beginning of each meeting emphasized the value of expressing different views , even though this was difficult for many participants . lecture public members about proper health - oriented behaviors ( ie , tobacco cessation ) , which made it more difficult to voice concerns raised in the public - only preparation meeting , in which a number of public members criticized tobacco cessation programs . during the deliberations , the moderators played a pivotal role in ensuring that the public members engaged in debates , by actively seeking their dissenting views : physician : i have a lot of difficulty with [ the indicator measured from ] the patients perceptions . i would be interested to hear from someone who has the opposite opinion?manager : i disagree with dr . smith on the uselessness of patients perceptions . if we measure it as a general tendency , it is important.public : i agree with dr . ( site c ) physician : i have a lot of difficulty with [ the indicator measured from ] the patients perceptions . i would be interested to hear from someone who has the opposite opinion ? manager : ( site c ) moderation techniques that actively sought the expression of marginal voices ( eg , i would be interested to hear from someone with the opposite opinion ) encouraged the active involvement of public representatives by offering a safe opportunity to express dissent with powerful participants ( i agree with dr . however , ) . whereas our lead moderator was good at facilitating the group process and the expression of divergent opinions , the 2 health professionals acting as comoderators tended to focus more on discussion content . these content - expert moderators sometimes fell into a participant role , arguing , for example , in favor of the robustness of certain indicators as a strategy to build consensus . as a result , we noticed that our lead moderator was somewhat more effective in supporting public participation , because of her focus on listening to and facilitating the group process rather than being distracted by or trying to contribute to the subject matter . although public participation alone brought some change in the professionals priorities , we observed that it was somewhat ineffective in challenging more entrenched opinions ( table 2 ) . part of the public members continuing difficulty was legitimizing their claim of speaking on behalf of the wider population rather than relying on anecdotal personal experiences . these criticisms of public members representativeness became more pointed when disagreements with professionals could not be resolved . for instance , one manager argued that comments from those people here [ pointing at public members ] gave her little information about what our population wants the public members baseline priorities collected at the beginning of the study were contrasted with those of the professionals , and descriptive statistics were presented for the overall region and for each local intervention site . the professionals saw the public consultation feedback as contributing to a better understanding of their population needs , and it played various roles in subsequent deliberations . first , it made more visible the gap between the professionals and the public members priorities . second , it added weight to individual public members claims of representing a wider group of people and opened the door to exploring differences of opinion : public 1 : i will tell you frankly , i am surprised that there are 3 indicators on which we agreed and the rest for me as a member of the public.public 2 : there are important things that have been left aside . i will tell you frankly , i am surprised that there are 3 indicators on which we agreed and the rest for me as a member of the public . ( site c ) third , because the public representatives who had been consulted at baseline were also present to discuss statistical findings with professionals , they could claim specific expertise in the interpretation of results . the public members participation in the intervention sites shaped in different ways the interpretation of the credibility of statistical summaries . in some instances , the public members kept their distance from the public consultation findings ( self - care support , in reality , i participated in this consultation [ ] maybe we did not understand the question and did not put enough importance on this [ public , site b ] ) , while in other cases , their presence supported the credibility of the findings and ensured that they were not simply tossed aside by the professionals : public : there is an enormous gap between public and professionals priorities.moderator : do you think this is a [ public ] misunderstanding problem?public : not at all . ( site a ) public : there is an enormous gap between public and professionals priorities . moderator : do you think this is a [ public ] misunderstanding problem ? public : not at all . ( site a ) overall , the statistical feedback of the public consultation prompted the participants to negotiate further any differences of opinions . the intervention 's public consultation component also added legitimacy and credibility to the public members role in setting priorities . more precisely , the combination of public consultation and direct public participation acted synergistically and increased the public members influence on the professionals . this synergy was particularly visible in intervention site a , where the introduction of the public consultation feedback altered the balance of power and shifted professionals opinions in favor of public priorities on access to a primary care professional . as underscored at the beginning of this article , the current literature on public involvement suggests that issues of credibility and legitimacy are subject to ongoing negotiations among participants and stakeholders whose interests and power may be supported or challenged by public involvement.19,45,4850,59 as our process evaluation findings illustrate , exploring these tensions requires opening the black box of public involvement to understand why and how certain components can address the imbalance of power and expertise . results from this study unpack some of the key ingredients that structured and supported productive deliberations between members of the public and professionals . our findings point at how specific components of involvement interventions foster public members legitimacy , credibility , and power to influence health care improvement and policy decisions ( figure 1 ) . one contribution of our study is showing how both technocratic ( public credibility ) and democratic process issues ( their legitimacy to represent others ) underlie the unfolding of a public involvement intervention and shape its impact on collective decisions . in the literature , technocratic discussions about public expertise largely focus on the notion of technical competence and assume that lay members of the public do not always understand scientifically valid evidence.60,61 our findings point more specifically toward the importance of credibility as a condition for successful involvement , that is , the perception that professionals and public members have of their respective expertise in important areas of deliberation . this means that public involvement interventions not only must give public participants enough information to understand the technical language used by professionals but also must support their ability to become a credible source of knowledge for professionals . it is interesting to observe empirically that such public expertise comes partly from individual ( shared ) experiences and partly from the public 's ability to access population - based data . in our study , the recruitment of patients and caregivers with direct personal experience of chronic disease , as well as the opportunity provided to broaden their knowledge base by interacting with other community members helped develop a specific public expertise.47 as they became more solidly grounded in their roles , both the public members and the professionals also became more aware of the limits of their own expertise and actively engaged in a process of mutual learning and influence , as postulated by deliberation theory.46,53,56,6265 from a democratic perspective , our findings point toward the need to better distinguish the statistical representativeness of a group from the representation role of individual participants.26,51 statistical representativeness the correspondence between the descriptive characteristics of a sample and those of the population from which they are drawn is only one aspect of public members legitimacy and is most applicable to public consultation strategies in which large groups of participants can be recruited . in contrast , the logic of direct public participation in small - group decision making is mainly one of representation , in which individual participants are asked to speak for a wider constituency . our findings highlight the need to reframe the debate about the recruitment of real patients or ordinary citizens and instead explore how public involvement interventions can support public members legitimacy to represent others , in both their own eyes and those of health professionals.21,66 in our study , a balanced recruitment strategy and the use of a preparation meeting supported participants ' ability to legitimately represent their community . finally , our findings suggest that different components of public involvement interventions can modulate the power imbalance between professionals and the public . process - oriented group moderation can play an important role in this regard through strategies like seating plans , the establishment of ground rules with participants , and agenda setting . the degree of power over collective health care decisions also is contingent on ongoing negotiations among stakeholders on how the public will be involved and supported in practice . our observations also confirm findings from other studies suggesting that there is strength in numbers , meaning that public power can be mediated by the number of public members ( through their impact in voting ) , their ability to build strategic alliances with other group members , and the possibility of supporting their claims based on population surveys and public consultation.45,55,59 observations from our study indicate that restricting the public 's involvement to offering a seat at the table to one or two individuals without appropriate support is unlikely to change health care and policy decisions . indeed , many of the key ingredients identified in this study appear to be absent from existing public involvement programs.25 accordingly , a number of our study 's actionable policy implications can be used to increase the potential effectiveness of public involvement . first , questions about the design of public involvement interventions are often reduced to identifying the right participants who seem competent to understand the conversation while still being seen as representative of ordinary patients or lay public members . as a result , more emphasis is put on recruitment and sampling strategies and less on other intervention components , such as who identifies public members , what opportunities they have to interact with one another , and how they can access population - based evidence . our results also point to the importance of preparation meetings that go beyond a basic understanding of technical terms and encourage the development of a specific public expertise . an important observation from our trial is the synergy of the preparation , participation , and consultation components of the intervention , each contributing to the public 's credibility , legitimacy , and power . in the academic literature , a theoretical divide tends to separate proponents of participation methods ( based on deliberative theory and political sciences ) , consultation methods ( based on epidemiological methods and health economics ) , and communication methods ( focused on risk communication , behavioral change theories , and clinical decision making ) . this academic fragmentation has so far supported a piecemeal approach to the development of public involvement interventions that may have hampered their practical effectiveness . our findings provide more comprehensive guidance for policymakers who wish to develop and implement public involvement interventions that integrate consultation , participation , and communication methods . collective health care decision - making processes like guideline development , health technology assessment , and clinical priority setting are often chaired by content experts and people in a hierarchical position of power ( eg , lead clinician and ceo).25 our findings show that group moderation by an expert in group processes ( rather than by a content expert ) could help even out existing power differences , facilitate more fruitful deliberation , and support professionals and public members mutual understanding and influence . as we observed in this article , the goals and actual process of involvement were subject to ongoing negotiations throughout the design and implementation of the intervention . during the study 's pilot phase , such negotiations were arbitrated by the research team , who ultimately decided on the format of the public involvement intervention tested in the trial . we speculate that more naturalistic involvement interventions might have tipped the negotiations in another direction and resulted in rules of engagement that may not have been favorable to public influence on collective choices . some settings also may simply be too polarized to agree on public involvement procedures . because the rhetoric of public involvement commands such widespread support , it may be tempting to gloss over the tensions regarding the competing goals and roles that the public members are expected to play . however , without negotiating and building policy support for public involvement , the search for effective involvement interventions will likely remain elusive . consequently , policymakers should seek to apply broad but clear and consistent principles enabling the development of more effective involvement interventions . the strength of this study is the detailed process evaluation data collected from a real - world experimental study . informed by theory , our evaluation of an intervention in which both productive disagreements and mutual learning took place offers a concrete example of integrating quantitative outcome results and qualitative process analysis . this methodological approach reveals the ingredients that hold together public involvement interventions and help them succeed . the generalizability of our findings could be limited because of the diversity of public involvement interventions and the influence of the sociopolitical contexts in which they are implemented . but by separating the process analysis of our intervention into its different components , we can more easily assess the generalizability of our findings to similar public involvement interventions.31 our study focused on the influence of components inside a public involvement intervention rather than on the role of external contextual factors ( eg , health care organizations characteristics , types of policy decisions , and community characteristics ) . research on contextual factors affecting the effectiveness of public involvement interventions is still in its infancy , and experts in the field have called for stronger evidence about what works in which context.67,68 our findings point to the limits of public involvement interventions alone and to the importance of external factors shaping public members credibility , legitimacy , and influence on collective health care decisions . for example , quality indicators measured from the patient 's perspective ( eg , respect and empathy ) were difficult for the public members to promote , partly because less research has been conducted on quality indicators measured from patients experience , as opposed to technical , disease - specific indicators like blood pressure control . research decisions made before the involvement intervention could therefore have affected the credibility of public priorities and their ability to influence professionals . this suggests that the impact of public involvement is shaped not only by the involvement intervention itself but also by contextual factors outside the participants and sponsors control . finally , seeking the active ingredients of public involvement , rather than simply describing how the intervention is unfolding in practice , implies certain normative judgments about the desirable outcomes of public involvement interventions . in our study , we assumed that interventions supporting the public members influence on collective decisions could be considered effective . we , however , recognize that other normative models of what constitutes effective involvement exist ( eg , considering public involvement as an intrinsic good , independently of its impact on collective decisions ) and could have resulted in different interpretations.55 as underscored at the beginning of this article , the current literature on public involvement suggests that issues of credibility and legitimacy are subject to ongoing negotiations among participants and stakeholders whose interests and power may be supported or challenged by public involvement.19,45,4850,59 as our process evaluation findings illustrate , exploring these tensions requires opening the black box of public involvement to understand why and how certain components can address the imbalance of power and expertise . results from this study unpack some of the key ingredients that structured and supported productive deliberations between members of the public and professionals . our findings point at how specific components of involvement interventions foster public members legitimacy , credibility , and power to influence health care improvement and policy decisions ( figure 1 ) . one contribution of our study is showing how both technocratic ( public credibility ) and democratic process issues ( their legitimacy to represent others ) underlie the unfolding of a public involvement intervention and shape its impact on collective decisions . in the literature , technocratic discussions about public expertise largely focus on the notion of technical competence and assume that lay members of the public do not always understand scientifically valid evidence.60,61 our findings point more specifically toward the importance of credibility as a condition for successful involvement , that is , the perception that professionals and public members have of their respective expertise in important areas of deliberation . this means that public involvement interventions not only must give public participants enough information to understand the technical language used by professionals but also must support their ability to become a credible source of knowledge for professionals . it is interesting to observe empirically that such public expertise comes partly from individual ( shared ) experiences and partly from the public 's ability to access population - based data . in our study , the recruitment of patients and caregivers with direct personal experience of chronic disease , as well as the opportunity provided to broaden their knowledge base by interacting with other community members helped develop a specific public expertise.47 as they became more solidly grounded in their roles , both the public members and the professionals also became more aware of the limits of their own expertise and actively engaged in a process of mutual learning and influence , as postulated by deliberation theory.46,53,56,6265 from a democratic perspective , our findings point toward the need to better distinguish the statistical representativeness of a group from the representation role of individual participants.26,51 statistical representativeness the correspondence between the descriptive characteristics of a sample and those of the population from which they are drawn is only one aspect of public members legitimacy and is most applicable to public consultation strategies in which large groups of participants can be recruited . in contrast , the logic of direct public participation in small - group decision making is mainly one of representation , in which individual participants are asked to speak for a wider constituency . our findings highlight the need to reframe the debate about the recruitment of real patients or ordinary citizens and instead explore how public involvement interventions can support public members legitimacy to represent others , in both their own eyes and those of health professionals.21,66 in our study , a balanced recruitment strategy and the use of a preparation meeting supported participants ' ability to legitimately represent their community . finally , our findings suggest that different components of public involvement interventions can modulate the power imbalance between professionals and the public . process - oriented group moderation can play an important role in this regard through strategies like seating plans , the establishment of ground rules with participants , and agenda setting . the degree of power over collective health care decisions also is contingent on ongoing negotiations among stakeholders on how the public will be involved and supported in practice . our observations also confirm findings from other studies suggesting that there is strength in numbers , meaning that public power can be mediated by the number of public members ( through their impact in voting ) , their ability to build strategic alliances with other group members , and the possibility of supporting their claims based on population surveys and public consultation.45,55,59 observations from our study indicate that restricting the public 's involvement to offering a seat at the table to one or two individuals without appropriate support is unlikely to change health care and policy decisions . indeed , many of the key ingredients identified in this study appear to be absent from existing public involvement programs.25 accordingly , a number of our study 's actionable policy implications can be used to increase the potential effectiveness of public involvement . first , questions about the design of public involvement interventions are often reduced to identifying the right participants who seem competent to understand the conversation while still being seen as representative of ordinary patients or lay more emphasis is put on recruitment and sampling strategies and less on other intervention components , such as who identifies public members , what opportunities they have to interact with one another , and how they can access population - based evidence . our results also point to the importance of preparation meetings that go beyond a basic understanding of technical terms and encourage the development of a specific public expertise . an important observation from our trial is the synergy of the preparation , participation , and consultation components of the intervention , each contributing to the public 's credibility , legitimacy , and power . in the academic literature , a theoretical divide tends to separate proponents of participation methods ( based on deliberative theory and political sciences ) , consultation methods ( based on epidemiological methods and health economics ) , and communication methods ( focused on risk communication , behavioral change theories , and clinical decision making ) . this academic fragmentation has so far supported a piecemeal approach to the development of public involvement interventions that may have hampered their practical effectiveness . our findings provide more comprehensive guidance for policymakers who wish to develop and implement public involvement interventions that integrate consultation , participation , and communication methods . collective health care decision - making processes like guideline development , health technology assessment , and clinical priority setting are often chaired by content experts and people in a hierarchical position of power ( eg , lead clinician and ceo).25 our findings show that group moderation by an expert in group processes ( rather than by a content expert ) could help even out existing power differences , facilitate more fruitful deliberation , and support professionals and public members mutual understanding and influence . as we observed in this article , the goals and actual process of involvement were subject to ongoing negotiations throughout the design and implementation of the intervention . during the study 's pilot phase , such negotiations were arbitrated by the research team , who ultimately decided on the format of the public involvement intervention tested in the trial . we speculate that more naturalistic involvement interventions might have tipped the negotiations in another direction and resulted in rules of engagement that may not have been favorable to public influence on collective choices . some settings also may simply be too polarized to agree on public involvement procedures . because the rhetoric of public involvement commands such widespread support , it may be tempting to gloss over the tensions regarding the competing goals and roles that the public members are expected to play . however , without negotiating and building policy support for public involvement , the search for effective involvement interventions will likely remain elusive . consequently , policymakers should seek to apply broad but clear and consistent principles enabling the development of more effective involvement interventions . the strength of this study is the detailed process evaluation data collected from a real - world experimental study . informed by theory , our evaluation of an intervention in which both productive disagreements and mutual learning took place offers a concrete example of integrating quantitative outcome results and qualitative process analysis . this methodological approach reveals the ingredients that hold together public involvement interventions and help them succeed . the generalizability of our findings could be limited because of the diversity of public involvement interventions and the influence of the sociopolitical contexts in which they are implemented . but by separating the process analysis of our intervention into its different components , we can more easily assess the generalizability of our findings to similar public involvement interventions.31 our study focused on the influence of components inside a public involvement intervention rather than on the role of external contextual factors ( eg , health care organizations characteristics , types of policy decisions , and community characteristics ) . research on contextual factors affecting the effectiveness of public involvement interventions is still in its infancy , and experts in the field have called for stronger evidence about what works in which context.67,68 our findings point to the limits of public involvement interventions alone and to the importance of external factors shaping public members credibility , legitimacy , and influence on collective health care decisions . for example , quality indicators measured from the patient 's perspective ( eg , respect and empathy ) were difficult for the public members to promote , partly because less research has been conducted on quality indicators measured from patients experience , as opposed to technical , disease - specific indicators like blood pressure control . research decisions made before the involvement intervention could therefore have affected the credibility of public priorities and their ability to influence professionals . this suggests that the impact of public involvement is shaped not only by the involvement intervention itself but also by contextual factors outside the participants and sponsors control . finally , seeking the active ingredients of public involvement , rather than simply describing how the intervention is unfolding in practice , implies certain normative judgments about the desirable outcomes of public involvement interventions . in our study , we assumed that interventions supporting the public members influence on collective decisions could be considered effective . we , however , recognize that other normative models of what constitutes effective involvement exist ( eg , considering public involvement as an intrinsic good , independently of its impact on collective decisions ) and could have resulted in different interpretations.55 public involvement calls for well - thought - out interventions that incorporate a number of interacting active ingredients . in this study , the public members credibility , legitimacy , and power increased their ability to influence group decisions . this was framed and strengthened by the recruitment of a balanced group of participants , structured training , opportunities to draw from others experiences , moderation techniques focused on effective group processes , and the combination of broad public consultation with public participation in small - group deliberation . the engagement of key stakeholders in negotiating the design and implementation of the intervention helped build policy support for public involvement . we need to better distinguish statistical representativeness from representation roles in discussions about public legitimacy . we also must expand our notion of public members competence beyond their understanding of technical terms in order to support the development of a contributory public expertise . greater attention to these ingredients could lead to more effective public involvement interventions and increase public members influence on health care improvement and policy decisions .

contextin the past 50 years , individual patient involvement at the clinical consultation level has received considerable attention . more recently , patients and the public have increasingly been involved in collective decisions concerning the improvement of health care and policymaking . however , rigorous evaluation guiding the development and implementation of effective public involvement interventions is lacking . this article describes those key ingredients likely to affect public members ability to deliberate productively with professionals and influence collective health care choices.methodwe conducted a trial process evaluation of public involvement in setting priorities for health care improvement . in all , 172 participants ( including 83 patients and public members and 89 professionals ) from 6 health and social services centers in canada participated in the trial . we videorecorded 14 one - day meetings , and 2 nonparticipant observers took structured notes . using qualitative analysis , we show how public members influenced health care improvement priorities.findingslegitimacy , credibility , and power explain the variations in the public members influence . their credibility was supported by their personal experience as patients and caregivers , the provision of a structured preparation meeting , and access to population - based data from their community . legitimacy was fostered by the recruitment of a balanced group of participants and by the public members opportunities to draw from one another 's experience . the combination of small - group deliberations , wider public consultation , and a moderation style focused on effective group process helped level out the power differences between professionals and the public . the engagement of key stakeholders in the intervention design and implementation helped build policy support for public involvement.conclusionsa number of interacting active ingredients structure and foster the public 's legitimacy , credibility , and power . by paying greater attention to them , policymakers could develop and implement more effective public involvement interventions .

Theoretical Assumptions Underpinning Public Involvement Interventions Methods Box 1. Trial Overview Results Building a Policy Coalition Supportive of Public Involvement Recruitment: Representing Legitimate Groups and Perspectives Preparation: Supporting Public Representation Role and Expertise Public Participation: Rational Deliberation, Collective Voices, and Strategic Alliances Moderation: Leveling Power Differences and Legitimizing Marginal Voices Public Consultation: Bringing the Population's View Discussion Key Findings and Contribution to the Existing Literature Policy Implications Strengths and Limitations of Our Study Conclusion

existing theories of public involvement embody certain assumptions and hypotheses about what public involvement interventions are made of ( their principal components ) and how they are expected to work ( their internal dynamic explaining outcomes).3640 three main theoretical constructs have been proposed to explain the public 's influence on collective health care decisions : ( 1 ) public members credibility and ability to contribute knowledge that is considered valid and relevant to inform collective health care decisions , ( 2 ) their legitimacy to speak on behalf of people affected directly or indirectly by health care services and policies , and ( 3 ) their power and ability to influence collective health care choices.21 technocratic theories of public involvement stress the importance of the public 's ability to demonstrate credible expertise . rowe and frewer contend that information sharing is the core process underlying public involvement and classify interventions according to the flow of information between professionals and the public : communication methods ( in which information is communicated to the public ) , consultation ( information is collected from the public ) , and participation ( information is exchanged between professionals and the public).41 such an information flow perspective is particularly well developed in deliberative theories , which anchor public involvement around the careful weighing of reasons for and against collective action propositions.42 the core hypothesis of deliberative theories is that the exchange of reasonable and credible arguments should result in mutual learning and in the generation of solutions that can be rationally justified to those affected by it.4346 from this perspective , access to the best available evidence through training and preparation is a critical component of effective public involvement interventions , ensuring that valid and relevant information is collected and exchanged among competent participants.35,41 collins and evans contend that members of the general public are experience - based experts whose knowledge is based mainly on their personal experiences rather than on their degrees and professional qualifications.47 these authors further distinguish among 3 levels of public expertise . democratic theories emphasize the importance of the public 's legitimacy to speak on behalf of others ; indeed , it is seen as a critical component of effective public involvement.27,45,4851 judging the public members legitimacy requires paying close attention to the social practice of public involvement and the political and organizational context in which divergent notions of representativeness are deployed in pursuit of differing roles for public participation.48 observations of real - life involvement interventions suggest that professionals use different criteria to support ( or question ) the public 's legitimacy , such as the participants statistical representativeness , the use of formal public representatives selection and nomination procedures , and the participants accountability to the community members they represent.48,49,51 finally , a number of authors have highlighted the importance of power as a core ingredient for explaining the public 's relative influence on collective decision making . for example , research on small - group decision making suggests that collective decisions often tend to move toward the majority 's view.53,54 in the context of collective health care improvement and policy decisions , power may also be determined by professional and hierarchical status.27,48 in its more subtle and discursive forms , power may be exerted through the types of assumptions that are taken for granted and accepted by the group.45 finally , power struggles can be woven into the fabric of a public involvement intervention itself , shaping the selected method of involvement , who is asked to participate , and how its sponsors define effectiveness.55 while power is closely connected to and contingent on the public 's credibility and legitimacy , it also points to other important components of public involvement interventions , including control over agenda and procedures , and decisions about how public recommendations will be incorporated in actual policy decisions.56 in summary , the theoretical literature points to the need to look closely at how public involvement interventions frame and foster public members ' legitimacy , credibility , and power , in order to explain their ability to influence collective decisions . details of the development and testing of the public involvement intervention have been published elsewhere.33,57 in a nutshell , we randomized health and social services centers in a canadian health region in intervention and control sites . in the intervention sites , the public members ( 1 ) attended a 1-day preparation meeting , ( 2 ) were consulted by vote on their local priorities for improvement , and ( 3 ) participated in a 2-day meeting to deliberate with professionals and reach agreement with them on local health care improvement priorities . design : cluster randomized trial.context : health and social services centers in a canadian region ( n = 6 ) were required to select health care improvement priorities to be incorporated in their financial accountability contract with the regional health authority . health and social services centers could choose from a list of 37 validated quality indicators for chronic disease prevention and management in primary care ( including indicators on access , integration of services , technical quality of care , quality of interpersonal relationships , and health outcomes).baseline public priorities : public members ( n = 83 ) , including chronic disease patients , caregivers , and healthy adults , were recruited from all participating sites . all public members participated in a 1-day preparation meeting and were consulted by vote on their local priorities for improvement.randomization : health and social services centers were randomized in intervention ( n = 3 ) and control sites ( n = 3 ) to compare health care improvement priority setting with and without public involvement.intervention : health care improvement priorities were selected by both professionals and public members . professionals did not receive feedback about the public consultation and deliberated among themselves , without public representatives.participants : in all , 172 individuals from 6 health and social services centers participated in the study . professionals choices moved toward indicators prioritized by the public ( eg , access ) , and public representatives choices moved toward indicators prioritized by professionals ( eg , self - care support).participants ' perception of the quality of the deliberation process scored high in all domains ( quality of the information received , procedures and moderation , interaction among participants , and overall satisfaction).there was no significant difference in professionals intentions to use the selected quality indicators for health care improvement , which scored high in both intervention and control groups . context : health and social services centers in a canadian region ( n = 6 ) were required to select health care improvement priorities to be incorporated in their financial accountability contract with the regional health authority . participants : in all , 172 individuals from 6 health and social services centers participated in the study . professionals choices moved toward indicators prioritized by the public ( eg , access ) , and public representatives choices moved toward indicators prioritized by professionals ( eg , self - care support).participants ' perception of the quality of the deliberation process scored high in all domains ( quality of the information received , procedures and moderation , interaction among participants , and overall satisfaction).there was no significant difference in professionals intentions to use the selected quality indicators for health care improvement , which scored high in both intervention and control groups . davies and colleagues showed the value of combining videorecording and direct observations to explore both the content of the deliberations and the participants social interactions during a public involvement intervention.45 we videorecorded 14 one - day meetings , and 2 independent , nonparticipant observers used structured observation charts to describe the deliberation content , the types of arguments used , and the participants interactions . design : cluster randomized trial.context : health and social services centers in a canadian region ( n = 6 ) were required to select health care improvement priorities to be incorporated in their financial accountability contract with the regional health authority . health and social services centers could choose from a list of 37 validated quality indicators for chronic disease prevention and management in primary care ( including indicators on access , integration of services , technical quality of care , quality of interpersonal relationships , and health outcomes).baseline public priorities : public members ( n = 83 ) , including chronic disease patients , caregivers , and healthy adults , were recruited from all participating sites . all public members participated in a 1-day preparation meeting and were consulted by vote on their local priorities for improvement.randomization : health and social services centers were randomized in intervention ( n = 3 ) and control sites ( n = 3 ) to compare health care improvement priority setting with and without public involvement.intervention : health care improvement priorities were selected by both professionals and public members . professionals did not receive feedback about the public consultation and deliberated among themselves , without public representatives.participants : in all , 172 individuals from 6 health and social services centers participated in the study . professionals choices moved toward indicators prioritized by the public ( eg , access ) , and public representatives choices moved toward indicators prioritized by professionals ( eg , self - care support).participants ' perception of the quality of the deliberation process scored high in all domains ( quality of the information received , procedures and moderation , interaction among participants , and overall satisfaction).there was no significant difference in professionals intentions to use the selected quality indicators for health care improvement , which scored high in both intervention and control groups . context : health and social services centers in a canadian region ( n = 6 ) were required to select health care improvement priorities to be incorporated in their financial accountability contract with the regional health authority . participants : in all , 172 individuals from 6 health and social services centers participated in the study . professionals choices moved toward indicators prioritized by the public ( eg , access ) , and public representatives choices moved toward indicators prioritized by professionals ( eg , self - care support).participants ' perception of the quality of the deliberation process scored high in all domains ( quality of the information received , procedures and moderation , interaction among participants , and overall satisfaction).there was no significant difference in professionals intentions to use the selected quality indicators for health care improvement , which scored high in both intervention and control groups . in the next section key components of a public involvement intervention during the design and pilot phase of the study , we consulted various stakeholders about the proposed public involvement intervention , including regional and health and social services center directors , medical leaders , as well as public representatives sitting on local and regional health authorities boards . clinicians and medical leaders saw the intervention as a way to promote patients responsibility for their own individual care : it is winning to involve people in taking care of themselves , that is why it is important that they be present when big decisions are made public members motivation to support and participate in the trial was either to improve local services or to learn about health and health care : i am curious to see what we will learn ; i want to bring my experience to improve services our tested intervention was therefore supported by a coalition of organizations and individuals with different expectations about the actual goals of public involvement . finally , the impact of the public participation component of the intervention also appeared to be mediated by informal social interactions between professionals and public members , which led to greater mutual understanding and helped lessen the power differential . in sum , direct public participation in deliberation meetings with professionals supported the public members influence on group decisions through their ability to display rational arguments , their use of collective speech strategies , and the establishment of strategic alliances with professionals . more precisely , the combination of public consultation and direct public participation acted synergistically and increased the public members influence on the professionals . clinicians and medical leaders saw the intervention as a way to promote patients responsibility for their own individual care : it is winning to involve people in taking care of themselves , that is why it is important that they be present when big decisions are made public members motivation to support and participate in the trial was either to improve local services or to learn about health and health care : i am curious to see what we will learn ; i want to bring my experience to improve services our tested intervention was therefore supported by a coalition of organizations and individuals with different expectations about the actual goals of public involvement . finally , the impact of the public participation component of the intervention also appeared to be mediated by informal social interactions between professionals and public members , which led to greater mutual understanding and helped lessen the power differential . lecture public members about proper health - oriented behaviors ( ie , tobacco cessation ) , which made it more difficult to voice concerns raised in the public - only preparation meeting , in which a number of public members criticized tobacco cessation programs . more precisely , the combination of public consultation and direct public participation acted synergistically and increased the public members influence on the professionals . as underscored at the beginning of this article , the current literature on public involvement suggests that issues of credibility and legitimacy are subject to ongoing negotiations among participants and stakeholders whose interests and power may be supported or challenged by public involvement.19,45,4850,59 as our process evaluation findings illustrate , exploring these tensions requires opening the black box of public involvement to understand why and how certain components can address the imbalance of power and expertise . our findings point at how specific components of involvement interventions foster public members legitimacy , credibility , and power to influence health care improvement and policy decisions ( figure 1 ) . in our study , the recruitment of patients and caregivers with direct personal experience of chronic disease , as well as the opportunity provided to broaden their knowledge base by interacting with other community members helped develop a specific public expertise.47 as they became more solidly grounded in their roles , both the public members and the professionals also became more aware of the limits of their own expertise and actively engaged in a process of mutual learning and influence , as postulated by deliberation theory.46,53,56,6265 from a democratic perspective , our findings point toward the need to better distinguish the statistical representativeness of a group from the representation role of individual participants.26,51 statistical representativeness the correspondence between the descriptive characteristics of a sample and those of the population from which they are drawn is only one aspect of public members legitimacy and is most applicable to public consultation strategies in which large groups of participants can be recruited . our findings highlight the need to reframe the debate about the recruitment of real patients or ordinary citizens and instead explore how public involvement interventions can support public members legitimacy to represent others , in both their own eyes and those of health professionals.21,66 in our study , a balanced recruitment strategy and the use of a preparation meeting supported participants ' ability to legitimately represent their community . finally , our findings suggest that different components of public involvement interventions can modulate the power imbalance between professionals and the public . our observations also confirm findings from other studies suggesting that there is strength in numbers , meaning that public power can be mediated by the number of public members ( through their impact in voting ) , their ability to build strategic alliances with other group members , and the possibility of supporting their claims based on population surveys and public consultation.45,55,59 observations from our study indicate that restricting the public 's involvement to offering a seat at the table to one or two individuals without appropriate support is unlikely to change health care and policy decisions . collective health care decision - making processes like guideline development , health technology assessment , and clinical priority setting are often chaired by content experts and people in a hierarchical position of power ( eg , lead clinician and ceo).25 our findings show that group moderation by an expert in group processes ( rather than by a content expert ) could help even out existing power differences , facilitate more fruitful deliberation , and support professionals and public members mutual understanding and influence . research on contextual factors affecting the effectiveness of public involvement interventions is still in its infancy , and experts in the field have called for stronger evidence about what works in which context.67,68 our findings point to the limits of public involvement interventions alone and to the importance of external factors shaping public members credibility , legitimacy , and influence on collective health care decisions . we , however , recognize that other normative models of what constitutes effective involvement exist ( eg , considering public involvement as an intrinsic good , independently of its impact on collective decisions ) and could have resulted in different interpretations.55 as underscored at the beginning of this article , the current literature on public involvement suggests that issues of credibility and legitimacy are subject to ongoing negotiations among participants and stakeholders whose interests and power may be supported or challenged by public involvement.19,45,4850,59 as our process evaluation findings illustrate , exploring these tensions requires opening the black box of public involvement to understand why and how certain components can address the imbalance of power and expertise . our findings point at how specific components of involvement interventions foster public members legitimacy , credibility , and power to influence health care improvement and policy decisions ( figure 1 ) . in our study , the recruitment of patients and caregivers with direct personal experience of chronic disease , as well as the opportunity provided to broaden their knowledge base by interacting with other community members helped develop a specific public expertise.47 as they became more solidly grounded in their roles , both the public members and the professionals also became more aware of the limits of their own expertise and actively engaged in a process of mutual learning and influence , as postulated by deliberation theory.46,53,56,6265 from a democratic perspective , our findings point toward the need to better distinguish the statistical representativeness of a group from the representation role of individual participants.26,51 statistical representativeness the correspondence between the descriptive characteristics of a sample and those of the population from which they are drawn is only one aspect of public members legitimacy and is most applicable to public consultation strategies in which large groups of participants can be recruited . our findings highlight the need to reframe the debate about the recruitment of real patients or ordinary citizens and instead explore how public involvement interventions can support public members legitimacy to represent others , in both their own eyes and those of health professionals.21,66 in our study , a balanced recruitment strategy and the use of a preparation meeting supported participants ' ability to legitimately represent their community . finally , our findings suggest that different components of public involvement interventions can modulate the power imbalance between professionals and the public . our observations also confirm findings from other studies suggesting that there is strength in numbers , meaning that public power can be mediated by the number of public members ( through their impact in voting ) , their ability to build strategic alliances with other group members , and the possibility of supporting their claims based on population surveys and public consultation.45,55,59 observations from our study indicate that restricting the public 's involvement to offering a seat at the table to one or two individuals without appropriate support is unlikely to change health care and policy decisions . first , questions about the design of public involvement interventions are often reduced to identifying the right participants who seem competent to understand the conversation while still being seen as representative of ordinary patients or lay more emphasis is put on recruitment and sampling strategies and less on other intervention components , such as who identifies public members , what opportunities they have to interact with one another , and how they can access population - based evidence . collective health care decision - making processes like guideline development , health technology assessment , and clinical priority setting are often chaired by content experts and people in a hierarchical position of power ( eg , lead clinician and ceo).25 our findings show that group moderation by an expert in group processes ( rather than by a content expert ) could help even out existing power differences , facilitate more fruitful deliberation , and support professionals and public members mutual understanding and influence . research on contextual factors affecting the effectiveness of public involvement interventions is still in its infancy , and experts in the field have called for stronger evidence about what works in which context.67,68 our findings point to the limits of public involvement interventions alone and to the importance of external factors shaping public members credibility , legitimacy , and influence on collective health care decisions . in this study , the public members credibility , legitimacy , and power increased their ability to influence group decisions . this was framed and strengthened by the recruitment of a balanced group of participants , structured training , opportunities to draw from others experiences , moderation techniques focused on effective group processes , and the combination of broad public consultation with public participation in small - group deliberation . the engagement of key stakeholders in negotiating the design and implementation of the intervention helped build policy support for public involvement . greater attention to these ingredients could lead to more effective public involvement interventions and increase public members influence on health care improvement and policy decisions .

patient specimens used to develop and validate a prognostic signature were collected in five european institutes between 1985 and 2005 . triple negativity of tumor tissues was confirmed based on mrna expression using real - time quantitative polymerase chain reaction with a reported criterion of estrogen receptor ( esr1 ) less than 0.2 , progesterone receptor ( pgr ) less than 0.1 , and human epidermal growth factor receptor ( her2/erbb2 ) genes bb2 less than 18 ( 12,13 ) . with this criterion , we identified 271 and 61 tnbc tumor tissues from local tissue bank and european organization for research and treatment of cancer ( eortc ) collaborators , respectively . tissues used in this study were restricted to lnn patients who had not received systemic adjuvant therapy . patients who developed distant metastasis as first event within 5 years after removal of the primary tumor were considered to have poor prognosis , whereas patients who remained free of distant metastasis for 5 years were defined as having good prognosis . in addition , inclusion for microdissection and mass spectrometry ( ms ) profiling depended on sufficient invasive tumor area for microdissection and morphological quality . with the above - mentioned criteria , samples were subsequently rejected on the basis of clinical reasons [ patients were diagnosed with positive lymph - nodes ( 92 ) , received adjuvant chemotherapy ( 3 ) , had insufficient clinical follow - up ( 19 ) , and developed local relapse before distant metastasis ( 26 ) ] and technical reasons [ insufficient tumor tissues ( 33 ) , indistinguishable morphology , and insufficient tumor area for microdissection ( 32 ) ] . as a consequence , 63 and 64 tumors from training and test sets were prepared for nlc - ms / ms profiling , respectively . clinical characteristics of the included ( n = 126 ) and excluded ( n = 206 ) subjects are summarized in supplementary table 1 ( available online ) . nlc - ms / ms data from 63 samples were used for development of the protein signature ( training set ) , and data from the other 63 samples were used for independent validation ( test set ) ( table 1 ; supplementary table 2 , available online ) . this study was approved by the local institutional medical ethics committee ( mec 02.953 ) , and wherever possible we adhered to the reporting recommendations for tumor marker prognostic studies ( 14 ) . emc = erasmus medical center ; eortc = european organization for research and treatment of cancer ; sd = standard deviation . the dedicated procedure of sample preparation was performed ( 15 ) ( supplementary methods , available online ) . tissue samples were cut into 8-m frozen sections and then microdissected to obtain approximately 4000 breast cancer epithelial cells . proteins were extracted from microdissected cells , and this was followed by denaturation , reduction , and alkylation . protein samples were digested at 37c for 4 hours using ms - grade trypsin ( promega , madison , wi ) at a 1:4 ( enzyme / protein ) ratio and then acidified for further analysis . global proteome profiles of the tnbc samples were recorded on an nlc hyphenated ltq - orbitrap - xl ms system ( thermoelectron , bremen , germany ) ( supplementary methods , available online ) . peptide mixtures were separated on the nlc system with a 3-hour binary gradient ( mobile phase a : water ; mobile phase b : acetonitrile ) in a 3-m c18 silica - packed 50-cm capillary column with 75-m inner diameter . mass spectra were acquired over a mass - to - charge ratio ( m / z ) range of 400 to 1800 at a resolving power of 30000 at 400 m / z . the five most intensive parent ions from the full scan were isolated and fragmented by collisional activated dissociation in the linear ion trap . recorded raw nlc - ms / ms data have been submitted to proteomexchange ( accession number : pxd000260 ) . the recorded ms spectra from the training and test set were separately analyzed in maxquant software ( free - ware available from www.maxquant.org , version 1.1.1.36 ) ( 16 ) . to identify a prognostic protein signature , cox regression analysis was performed to associate protein abundance with metastasis - free survival time of patients in the training set . weighted protein abundance of the prognostic proteins was computed by multiplication of their protein abundance and corresponding cox regression coefficients . a relapse score was calculated by summation of weighted protein abundance of all protein(s ) in a given predictive model , followed by z score transformation of summed score . multiple predictive models were constructed from the training set by starting with the protein with the lowest p value and stepwise adding one more protein in the next model . efficiency of these models was then assessed by summation of three parameters : 1 ) area under the curve ( auc ) of receiver operating characteristic ( roc ) curve ( 17 ) , 2 ) youden s index at 100% sensitivity ( sensitivity + specificity 1 ) , and 3 ) reversed model size ( 1 ni / n , where ni is the number of proteins in a defined model , and n is the total number of prognostic proteins ) . the cutoff of the prognostic protein signature was determined from the roc curve of the training set to ensure 100% sensitivity and highest specificity . meier survival curves and log - rank test were performed to evaluate the differences in the time to distant metastasis of predicted good and poor prognosis groups . univariate and multivariable analyses with cox proportional hazard model were used to assess the prognostic value of the protein signature with and without consideration of the individual clinical prognostic variables . pathway analysis was performed to interpret biological function of signature proteins using gene set enrichment analysis ( 18 ) . the supplementary methods ( available online ) describe detailed information about maxquant settings , statistical software packages , and pathway analysis . patient specimens used to develop and validate a prognostic signature were collected in five european institutes between 1985 and 2005 . triple negativity of tumor tissues was confirmed based on mrna expression using real - time quantitative polymerase chain reaction with a reported criterion of estrogen receptor ( esr1 ) less than 0.2 , progesterone receptor ( pgr ) less than 0.1 , and human epidermal growth factor receptor ( her2/erbb2 ) genes bb2 less than 18 ( 12,13 ) . with this criterion , we identified 271 and 61 tnbc tumor tissues from local tissue bank and european organization for research and treatment of cancer ( eortc ) collaborators , respectively . tissues used in this study were restricted to lnn patients who had not received systemic adjuvant therapy . patients who developed distant metastasis as first event within 5 years after removal of the primary tumor were considered to have poor prognosis , whereas patients who remained free of distant metastasis for 5 years were defined as having good prognosis . in addition , inclusion for microdissection and mass spectrometry ( ms ) profiling depended on sufficient invasive tumor area for microdissection and morphological quality . with the above - mentioned criteria , samples were subsequently rejected on the basis of clinical reasons [ patients were diagnosed with positive lymph - nodes ( 92 ) , received adjuvant chemotherapy ( 3 ) , had insufficient clinical follow - up ( 19 ) , and developed local relapse before distant metastasis ( 26 ) ] and technical reasons [ insufficient tumor tissues ( 33 ) , indistinguishable morphology , and insufficient tumor area for microdissection ( 32 ) ] . as a consequence , 63 and 64 tumors from training and test sets were prepared for nlc - ms / ms profiling , respectively . clinical characteristics of the included ( n = 126 ) and excluded ( n = 206 ) subjects are summarized in supplementary table 1 ( available online ) . nlc - ms / ms data from 63 samples were used for development of the protein signature ( training set ) , and data from the other 63 samples were used for independent validation ( test set ) ( table 1 ; supplementary table 2 , available online ) . this study was approved by the local institutional medical ethics committee ( mec 02.953 ) , and wherever possible we adhered to the reporting recommendations for tumor marker prognostic studies ( 14 ) . emc = erasmus medical center ; eortc = european organization for research and treatment of cancer ; sd = standard deviation . the dedicated procedure of sample preparation was performed ( 15 ) ( supplementary methods , available online ) . tissue samples were cut into 8-m frozen sections and then microdissected to obtain approximately 4000 breast cancer epithelial cells . proteins were extracted from microdissected cells , and this was followed by denaturation , reduction , and alkylation . protein samples were digested at 37c for 4 hours using ms - grade trypsin ( promega , madison , wi ) at a 1:4 ( enzyme / protein ) ratio and then acidified for further analysis . global proteome profiles of the tnbc samples were recorded on an nlc hyphenated ltq - orbitrap - xl ms system ( thermoelectron , bremen , germany ) ( supplementary methods , available online ) . peptide mixtures were separated on the nlc system with a 3-hour binary gradient ( mobile phase a : water ; mobile phase b : acetonitrile ) in a 3-m c18 silica - packed 50-cm capillary column with 75-m inner diameter . mass spectra were acquired over a mass - to - charge ratio ( m / z ) range of 400 to 1800 at a resolving power of 30000 at 400 m / z . the five most intensive parent ions from the full scan were isolated and fragmented by collisional activated dissociation in the linear ion trap . recorded raw nlc - ms / ms data have been submitted to proteomexchange ( accession number : pxd000260 ) . the recorded ms spectra from the training and test set were separately analyzed in maxquant software ( free - ware available from www.maxquant.org , version 1.1.1.36 ) ( 16 ) . to identify a prognostic protein signature , cox regression analysis was performed to associate protein abundance with metastasis - free survival time of patients in the training set . weighted protein abundance of the prognostic proteins was computed by multiplication of their protein abundance and corresponding cox regression coefficients . a relapse score was calculated by summation of weighted protein abundance of all protein(s ) in a given predictive model , followed by z score transformation of summed score . multiple predictive models were constructed from the training set by starting with the protein with the lowest p value and stepwise adding one more protein in the next model . efficiency of these models was then assessed by summation of three parameters : 1 ) area under the curve ( auc ) of receiver operating characteristic ( roc ) curve ( 17 ) , 2 ) youden s index at 100% sensitivity ( sensitivity + specificity 1 ) , and 3 ) reversed model size ( 1 ni / n , where ni is the number of proteins in a defined model , and n is the total number of prognostic proteins ) . the cutoff of the prognostic protein signature was determined from the roc curve of the training set to ensure 100% sensitivity and highest specificity . meier survival curves and log - rank test were performed to evaluate the differences in the time to distant metastasis of predicted good and poor prognosis groups . univariate and multivariable analyses with cox proportional hazard model were used to assess the prognostic value of the protein signature with and without consideration of the individual clinical prognostic variables . pathway analysis was performed to interpret biological function of signature proteins using gene set enrichment analysis ( 18 ) . the supplementary methods ( available online ) describe detailed information about maxquant settings , statistical software packages , and pathway analysis . the training and test samples were profiled by nlc - ms / ms in 2010 and 2012 , respectively ( figure 1 ) . the baseline clinical features of patients were similar between the erasmus university medical center ( emc ) training and multicenter test set , although patients were slightly older and more patients were classified with grade 2 tumors in the test set ( table 1 ) . the median follow - ups for the good - prognosis patients in the training and test set were 117 ( range = 61257 ) and 108 ( range = 61234 ) months , respectively . emc = erasmus university medical center ; eortc = european organization for research and treatment of cancer ; lcm = laser capture microdissection ; nlc - ms / ms = nanoscale liquid chromatography and tandem mass spectrometry . proteome profiles for training and test samples were independently generated by nlc - ms / ms ( see supplementary methods , available online ) . a stringent filtering procedure resulted in a total of 981 proteins for development of a prognostic signature ( figure 1 ; supplementary figure 1 , available online ) . good reproducibility and quantitative precision were observed from quantified proteins in replicate samples by performing the same filtering criteria ( see supplementary methods , available online ) . the 981 proteins were used to develop a prognostic signature in the training set . using univariate cox analysis , a panel of 23 proteins was statistically significantly associated with metastasis - free survival of patients ( p < .01 ) ( figure 1 ; supplementary figure 1 , available online ) . twenty - three prognostic models were derived from these 23 proteins using a multivariable linear regression model , from which an 11-protein model performed fairly comparably with the models with 15 or more proteins based on an auc of roc curve and specificity at 100% sensitivity to predict poor - prognosis patients . by further considering model size , we calculated efficiency for all 23 models , and the 11-protein model showed the highest model efficiency at 2.31 ( figure 2 ) . detailed information for these 11 proteins is listed in table 2 and supplementary table 3 ( available online ) . of these 11 proteins , 10 proteins were upregulated ( cmpk1 , aifm1 , fth1 , eml4 , ganab , ctnna1 , ap1g1 , stx12 , ap1m1 , and capzb ) , whereas one protein was downregulated ( mthfd1 ) in good - prognosis patients in the training set . the roc curve derived from the 11-protein signature showed good sensitivity and specificity with auc of 0.95 ( figure 3a ) . a cutoff was selected with a relapse score of zero to identify good - prognosis patients ( negative score ) at which maximal specificity was reached at 100% sensitivity ( figure 3a , green dot ) . eleven - signature proteins and their prognostic information in training set * adapted from uniprot knowledgebase . p values were computed by cox regression analysis , and the corresponding false discovery rate ( fdr ) of the proteins is reported in parentheses . selection of the best predictive signature from 23 prognostic models developed in the training set . the different models were created from 23 prognostic proteins ( cox regression analysis : p < .01 ) , starting with the protein with the lowest p value and gradually adding one more protein at a time , thereby constructing 23 different prognostic models . model efficiency was considered by three aspects : 1 ) area under the curve ( auc ) of the receiver operating characteristic ( roc ) curve , 2 ) specificity at 100% sensitivity , and 3 ) reversed model size ( 1 1/n , where n is number of used protein[s ] for the model ) . the model with the highest model efficiency was considered as the best model , resulting in selection of the 11-protein signature ( model efficiency = 2.31 ) for validation . a ) receiver operating characteristic ( roc ) curve of the 11-protein signature in the training set ( red solid line : area under the curve [ auc ] ) . a cutoff was chosen to ensure maximal sensitivity to identify the good - prognosis patients with the highest possible specificity in the training set ( green dot ) . other models developed from 23 prognostic proteins were also plotted as roc curves ( black dashed lines ) . meier analysis shows that the 11-protein signature is strongly predictive of metastasis - free survival ( c ) and breast cancer ( bc)related survival ( d ) ci = confidence interval ; hr = hazard ratio . e ) waterfall plot stratifies two groups of triple - negative breast cancer patients with different predicted prognosis in the test set . the 11-protein signature was validated on the test cohort , resulting in an roc curve with auc of 0.83 ( figure 3b ) . using the predetermined cutoff ( relapse score = 0 ) , patients predicted as having a poor prognosis ( positive score ) had much worse 5-year metastasis - free survival ( log - rank p < .001 ) ( figure 3c ) and breast cancer related survival ( log - rank p < .001 ) ( figure 3d ) than predicted good - prognosis patients . in the predicted good - prognosis group , 31 of 33 patients did not develop distant metastasis ( negative predictive value = 93.9% ; 95% confidence interval [ ci ] = 82.3% to 98.9% ) ( figure 3 , c and e ) . in contrast , 17 of 30 patients developed distant metastasis in the predicted poor - prognosis group ( positive predictive value = 56.7% ; 95% ci = 43.8% to 62.1% ) ( figure 3 , c and e ) . overall , our signature results in a sensitivity of 89.5% ( 95% ci = 69.2% to 98.1% ) and specificity of 70.5% ( 95% ci = 61.7% to 74.2% ) to predict poor - prognosis patients in the test set . in univariate cox analyses , the poor - prognosis patients also have statistically significantly higher risk of developing distant metastases ( hazard ratio [ hr ] = 13.15 ; 95% ci = 3.03 to 57.07 ; p = .001 ) ( figure 3c ; table 3 ) and breast cancer related death ( hr = 22.78 ; 95% ci = 3.00 to 173.08 ; p = .003 ) ( figure 3d ; table 3 ) than those predicted to have good prognosis . the 11-protein signature was also prognostic in subgroups of patients with different menopausal status ( figure 4 , a and b ) and tumor size ( figure 4 , c and d ) . furthermore , the 11-protein signature was a strong independent prognostic factor to predict risk of distant metastasis ( hr = 12.45 ; 95% ci = 2.67 to 58.11 ; p = .001 ) and of breast cancer related death ( hr = 36.08 ; 95% ci = 4.00 to 325.67 ; p = .001 ) in multivariable cox regression model after correction for the contribution of traditional prognostic factors ( table 3 ) . no other prognostic factors , such as age , menopausal status , tumor size , or tumor grade of the patients , were statistically significantly associated with metastasis - free survival in univariate and multivariable analyses ( table 3 ) . univariate and multivariable analyses * of the 11-protein signature for its prognostic value ( n = 63 ) * univariate and multivariable analyses were performed based on cox regression model . kaplan meier analysis of metastasis - free survival in subgroups of triple - negative breast cancer patients in the test set . hazard ratio ( hr ) and 95% confidence interval ( ci ) could not be computed because of the absence of metastatic events in one of the tested groups . hazard ratio and 95% confidence interval could not be computed because of the absence of metastatic events in one of the tested groups . two clinical consensus criteria , st . gallen ( 19 ) and national institutes of health ( nih ) ( 20 ) , are often applied to guide treatment of breast cancer cases . in our test set , 91% ( n = 40 of 44 ) and 95% ( n = 38 of 40 ) of the good - prognosis patients would be classified as high risk and therefore would be guided to receive apparently unnecessary adjuvant chemotherapy using st . gallen and nih criteria , respectively ( table 4 ) . on the other hand , only 30% ( n = 13 of 44 ) of the good - prognosis patients would be classified for adjuvant chemotherapy using the 11-protein signature ( table 4 ) . therefore , more than 60% of patients in the test set would have been exempted from unnecessary adjuvant chemotherapy using the 11-protein signature as guidance , compared with st . comparison of the 11-protein signature with currently applied clinical consensus criteria on treatment of breast cancer * st . gallen consensus criteria : tumor 2 cm , esr1 negative , grade 23 , patient aged < 35 years ( one of these criteria ) . patients with missing clinical information were excluded from these analyses . a statistically significant improvement . the function of the 11 signature proteins related to progression of tnbc was interpreted in silico by gene set enrichment analysis . to overcome low identification rate of our proteomic data ( see supplementary methods , available online ) , we used gene expression data from 63 in - house available tnbc samples , of which 47 samples were also included in our global proteome profiling . an overall statistically significant correlation between transcriptome and proteome in the 47 samples indicated the validity of using gene expression to interpret the molecular functions related to tnbc progression and expression of signature proteins ( see supplementary data , available online ) . in total , biological pathways related to good prognosis of the tnbc patients were mainly associated with immune response ( eg , modulation of cytokines , antigen processing and presentation , and activation of t cells , b cells , and natural killer cells ) and cell death ( eg , ceramide signaling , tumor necrosis factor mediated apoptosis , fas - fas ligand mediated apoptosis , and caspase cascade ) . nine of 10 genes were associated with good prognosis of tnbc patients ( cmpk1 , aifm1 , fth1 , eml4 , ganab , ctnna1 , ap1g1 , stx12 , and capzb ) , of which fth1 , ganab , and stx12 were associated with 58 , 18 , and three enriched pathways related to immune response or cell death with recommended false discovery rate less than 0.25 ( supplementary table 4 , available online ) . on the other hand , cell metabolism ( eg , metabolism of nucleotides and noncoding rna ) and transport of macromolecules ( eg , transport of mature transcripts and ribonucleoproteins and export of proteins ) were key pathways related to poor prognosis of tnbc patients . mthfd1 was the only protein upregulated in tnbc patients with poor prognosis and was associated with two metabolic pathways ( metabolism of nucleotides and noncoding rna ) with false discovery rate less than 0.25 ( supplementary table 4 , available online ) . proteome profiles for training and test samples were independently generated by nlc - ms / ms ( see supplementary methods , available online ) . a stringent filtering procedure resulted in a total of 981 proteins for development of a prognostic signature ( figure 1 ; supplementary figure 1 , available online ) . good reproducibility and quantitative precision were observed from quantified proteins in replicate samples by performing the same filtering criteria ( see supplementary methods , available online ) . using univariate cox analysis , a panel of 23 proteins was statistically significantly associated with metastasis - free survival of patients ( p < .01 ) ( figure 1 ; supplementary figure 1 , available online ) . twenty - three prognostic models were derived from these 23 proteins using a multivariable linear regression model , from which an 11-protein model performed fairly comparably with the models with 15 or more proteins based on an auc of roc curve and specificity at 100% sensitivity to predict poor - prognosis patients . by further considering model size , we calculated efficiency for all 23 models , and the 11-protein model showed the highest model efficiency at 2.31 ( figure 2 ) . detailed information for these 11 proteins is listed in table 2 and supplementary table 3 ( available online ) . of these 11 proteins , 10 proteins were upregulated ( cmpk1 , aifm1 , fth1 , eml4 , ganab , ctnna1 , ap1g1 , stx12 , ap1m1 , and capzb ) , whereas one protein was downregulated ( mthfd1 ) in good - prognosis patients in the training set . the roc curve derived from the 11-protein signature showed good sensitivity and specificity with auc of 0.95 ( figure 3a ) . a cutoff was selected with a relapse score of zero to identify good - prognosis patients ( negative score ) at which maximal specificity was reached at 100% sensitivity ( figure 3a , green dot ) . eleven - signature proteins and their prognostic information in training set * adapted from uniprot knowledgebase . p values were computed by cox regression analysis , and the corresponding false discovery rate ( fdr ) of the proteins is reported in parentheses . selection of the best predictive signature from 23 prognostic models developed in the training set . the different models were created from 23 prognostic proteins ( cox regression analysis : p < .01 ) , starting with the protein with the lowest p value and gradually adding one more protein at a time , thereby constructing 23 different prognostic models . model efficiency was considered by three aspects : 1 ) area under the curve ( auc ) of the receiver operating characteristic ( roc ) curve , 2 ) specificity at 100% sensitivity , and 3 ) reversed model size ( 1 1/n , where n is number of used protein[s ] for the model ) . the model with the highest model efficiency was considered as the best model , resulting in selection of the 11-protein signature ( model efficiency = 2.31 ) for validation . a ) receiver operating characteristic ( roc ) curve of the 11-protein signature in the training set ( red solid line : area under the curve [ auc ] ) . a cutoff was chosen to ensure maximal sensitivity to identify the good - prognosis patients with the highest possible specificity in the training set ( green dot ) . other models developed from 23 prognostic proteins were also plotted as roc curves ( black dashed lines ) . meier analysis shows that the 11-protein signature is strongly predictive of metastasis - free survival ( c ) and breast cancer ( bc)related survival ( d ) ci = confidence interval ; hr = hazard ratio . e ) waterfall plot stratifies two groups of triple - negative breast cancer patients with different predicted prognosis in the test set . the 11-protein signature was validated on the test cohort , resulting in an roc curve with auc of 0.83 ( figure 3b ) . using the predetermined cutoff ( relapse score = 0 ) , patients predicted as having a poor prognosis ( positive score ) had much worse 5-year metastasis - free survival ( log - rank p < .001 ) ( figure 3c ) and breast cancer related survival ( log - rank p < .001 ) ( figure 3d ) than predicted good - prognosis patients . in the predicted good - prognosis group , 31 of 33 patients did not develop distant metastasis ( negative predictive value = 93.9% ; 95% confidence interval [ ci ] = 82.3% to 98.9% ) ( figure 3 , c and e ) . in contrast , 17 of 30 patients developed distant metastasis in the predicted poor - prognosis group ( positive predictive value = 56.7% ; 95% ci = 43.8% to 62.1% ) ( figure 3 , c and e ) . overall , our signature results in a sensitivity of 89.5% ( 95% ci = 69.2% to 98.1% ) and specificity of 70.5% ( 95% ci = 61.7% to 74.2% ) to predict poor - prognosis patients in the test set . in univariate cox analyses , the poor - prognosis patients also have statistically significantly higher risk of developing distant metastases ( hazard ratio [ hr ] = 13.15 ; 95% ci = 3.03 to 57.07 ; p = .001 ) ( figure 3c ; table 3 ) and breast cancer related death ( hr = 22.78 ; 95% ci = 3.00 to 173.08 ; p = .003 ) ( figure 3d ; table 3 ) than those predicted to have good prognosis . the 11-protein signature was also prognostic in subgroups of patients with different menopausal status ( figure 4 , a and b ) and tumor size ( figure 4 , c and d ) . furthermore , the 11-protein signature was a strong independent prognostic factor to predict risk of distant metastasis ( hr = 12.45 ; 95% ci = 2.67 to 58.11 ; p = .001 ) and of breast cancer related death ( hr = 36.08 ; 95% ci = 4.00 to 325.67 ; p = .001 ) in multivariable cox regression model after correction for the contribution of traditional prognostic factors ( table 3 ) . no other prognostic factors , such as age , menopausal status , tumor size , or tumor grade of the patients , were statistically significantly associated with metastasis - free survival in univariate and multivariable analyses ( table 3 ) . univariate and multivariable analyses * of the 11-protein signature for its prognostic value ( n = 63 ) * univariate and multivariable analyses were performed based on cox regression model . kaplan meier analysis of metastasis - free survival in subgroups of triple - negative breast cancer patients in the test set . hazard ratio ( hr ) and 95% confidence interval ( ci ) could not be computed because of the absence of metastatic events in one of the tested groups . hazard ratio and 95% confidence interval could not be computed because of the absence of metastatic events in one of the tested groups . two clinical consensus criteria , st . gallen ( 19 ) and national institutes of health ( nih ) ( 20 ) , are often applied to guide treatment of breast cancer cases . in our test set , 91% ( n = 40 of 44 ) and 95% ( n = 38 of 40 ) of the good - prognosis patients would be classified as high risk and therefore would be guided to receive apparently unnecessary adjuvant chemotherapy using st . gallen and nih criteria , respectively ( table 4 ) . on the other hand , only 30% ( n = 13 of 44 ) of the good - prognosis patients would be classified for adjuvant chemotherapy using the 11-protein signature ( table 4 ) . therefore , more than 60% of patients in the test set would have been exempted from unnecessary adjuvant chemotherapy using the 11-protein signature as guidance , compared with st . comparison of the 11-protein signature with currently applied clinical consensus criteria on treatment of breast cancer * st . gallen consensus criteria : tumor 2 cm , esr1 negative , grade 23 , patient aged < 35 years ( one of these criteria ) . patients with missing clinical information were excluded from these analyses . a statistically significant improvement . the function of the 11 signature proteins related to progression of tnbc was interpreted in silico by gene set enrichment analysis . to overcome low identification rate of our proteomic data ( see supplementary methods , available online ) , we used gene expression data from 63 in - house available tnbc samples , of which 47 samples were also included in our global proteome profiling . an overall statistically significant correlation between transcriptome and proteome in the 47 samples indicated the validity of using gene expression to interpret the molecular functions related to tnbc progression and expression of signature proteins ( see supplementary data , available online ) . in total biological pathways related to good prognosis of the tnbc patients were mainly associated with immune response ( eg , modulation of cytokines , antigen processing and presentation , and activation of t cells , b cells , and natural killer cells ) and cell death ( eg , ceramide signaling , tumor necrosis factor mediated apoptosis , fas - fas ligand mediated apoptosis , and caspase cascade ) . nine of 10 genes were associated with good prognosis of tnbc patients ( cmpk1 , aifm1 , fth1 , eml4 , ganab , ctnna1 , ap1g1 , stx12 , and capzb ) , of which fth1 , ganab , and stx12 were associated with 58 , 18 , and three enriched pathways related to immune response or cell death with recommended false discovery rate less than 0.25 ( supplementary table 4 , available online ) . on the other hand , cell metabolism ( eg , metabolism of nucleotides and noncoding rna ) and transport of macromolecules ( eg , transport of mature transcripts and ribonucleoproteins and export of proteins ) were key pathways related to poor prognosis of tnbc patients . mthfd1 was the only protein upregulated in tnbc patients with poor prognosis and was associated with two metabolic pathways ( metabolism of nucleotides and noncoding rna ) with false discovery rate less than 0.25 ( supplementary table 4 , available online ) . prognosis of lnn tnbc patients is extremely heterogeneous and often not associated with conventional prognostic parameters ( patient age , tumor size , and tumor grade ) ( 21 ) , concordant with this study . therefore , a substantial proportion of patients are being overtreated with systemic adjuvant therapy . in this study , we identified and independently validated a clinically relevant prognostic 11-protein signature for tnbc , which could be used to reduce the number of patients that would receive unnecessary adjuvant chemotherapy . to translate the 11-protein signature into a clinically useful assay , first , a quantitative , targeted , and ready - to - use assay needs to be developed for absolute quantification of the signature proteins . the most promising candidate for such an assay would be a mass spectrometry based selected reaction monitoring assay ( 22 ) . second , such a targeted assay can then be incorporated into future clinical trials and subsequently used for clinical decision making for treatment options . furthermore , some of the signature proteins may serve as potential targets for novel therapies . some signature proteins have been reported to be prognostic in breast cancer or other cancers . for instance , decreased levels of fth1 protein have been associated with lymph node metastasis in colorectal cancer ( 23 ) . similarly , abnormal expression of ctnna1 protein has been related with poor patient survival in invasive breast cancer ( 24 ) . also , association has been observed between increased expression of ganab protein and favorable prognosis in head and neck cancer ( 25 ) . single nucleotide polymorphisms of cmpk1 have been associated with prognosis of non small cell lung cancer ( 26 ) and pancreatic cancer ( 27 ) patients treated with gemcitabine - based chemotherapeutics . a correlation has also been reported between a 1958aa single nucleotide polymorphism of mthfd1 gene and poor clinical outcome of premenopausal breast cancer patients ( 28 ) . moreover eml - alk fusion gene has been suggested to be associated with relatively favorable prognosis of non small cell lung cancer patients ( 29 ) . we speculate that there may be a correlation between expression of these proteins and their genetic variance , which needs to be further investigated . pathway analysis showed that immune response , cell death , cell metabolism , and transport of macromolecules are the major underlying molecular mechanisms of the signature proteins related to prognosis of tnbc patients . lehmann and colleagues identified an immunomodulatory subtype that was associated with favorable clinical outcome ( 30 ) . rody and colleagues identified a signature of high b - cell and low il-8 metagenes that predicted good prognosis of tnbc patients ( 5 ) , and yau and coworkers reported a 14-gene signature linked to immune / inflammatory cytokine regulation in which the majority of genes were associated with good prognosis of tnbc patients ( 8) . in our prognostic signature , three proteins ( fth1 , ganab , and stx12 ) are associated with immunomodulation and cell death associated pathways . fth1 is an iron storage protein and indirectly regulates the ratio of cd4 t cells and cd8 t cells by altering iron distribution ( 31 ) . increased cd8 t cells may help to eliminate tumor cells ( 32 ) , which would be favorable for survival of tnbc patients . molecular functions of ganab and stx12 related to immune response and cell death have not been well studied . therefore , further studies are required to understand the functions of these proteins in disease progression . on the other hand , it is known that the luminal androgen receptor subtype of tnbc , with aberrant alteration of cell metabolism , is related to adverse clinical outcome of tnbc patients ( 30 ) . in our signature , mthfd1 is associated with poor prognosis and is involved in folate metabolism . it has been suggested that increased concentration of folate resulted in a dose - dependent downregulation of tumor suppressor genes in breast cancer cell lines due to increased dna methylation of their promoter region ( 33 ) , which may indicate the importance of mthfd1 in tnbc progression . first , microdissection - based sample preparation is tedious and may introduce additional biases between diagnostic individuals and laboratories . second , the nlc - ms / ms based platform is difficult to apply in routine clinical practice . therefore , our future work will focus on developing simple sampling strategies and high - throughput selected reaction monitoring assay to reliably measure the 11-protein signature . in summary , we have developed an 11-protein signature to predict tnbc patients who develop a distant metastasis with high sensitivity , specificity , positive predictive value , and negative predictive value ( 82.3% to 98.9% ) . therefore , our signature could aid in clinical practice to avoid unnecessary treatment with adjuvant chemotherapy of lnn tnbc patients . future prospective clinical trials are needed to further consolidate the validity of the 11-protein signature . this study was financially supported by the netherlands genomics initiative / netherlands organisation for scientific research ( the hague , the netherlands ) , center for translational molecular medicine ( eindhoven , the netherlands ) breast care project 030 - 104 , and dutch cancer society , ( amsterdam , the netherlands ) project number emcr 20094319 .

backgroundclinical outcome of patients with triple - negative breast cancer ( tnbc ) is highly variable . this study aims to identify and validate a prognostic protein signature for tnbc patients to reduce unnecessary adjuvant systemic therapy.methodsfrozen primary tumors were collected from 126 lymph node negative and adjuvant therapy naive tnbc patients . these samples were used for global proteome profiling in two series : an in - house training ( n = 63 ) and a multicenter test ( n = 63 ) set . patients who remained free of distant metastasis for a minimum of 5 years after surgery were defined as having good prognosis . cox regression analysis was performed to develop a prognostic signature , which was independently validated . all statistical tests were two-sided.resultsan 11-protein signature was developed in the training set ( median follow - up for good - prognosis patients = 117 months ) and subsequently validated in the test set ( median follow - up for good - prognosis patients = 108 months ) showing 89.5% sensitivity ( 95% confidence interval [ ci ] = 69.2% to 98.1% ) , 70.5% specificity ( 95% ci = 61.7% to 74.2% ) , 56.7% positive predictive value ( 95% ci = 43.8% to 62.1% ) , and 93.9% negative predictive value ( 95% ci = 82.3% to 98.9% ) for poor - prognosis patients . the predicted poor - prognosis patients had higher risk to develop distant metastasis than the predicted good - prognosis patients in univariate ( hazard ratio [ hr ] = 13.15 ; 95% ci = 3.03 to 57.07 ; p = .001 ) and multivariable ( hr = 12.45 ; 95% ci = 2.67 to 58.11 ; p = .001 ) analysis . furthermore , the predicted poor - prognosis group had statistically significantly more breast cancer specific mortality . using our signature as guidance , more than 60% of patients would have been exempted from unnecessary adjuvant chemotherapy compared with conventional prognostic guidelines.conclusionswe report the first validated proteomic signature to assess the natural course of clinical tnbc .

Methods Study Design Experimental Procedures Statistical Analysis Results Development of a Proteomic Prognostic Signature for TNBC Patients Validation of the 11-Protein Signature in a Multicenter Cohort of TNBC Patients Pathway Analysis of Prognostic Signature Discussion Funding

patient specimens used to develop and validate a prognostic signature were collected in five european institutes between 1985 and 2005 . tissues used in this study were restricted to lnn patients who had not received systemic adjuvant therapy . patients who developed distant metastasis as first event within 5 years after removal of the primary tumor were considered to have poor prognosis , whereas patients who remained free of distant metastasis for 5 years were defined as having good prognosis . with the above - mentioned criteria , samples were subsequently rejected on the basis of clinical reasons [ patients were diagnosed with positive lymph - nodes ( 92 ) , received adjuvant chemotherapy ( 3 ) , had insufficient clinical follow - up ( 19 ) , and developed local relapse before distant metastasis ( 26 ) ] and technical reasons [ insufficient tumor tissues ( 33 ) , indistinguishable morphology , and insufficient tumor area for microdissection ( 32 ) ] . nlc - ms / ms data from 63 samples were used for development of the protein signature ( training set ) , and data from the other 63 samples were used for independent validation ( test set ) ( table 1 ; supplementary table 2 , available online ) . to identify a prognostic protein signature , cox regression analysis was performed to associate protein abundance with metastasis - free survival time of patients in the training set . efficiency of these models was then assessed by summation of three parameters : 1 ) area under the curve ( auc ) of receiver operating characteristic ( roc ) curve ( 17 ) , 2 ) youden s index at 100% sensitivity ( sensitivity + specificity 1 ) , and 3 ) reversed model size ( 1 ni / n , where ni is the number of proteins in a defined model , and n is the total number of prognostic proteins ) . the cutoff of the prognostic protein signature was determined from the roc curve of the training set to ensure 100% sensitivity and highest specificity . meier survival curves and log - rank test were performed to evaluate the differences in the time to distant metastasis of predicted good and poor prognosis groups . univariate and multivariable analyses with cox proportional hazard model were used to assess the prognostic value of the protein signature with and without consideration of the individual clinical prognostic variables . patient specimens used to develop and validate a prognostic signature were collected in five european institutes between 1985 and 2005 . patients who developed distant metastasis as first event within 5 years after removal of the primary tumor were considered to have poor prognosis , whereas patients who remained free of distant metastasis for 5 years were defined as having good prognosis . with the above - mentioned criteria , samples were subsequently rejected on the basis of clinical reasons [ patients were diagnosed with positive lymph - nodes ( 92 ) , received adjuvant chemotherapy ( 3 ) , had insufficient clinical follow - up ( 19 ) , and developed local relapse before distant metastasis ( 26 ) ] and technical reasons [ insufficient tumor tissues ( 33 ) , indistinguishable morphology , and insufficient tumor area for microdissection ( 32 ) ] . nlc - ms / ms data from 63 samples were used for development of the protein signature ( training set ) , and data from the other 63 samples were used for independent validation ( test set ) ( table 1 ; supplementary table 2 , available online ) . to identify a prognostic protein signature , cox regression analysis was performed to associate protein abundance with metastasis - free survival time of patients in the training set . multiple predictive models were constructed from the training set by starting with the protein with the lowest p value and stepwise adding one more protein in the next model . efficiency of these models was then assessed by summation of three parameters : 1 ) area under the curve ( auc ) of receiver operating characteristic ( roc ) curve ( 17 ) , 2 ) youden s index at 100% sensitivity ( sensitivity + specificity 1 ) , and 3 ) reversed model size ( 1 ni / n , where ni is the number of proteins in a defined model , and n is the total number of prognostic proteins ) . the cutoff of the prognostic protein signature was determined from the roc curve of the training set to ensure 100% sensitivity and highest specificity . meier survival curves and log - rank test were performed to evaluate the differences in the time to distant metastasis of predicted good and poor prognosis groups . univariate and multivariable analyses with cox proportional hazard model were used to assess the prognostic value of the protein signature with and without consideration of the individual clinical prognostic variables . pathway analysis was performed to interpret biological function of signature proteins using gene set enrichment analysis ( 18 ) . the baseline clinical features of patients were similar between the erasmus university medical center ( emc ) training and multicenter test set , although patients were slightly older and more patients were classified with grade 2 tumors in the test set ( table 1 ) . the median follow - ups for the good - prognosis patients in the training and test set were 117 ( range = 61257 ) and 108 ( range = 61234 ) months , respectively . the 981 proteins were used to develop a prognostic signature in the training set . twenty - three prognostic models were derived from these 23 proteins using a multivariable linear regression model , from which an 11-protein model performed fairly comparably with the models with 15 or more proteins based on an auc of roc curve and specificity at 100% sensitivity to predict poor - prognosis patients . of these 11 proteins , 10 proteins were upregulated ( cmpk1 , aifm1 , fth1 , eml4 , ganab , ctnna1 , ap1g1 , stx12 , ap1m1 , and capzb ) , whereas one protein was downregulated ( mthfd1 ) in good - prognosis patients in the training set . a cutoff was selected with a relapse score of zero to identify good - prognosis patients ( negative score ) at which maximal specificity was reached at 100% sensitivity ( figure 3a , green dot ) . p values were computed by cox regression analysis , and the corresponding false discovery rate ( fdr ) of the proteins is reported in parentheses . selection of the best predictive signature from 23 prognostic models developed in the training set . a ) receiver operating characteristic ( roc ) curve of the 11-protein signature in the training set ( red solid line : area under the curve [ auc ] ) . a cutoff was chosen to ensure maximal sensitivity to identify the good - prognosis patients with the highest possible specificity in the training set ( green dot ) . meier analysis shows that the 11-protein signature is strongly predictive of metastasis - free survival ( c ) and breast cancer ( bc)related survival ( d ) ci = confidence interval ; hr = hazard ratio . e ) waterfall plot stratifies two groups of triple - negative breast cancer patients with different predicted prognosis in the test set . using the predetermined cutoff ( relapse score = 0 ) , patients predicted as having a poor prognosis ( positive score ) had much worse 5-year metastasis - free survival ( log - rank p < .001 ) ( figure 3c ) and breast cancer related survival ( log - rank p < .001 ) ( figure 3d ) than predicted good - prognosis patients . in the predicted good - prognosis group , 31 of 33 patients did not develop distant metastasis ( negative predictive value = 93.9% ; 95% confidence interval [ ci ] = 82.3% to 98.9% ) ( figure 3 , c and e ) . in contrast , 17 of 30 patients developed distant metastasis in the predicted poor - prognosis group ( positive predictive value = 56.7% ; 95% ci = 43.8% to 62.1% ) ( figure 3 , c and e ) . overall , our signature results in a sensitivity of 89.5% ( 95% ci = 69.2% to 98.1% ) and specificity of 70.5% ( 95% ci = 61.7% to 74.2% ) to predict poor - prognosis patients in the test set . in univariate cox analyses , the poor - prognosis patients also have statistically significantly higher risk of developing distant metastases ( hazard ratio [ hr ] = 13.15 ; 95% ci = 3.03 to 57.07 ; p = .001 ) ( figure 3c ; table 3 ) and breast cancer related death ( hr = 22.78 ; 95% ci = 3.00 to 173.08 ; p = .003 ) ( figure 3d ; table 3 ) than those predicted to have good prognosis . the 11-protein signature was also prognostic in subgroups of patients with different menopausal status ( figure 4 , a and b ) and tumor size ( figure 4 , c and d ) . furthermore , the 11-protein signature was a strong independent prognostic factor to predict risk of distant metastasis ( hr = 12.45 ; 95% ci = 2.67 to 58.11 ; p = .001 ) and of breast cancer related death ( hr = 36.08 ; 95% ci = 4.00 to 325.67 ; p = .001 ) in multivariable cox regression model after correction for the contribution of traditional prognostic factors ( table 3 ) . no other prognostic factors , such as age , menopausal status , tumor size , or tumor grade of the patients , were statistically significantly associated with metastasis - free survival in univariate and multivariable analyses ( table 3 ) . univariate and multivariable analyses * of the 11-protein signature for its prognostic value ( n = 63 ) * univariate and multivariable analyses were performed based on cox regression model . kaplan meier analysis of metastasis - free survival in subgroups of triple - negative breast cancer patients in the test set . hazard ratio ( hr ) and 95% confidence interval ( ci ) could not be computed because of the absence of metastatic events in one of the tested groups . hazard ratio and 95% confidence interval could not be computed because of the absence of metastatic events in one of the tested groups . in our test set , 91% ( n = 40 of 44 ) and 95% ( n = 38 of 40 ) of the good - prognosis patients would be classified as high risk and therefore would be guided to receive apparently unnecessary adjuvant chemotherapy using st . on the other hand , only 30% ( n = 13 of 44 ) of the good - prognosis patients would be classified for adjuvant chemotherapy using the 11-protein signature ( table 4 ) . therefore , more than 60% of patients in the test set would have been exempted from unnecessary adjuvant chemotherapy using the 11-protein signature as guidance , compared with st . to overcome low identification rate of our proteomic data ( see supplementary methods , available online ) , we used gene expression data from 63 in - house available tnbc samples , of which 47 samples were also included in our global proteome profiling . in total , biological pathways related to good prognosis of the tnbc patients were mainly associated with immune response ( eg , modulation of cytokines , antigen processing and presentation , and activation of t cells , b cells , and natural killer cells ) and cell death ( eg , ceramide signaling , tumor necrosis factor mediated apoptosis , fas - fas ligand mediated apoptosis , and caspase cascade ) . nine of 10 genes were associated with good prognosis of tnbc patients ( cmpk1 , aifm1 , fth1 , eml4 , ganab , ctnna1 , ap1g1 , stx12 , and capzb ) , of which fth1 , ganab , and stx12 were associated with 58 , 18 , and three enriched pathways related to immune response or cell death with recommended false discovery rate less than 0.25 ( supplementary table 4 , available online ) . of these 11 proteins , 10 proteins were upregulated ( cmpk1 , aifm1 , fth1 , eml4 , ganab , ctnna1 , ap1g1 , stx12 , ap1m1 , and capzb ) , whereas one protein was downregulated ( mthfd1 ) in good - prognosis patients in the training set . a cutoff was selected with a relapse score of zero to identify good - prognosis patients ( negative score ) at which maximal specificity was reached at 100% sensitivity ( figure 3a , green dot ) . selection of the best predictive signature from 23 prognostic models developed in the training set . a ) receiver operating characteristic ( roc ) curve of the 11-protein signature in the training set ( red solid line : area under the curve [ auc ] ) . a cutoff was chosen to ensure maximal sensitivity to identify the good - prognosis patients with the highest possible specificity in the training set ( green dot ) . meier analysis shows that the 11-protein signature is strongly predictive of metastasis - free survival ( c ) and breast cancer ( bc)related survival ( d ) ci = confidence interval ; hr = hazard ratio . e ) waterfall plot stratifies two groups of triple - negative breast cancer patients with different predicted prognosis in the test set . the 11-protein signature was validated on the test cohort , resulting in an roc curve with auc of 0.83 ( figure 3b ) . using the predetermined cutoff ( relapse score = 0 ) , patients predicted as having a poor prognosis ( positive score ) had much worse 5-year metastasis - free survival ( log - rank p < .001 ) ( figure 3c ) and breast cancer related survival ( log - rank p < .001 ) ( figure 3d ) than predicted good - prognosis patients . in the predicted good - prognosis group , 31 of 33 patients did not develop distant metastasis ( negative predictive value = 93.9% ; 95% confidence interval [ ci ] = 82.3% to 98.9% ) ( figure 3 , c and e ) . in contrast , 17 of 30 patients developed distant metastasis in the predicted poor - prognosis group ( positive predictive value = 56.7% ; 95% ci = 43.8% to 62.1% ) ( figure 3 , c and e ) . overall , our signature results in a sensitivity of 89.5% ( 95% ci = 69.2% to 98.1% ) and specificity of 70.5% ( 95% ci = 61.7% to 74.2% ) to predict poor - prognosis patients in the test set . in univariate cox analyses , the poor - prognosis patients also have statistically significantly higher risk of developing distant metastases ( hazard ratio [ hr ] = 13.15 ; 95% ci = 3.03 to 57.07 ; p = .001 ) ( figure 3c ; table 3 ) and breast cancer related death ( hr = 22.78 ; 95% ci = 3.00 to 173.08 ; p = .003 ) ( figure 3d ; table 3 ) than those predicted to have good prognosis . the 11-protein signature was also prognostic in subgroups of patients with different menopausal status ( figure 4 , a and b ) and tumor size ( figure 4 , c and d ) . furthermore , the 11-protein signature was a strong independent prognostic factor to predict risk of distant metastasis ( hr = 12.45 ; 95% ci = 2.67 to 58.11 ; p = .001 ) and of breast cancer related death ( hr = 36.08 ; 95% ci = 4.00 to 325.67 ; p = .001 ) in multivariable cox regression model after correction for the contribution of traditional prognostic factors ( table 3 ) . no other prognostic factors , such as age , menopausal status , tumor size , or tumor grade of the patients , were statistically significantly associated with metastasis - free survival in univariate and multivariable analyses ( table 3 ) . univariate and multivariable analyses * of the 11-protein signature for its prognostic value ( n = 63 ) * univariate and multivariable analyses were performed based on cox regression model . kaplan meier analysis of metastasis - free survival in subgroups of triple - negative breast cancer patients in the test set . hazard ratio ( hr ) and 95% confidence interval ( ci ) could not be computed because of the absence of metastatic events in one of the tested groups . in our test set , 91% ( n = 40 of 44 ) and 95% ( n = 38 of 40 ) of the good - prognosis patients would be classified as high risk and therefore would be guided to receive apparently unnecessary adjuvant chemotherapy using st . on the other hand , only 30% ( n = 13 of 44 ) of the good - prognosis patients would be classified for adjuvant chemotherapy using the 11-protein signature ( table 4 ) . therefore , more than 60% of patients in the test set would have been exempted from unnecessary adjuvant chemotherapy using the 11-protein signature as guidance , compared with st . to overcome low identification rate of our proteomic data ( see supplementary methods , available online ) , we used gene expression data from 63 in - house available tnbc samples , of which 47 samples were also included in our global proteome profiling . in total biological pathways related to good prognosis of the tnbc patients were mainly associated with immune response ( eg , modulation of cytokines , antigen processing and presentation , and activation of t cells , b cells , and natural killer cells ) and cell death ( eg , ceramide signaling , tumor necrosis factor mediated apoptosis , fas - fas ligand mediated apoptosis , and caspase cascade ) . nine of 10 genes were associated with good prognosis of tnbc patients ( cmpk1 , aifm1 , fth1 , eml4 , ganab , ctnna1 , ap1g1 , stx12 , and capzb ) , of which fth1 , ganab , and stx12 were associated with 58 , 18 , and three enriched pathways related to immune response or cell death with recommended false discovery rate less than 0.25 ( supplementary table 4 , available online ) . prognosis of lnn tnbc patients is extremely heterogeneous and often not associated with conventional prognostic parameters ( patient age , tumor size , and tumor grade ) ( 21 ) , concordant with this study . in this study , we identified and independently validated a clinically relevant prognostic 11-protein signature for tnbc , which could be used to reduce the number of patients that would receive unnecessary adjuvant chemotherapy . rody and colleagues identified a signature of high b - cell and low il-8 metagenes that predicted good prognosis of tnbc patients ( 5 ) , and yau and coworkers reported a 14-gene signature linked to immune / inflammatory cytokine regulation in which the majority of genes were associated with good prognosis of tnbc patients ( 8) . increased cd8 t cells may help to eliminate tumor cells ( 32 ) , which would be favorable for survival of tnbc patients . in summary , we have developed an 11-protein signature to predict tnbc patients who develop a distant metastasis with high sensitivity , specificity , positive predictive value , and negative predictive value ( 82.3% to 98.9% ) . this study was financially supported by the netherlands genomics initiative / netherlands organisation for scientific research ( the hague , the netherlands ) , center for translational molecular medicine ( eindhoven , the netherlands ) breast care project 030 - 104 , and dutch cancer society , ( amsterdam , the netherlands ) project number emcr 20094319 .

the consolidation phase of motor skill learning involves the transformation of memories from a trace or engram [ 16 ] , established through an encoding or acquisition phase , to a more enhanced or stabile long - term memory [ 1 , 7 ] . motor memory plasticity appears to have a significant time - dependent component required for the consolidation of a trained skill or movement [ 8 , 9 ] and the potential interactions between exercise and sleep may well influence the consolidation process but are currently unknown . the interplay of factors during consolidation including rest , sleep , alternative tasks , and exercise is complicated as there also appears to be different temporal aspects of consolidation itself : synaptic and systems consolidation [ 3 , 10 , 11 ] . the different temporal phases of motor memory consolidation are therefore complex and studies are only beginning to provide evidence of how exercise may affect these phases and how the underlying cellular mechanisms are affected . it can be argued that exercise prior to motor skill acquisition mainly influences acquisition itself potentially via increased awareness [ 14 , 15 ] and/or a priming effect , but it can possibly affect consolidation , although this is hard to differentiate . however , exercise after acquisition can only affect consolidation . an acute bout of high intensity exercise has been shown to improve motor skill learning when carried out in the period following [ 13 , 17 , 18 ] and preceding acquisition of the skill when measured with delayed retention tests . exercise placed after acquisition demonstrates the greatest effect on the consolidation and the strengthening of this type of procedural memory [ 12 , 17 , 18 ] . increases in peripheral ( and central ) availability of neurotrophic factors , energy substrates , and signaling molecules are capable of influencing memory consolidation . transient changes in corticospinal excitability ( cse ) and intracortical inhibition and facilitation in the primary motor cortex , relating to motor skill learning , have also provided evidence to suggest that these mechanisms may be enhanced or protected by exercise [ 2022 ] . exercise , therefore , has the potential to influence memory processes by affecting the underlying cellular mechanisms and networks resulting in robust , long - term behavioural changes and thereby functioning as an endogenous neuroenhancement strategy to facilitate motor learning . the literature supports the idea that exercise intensity plays a central role for improvements in motor performance [ 15 , 24 ] , but the importance of the temporal placement of exercise in relation to motor practice has not been thoroughly investigated . muellbacher and coworkers demonstrated that repetitive transcranial magnetic stimulation ( rtms ) caused interference when placed immediately following motor learning but not 6 h later and more recently lundbye - jensen and coworkers reported a shorter period of 3 - 4 h after which rtms did not interfere with consolidation . following practice , the newly formed motor memory is susceptible to interference in a time - dependent manner from other motor tasks competing for the same neural resources and suprathreshold rtms , both resulting in impaired performance on subsequent retention test [ 11 , 25 ] . this raises the question as to whether there exists a temporal gradient relating to the effects of the exercise bout or perhaps a window of opportunity for affecting the transient plastic neural mechanisms , after which the effect disappears or is greatly reduced . the idea that this is the case has been proposed in various studies : so - called occlusion of long - term potentiation- ( ltp- ) like plasticity [ 26 , 27 ] where there is competition for neural resources . similarly , the synaptic tagging and capture hypothesis proposes that the initiation of ltp creates only the potential for lasting changes in neuronal structures [ 28 , 29 ] . in rodents , a recent study by siette and coworkers indicated that close temporal proximity of exercise was central to enhancing the acquisition , extinction , and reconsolidation of context conditioned fear compared to delayed exercise . memories created through fear - conditioning protocols in rodents differ , however , from memories created through motor skill practice in humans as in the present study . furthermore , xu and coworkers demonstrated in mice that a rapid ( < 1 h ) synaptic reorganization occurs immediately following motor skill learning . the results from these studies provide preliminary evidence for exercise placement in close temporal proximity to acquisition in order to have a positive effect . in order to investigate the role of exercise timing on motor memory consolidation , we varied the temporal proximity of a high intensity exercise bout after motor skill acquisition and then measured performance changes with delayed retention tests . the primary hypothesis for this study was that a temporal gradient exists relating to the positive effects of exercise on procedural memory . this is based on the studies showing that protein synthesis and ltp , relating to memory formation , occur transiently immediately following learning . therefore , we propose that the closer the temporal proximity of the acute exercise bout to acquisition , the larger the positive effect on the retention of the procedural memory . confirmation of this hypothesis would provide us with new important information relating to the importance of exercise timing in relation to aiding learning . subjects were required to visit the laboratory on four separate occasions with the aim of assessing the effects of a high intensity exercise bout on the consolidation and retention of a newly acquired motor skill . subjects performed a graded maximal exercise test on a bicycle ergometer to measure their maximal oxygen uptake ( vo2 peak ) . at least 1 d after the screening session , subjects returned to the laboratory to complete the main experimental session and were required to refrain from exercising during this period . subjects then returned to the laboratory exactly 1 d and 7 d after the main experiment to complete the retention tests , r1 and r7 , respectively . all sessions were carried out at the same time of day ( 2 h ) . randomization was stratified to ensure that the groups were matched for age , body mass index ( bmi ) , relative maximal oxygen consumption ( vo2 peak : ml o2minkg ) , and baseline score in the visuomotor accuracy tracking task ( vat ) . the subjects were divided between a resting control group ( con ) and three exercise groups ( ex90 , ex90 + 1 , and ex90 + 2 ) . data from subjects in the con and ex90 have been reported in a previous study focusing on the importance of exercise intensity . the subjects were required to abstain from physical activity 2 h before and 4 h after the test sessions . forty - eight able - bodied , healthy , right - handed males ( 24.0 2.5 yrs ) were recruited from the copenhagen area to participate in the study ( table 1 ) . right - handedness for each subject was evaluated with the edinburgh handedness inventory ( 87.4 2.7 ) . at the time of recruitment for the study all subjects were nave to the vat used to investigate motor skill learning and procedural memory . exclusion criteria for participation in the study included the following : age below 20 or over 35 , body mass index ( bmi ) above 30 , a history of neurological , psychiatric , or medical diseases , and a current intake of medication and/or recreational drugs , which could have an impact on learning and/or the central nervous system . the experiments were approved by the local ethics committee for the greater copenhagen area ( protocol h-2 - 2011 - 032 ) and the study was performed in accordance with the declaration of helsinki . the graded maximal exercise test was conducted in order to assess the subject 's aerobic fitness level and to collect blood lactate samples at various workloads . maximal oxygen consumption was determined when at least one of the following criteria was met : a plateau in the vo2 curve , a rer 1.1 , an inability to maintain 80 rpm , and/or volitional exhaustion . mean values for relative vo2 peak and wmax for each group can be seen in table 1 . all subjects performed standardized neuropsychological tests of spatial working memory ( swm ) and sustained attention ( rapid visual processing , rvp ) with cantab software ( cambridge cognition ltd . , uk ) . questionnaires were completed including the academic motivation scale ( ams ) , the task and ego orientation in sport questionnaire ( teosq ) , the international physical activity questionnaire- ( ipaq- ) long , flow proneness [ 38 , 39 ] , health background via a standardized general eligibility questionnaire , and positive and negative affective status ( panas ) . no differences were observed between groups for rvp ( sustained attention ) , swm ( table 2 ) , and ipaq levels at baseline ( table 1 ) . on arrival at the laboratory subjects were required to complete the panas to determine positive ( pas ) and negative ( nas ) affects before vat acquisition , r1 , and r7 , respectively . the vat involved 5 training blocks ( b1 , b2 , b3 , b4 , and b5 ) of 20 identical targets ( 4 min per block ) with feedback in the form of a performance score , separated by 2 min rest periods between blocks . following the completion of block 3 subjects completed a flow questionnaire relating to how they evaluated their mental state and performance during the vat . this was the danish version of the 13-item flow kurz skala , which has been previously used and described by this group . similarly an intrinsic motivation inventory ( imi ) was filled out on completion of the vat . at 20 min after vat subjects in group ex90 completed a standardized acute exercise bout on a cycle ergometer in an adjacent laboratory . at 1 h and 2 h after vat subjects in groups ex90 + 1 and ex90 + 2 completed the same exercise bout , respectively . in the period following completion of the vat and start of exercise , subjects sat quietly in an adjacent office and were allowed to read nonacademic literature ( newspapers and magazines were provided ) . the exercise bout consisted of a short warm - up followed by 3 3 min intervals at 90% of peak power output ( wmax ) for all exercise groups . blood lactate measurements were taken before and 5 min after exercise as well as in the last 30 s of each of the three intervals . the main experiment was concluded with completion of the montral sleep diary for the night preceding the main experiment . this was an adapted sleep questionnaire based on the pittsburgh sleep diary , which has been used in numerous studies [ 4648 ] . at 1 day after acquisition subjects were required to complete a retention test in the vat ( r1 ) , without feedback on motor performance . the removal of feedback at the retention tests was done to exclude any learning effects , which might relate to receiving feedback such as guidance and motivation . this format was also repeated at the 7 d retention test ( r7 ) and an additional training block ( r7tr ) in the vat with feedback was completed in order to check for continued learning potential . the exercise protocol was identical to the one used in the study by thomas and coworkers . the protocol was designed to ensure high levels of peak blood lactate ( 10 mmol / l [ 17 , 50 ] ) and the total duration of the exercise was limited ( 17 min ) in order to avoid excessive fatigue and/or dehydration , which could potentially have a negative effect on memory processing [ 51 , 52 ] . subjects performed a two - minute warm - up on a cycle ergometer ( ergomedic 939e , monark , sweden ) at 100 w followed by a further 2 min at 60% of wmax after which they completed 3 blocks of high intensity cycling ( 90% of wmax ) keeping a cadence of 80 rpm interspersed by a 2 min lower intensity active rest interval ( 60% of wmax ) also with a cadence of 80 rpm . heart rate , via heart rate monitors ( polar electro , kempele , finland ) , and rating of perceived exertion ( rpe ) values ( borg scale ) were recorded during exercise and blood lactate measurements ( accutrend plus system , roche diagnostics , switzerland ) were taken at rest prior to exercise ( pre ) , at completion of each work interval ( 1 , 2 , and 3 ) then again at 5 min after exercise completion ( post ) . the vat has been described in detail previously by this group [ 13 , 17 ] and the protocol used was identical . each vat trial consisted of a fixed target consisting of a modified triple sine wave curve . subjects were required to track the target as accurately as possible by moving a cursor trace up and down , respectively , with wrist extension moving the cursor upwards and flexion moving it downwards . at the end of each trial , augmented feedback on performance was presented as a numerical motor performance score and the subject 's trace presented with the target trace . the numerical score range was 0100 with 100 representing a perfect trace of the target . augmented feedback was only presented during motor skill acquisition , not during delayed retention tests . subjects performed the vat on four occasions : at the main experiment ( acquisition ) , at the 1 d retention test ( r1 ) , at the 7 d retention test ( r7 ) , and an additional motor skill training block at 7 d ( r7tr ) , which was included to check for a ceiling effect in the vat . the acquisition phase consisted of 5 blocks of 20 trials ( 100 trials in total ) with each block taking 4 min to complete , with rest periods of 2 min between blocks giving a total time of 28 min . the two retention tests ( r1 and r7 ) consisted of 1 block of 20 targets with mean scores for targets 220 used for analysis . the test at 7 d to check for a ceiling effect ( r7tr ) was 1 block of 20 targets under the same conditions as acquisition with the mean score for targets 220 used for analysis . two - way repeated measurements anova models were fitted by means of linear mixed models . for the vat parameters linear mixed models including group - time interactions as fixed effects and subject - specific random effects were fitted to account for the 4-by-7 two - way analysis of variance ( anova ) layout of the study ( 4 groups and 7 time points ) with repeated measurements per subjects . the random effects captured inherent variability between subjects and thus allowed removal of between - subject variation . these included separate analyses for acquisition ( 4-by-5 ) and retention ( 4-by-3 ) as two - way anova layouts . within these models we identified a limited number of comparisons corresponding to the research questions of interest in this study . model checking was based on residual plots and normal probability plots using the raw residuals . the same linear mixed model was also fitted to the exercise parameters for the three groups ex90 , ex90 + 1 , and ex90 + 2 with a 3-by-4 two - way anova layout . data from tests of sustained attention , spatial working memory , panas , sleepiness , physical activity level , and sleep were also analyzed using similar linear mixed models ( also with an 4-by-3 two - way anova layout ) . separate models were fitted for the vat acquisition phase and then subsequently the retention tests r1 and r7 . within the two - way anova layout of each model we identified a limited number of relevant comparisons between changes from baseline within intervention groups in order to reduce the problem of multiple testing . additionally , we also considered comparisons between groups at specific time points . all these pairwise comparisons were evaluated using model - based t - tests derived from the mixed models . the resulting p values for comparisons of changes were multiplicity adjusted using the computational single - step method , which provides a less conservative adjustment than the bonferroni adjustment through utilization of correlations between means entering in the pairwise comparisons . the p values for the additional comparisons between groups at specific time points were left unadjusted . likewise , model - based t - tests were used to evaluate differences in changes between group mean scores at the time points block 5 ( b5 ) , r1 , and r7 . data are reported as mean se unless otherwise stated , where appropriate data are reported with 95% c.i . all statistical analyses were carried out in r ( r core team , 2015 ) . in particular , linear mixed models were fitted using the functionality of the package lme4 , whereas multiple comparisons and adjusted p values were calculated using the single - step method provided by the package multcomp . mean scores ( se ) for all groups at all time points can be seen in figure 3(a ) . from b1 to b5 all groups showed significant improvement ( all p < 0.05 ) , with a mean increase of 39.4 1.2% equivalent to an increase of 20.6 0.5 in mean performance score . there were no between - group differences in performance score changes from b1 to b5 , indicating that skill improvements during acquisition and , more importantly , skill level at the end of acquisition ( b5 ) , were similar among groups ( figure 3(a ) ) . changes in mean performance score from b5 to r1 for ex90 were greater than con 3.14 0.82 ( p < 0.001 ) and this change for ex90 was also greater than the ex90 + 2 group there was a significant difference of 4.56 0.81 ( p < 0.001 ) between the ex90 and con groups as well as between the ex90 + 1 and con groups 2.54 0.81 ( p = 0.008 ) , between the con and ex90 + 2 groups 2.52 0.81 ( p = 0.008 ) , and between the ex90 + 2 and ex90 groups 2.04 0.81 ( p = 0.049 ) . there was a tendency towards a greater change between the ex90 group and the ex90 + 1 group between b5 and r7 2.03 0.81 ( p = 0.052 ) . figure 3(b ) shows the changes in mean vat scores at r1 and r7 relative to b5 . for the ex90 group there was a significant increase of 2.15 0.59 ( p < 0.001 ) in mean performance score at r1 compared to b5 and likewise from b5 to r7 3.47 0.57 ( p < 0.001 ) ( b5 70.48 1.26 ; r1 72.63 1.26 : r7 73.95 1.26 ) . there was a significant increase of 1.45 0.57 ( p = 0.011 ) in mean performance score for ex90 + 1 from b5 to r7 ( b5 72.79 1.26 : r7 74.24 1.26 ) and for ex90 + 2 1.43 0.57 ( p = 0.012 ) . at r1 a significant between - group difference of 3.69 1.79 ( p = 0.038 ) was observed between con and ex90 + 1 ( con 69.77 1.26 ; ex90 + 1 73.46 1.26 ) . at r7 significant between - group differences in mean scores of 4.29 1.78 ( p = 0.016 ) and 4.58 1.78 ( p = 0.010 ) were found between con and ex90 and con and ex90 + 1 ( con 69.66 1.26 ; ex90 73.30 1.26 ; ex90 + 1 74.24 1.26 ) , respectively , and between con and ex90 + 2 3.64 1.78 ( p = 0.041 ) . changes in performance score from r7 to r7tr for the con group were greater than the ex90 group 2.11 0.73 ( p = 0.017 ) and likewise the change for the ex90 + 2 group was greater than the ex90 group 1.96 0.73 ( p = 0.030 ) . there was a significant increase of 2.32 0.51 ( p < 0.001 ) for the con group between time points r7 and r7tr ( r7 69.66 1.22 ; r7tr 71.98 1.22 ) . likewise , for the ex90 + 1 group 1.94 0.51 ( p < 0.001 ) ( r7 74.24 1.22 ; r7tr 76.18 1.22 ) and the ex90 + 2 group 2.18 0.51 ( p < 0.001 ) ( r7 73.30 1.22 ; r7tr 75.48 1.22 ) demonstrating that learning was not saturated and that there was potential for further learning . however , this was not the case for ex90 ( r7 73.95 1.22 ; r7tr 74.17 1.22 ) . blood lactate levels for time points before exercise ( pre ) , intervals 1 , 2 , and 3 , and 5 min after exercise ( post ) are presented in figure 4 . the main finding of this study is that a temporal gradient exists in the positive effect of an acute high intensity exercise bout on the retention of a motor skill . exercise carried out 20 min after motor skill acquisition led to superior retention ( changes in performance scores ) compared to a resting control group and the delayed ( + 2 h ) exercise group at both 1 and 7 d. delaying exercise by 2 h appears to significantly diminish this effect , although changes in performance scores at 7 d were still greater than a resting control group . all groups were capable of improving performance in r7tr , suggesting that a potential ceiling effect for the vat did not exist for any group . these findings may further our understanding of how exercise plays a role as a potent amplifier of practice - dependent plasticity and demonstrates how we may be able to advance practical guidelines in rehabilitation , sports , ergonomics , and education settings in order to aid learning processes . the current literature supports the idea of a memory trace or motor engram , which is established through motor skill practice and is then susceptible to positive interference , such as exercise [ 13 , 1719 ] and sleep . negative interference can also occur via tasks that compete for the same neural resources or networks [ 7 , 25 , 57 , 58 ] or rtms [ 11 , 59 , 60 ] in the period following acquisition . the present findings are in accordance with recent studies [ 13 , 17 , 19 ] and support the existence of a temporal window in which motor memory consolidation can be influenced . a recent finding reported by rhee and coworkers included a delayed exercise condition ( ~1.75 h ) following a motor sequence task . this behavioural intervention resulted in a broad enhancement and protection of the offline gains when performed immediately prior to a second interfering task at 2 h. the performance level at 24 h was comparable to a control group with no interference and greater than an exercise condition immediately following the initial task . whereas exercise immediately after practicing a visuomotor precision task is beneficial for motor memory consolidation the findings by rhee et al . do however support the notion of beneficial effects of exercise following learning . additionally , the current findings demonstrate that enhancing memory consolidation through intense aerobic exercise may be restricted to a narrower timeframe compared to that of negative interference . motor skill learning is accompanied by a transient increase in corticospinal excitability ( cse ) observed as increased mep amplitude [ 10 , 25 , 62 , 63 ] . early work suggested a causal link between changes in cse and the retention of motor memory . more recently it has been suggested that the increase in cse relative to the potential for increases in cse , the so - called occlusion index , rather than the change itself relating to offline changes in motor memory [ 26 , 64 , 65 ] . this represents a limited ability for further ltp - like plasticity due to competition of neural resources and it is argued that the greater the extent of occlusion the more resilient the memory trace to retrograde interference . these results correspond to findings by tunovic and colleagues who observed that when cse decreases were abolished via theta burst tms following an implicit version of the serial reaction time task skill improvements were observed and these improvements corresponded to the changes in cse . since exercise increases cse and affects intracortical networks [ 20 , 21 ] this could be one marker for the underlying processes leading to offline changes in motor performance . the time - dependent processes involved in establishing motor memory are critical in relation to this study 's results . the synaptic tagging and capture hypothesis [ 28 , 29 ] propose that the cellular component of consolidation involves a temporary structural state of the synapse , which represents a permissive unlocking process without which protein synthesis and the supply of plasticity related proteins ( prps ) are incapable of stabilizing plasticity . exercise - induced transient increases in circulating levels of brain derived neurotrophic factor ( bdnf ) , norepinephrine , epinephrine , dopamine , and lactate [ 13 , 66 , 67 ] might support or even amplify the tagging process , which in turn could potentially stimulate and signal a variety of pathways supporting long - term potentiation ( ltp ) and an upregulation of protein synthesis . many studies have reported effects on cognitive function when the exercise bout is placed before or during the task , but here we focus on motor skill learning with exercise placed after acquisition to investigate motor memory consolidation . the transient and high ( > 10 mmol / l ) increase in blood lactate level , which in turn signals an upregulation of the monoamine carboxylate transporters , a release of bdnf , and increases in cse excitability , may represent an amplification effect that could explain the increase in the postacquisition susceptibility of the neural networks to be affected by the exercise bout . in combination with the transient increases in biomarkers such as dopamine , norepinephrine , epinephrine , and insulin - like growth factor 1 ( igf-1 ) there may be an interaction producing a favorable cellular and molecular environment for consolidation . the catecholamine hypothesis relating to the effects of acute exercise on cognition may also provide a neurochemical basis for the improvements in procedural memory . however , linking distinct brain functions across different experimental protocols in light of this study 's results should be done with caution until further evidence supports this . if the plastic mechanisms triggered by motor learning itself are transient and , as several theories suggest , are susceptible in a time frame of ~2 h after learning then it follows that the effects of exercise on consolidation would be the greatest when adding to these already upregulated processes at < 1 h after motor learning . it is , however , important to underline that these lines of argumentation are speculative and at best help explain our results on the basis of current knowledge . offline gains in motor memory have been demonstrated and investigated in order to try and understand the neurobiology of consolidation [ 1 , 3 , 4 , 7073 ] . different stages , fast and slow , are also proposed as well as parallel mechanisms including striatal and hippocampal systems : corticostriatal and corticocerebellar systems [ 76 , 77 ] . there are , furthermore , different aspects of the learning task : goal versus movement . whether exercise preferentially affects one or more of these mechanisms , or all at once via a global flooding of the signaling cascades involved , can only be speculated on at the current time . similarly , the role of sleep and the interaction between sleep and exercise is also currently poorly understood although sleep - dependent motor memory plasticity has been reported in the literature [ 8 , 9 , 14 , 56 ] . the registration of sleep and affect schedules ( panas ) in this study showed that there were similar levels between groups and across time points effectively excluding these parameters as confounders . the study design implied an element of repeated measurements per subject such that each subject served as his or her own control , which allowed us to eliminate some of the between - subject variation and thus also increase power . furthermore , any extrapolation of these results to other types of memory and learning / exercise paradigms must be done with caution . the results of this study show that the positive effects of an acute bout of high intensity exercise on the consolidation and retention of motor skill learning are greatest when placed in close temporal proximity following the acquisition of the skill with this positive effect diminishing as time to exercise increases . this temporal gradient would support the idea that in order to maximize the effects of exercise on motor memory consolidation , it should be placed immediately following acquisition of the skill / encoding of the memory ( < 1 h ) . the specific neurophysiological mechanisms , which are affected by exercise in this period , are currently not fully understood , but the systemic effect of an acute exercise bout may well amplify processes of neuroplasticity at a systemic , molecular , and cellular level .

high intensity aerobic exercise amplifies offline gains in procedural memory acquired during motor practice . this effect seems to be evident when exercise is placed immediately after acquisition , during the first stages of memory consolidation , but the importance of temporal proximity of the exercise bout used to stimulate improvements in procedural memory is unknown . the effects of three different temporal placements of high intensity exercise were investigated following visuomotor skill acquisition on the retention of motor memory in 48 young ( 24.0 2.5 yrs ) , healthy male subjects randomly assigned to one of four groups either performing a high intensity ( 90% maximal power output ) exercise bout at 20 min ( ex90 ) , 1 h ( ex90 + 1 ) , 2 h ( ex90 + 2 ) after acquisition or rested ( con ) . retention tests were performed at 1 d ( r1 ) and 7 d ( r7 ) . at r1 changes in performance scores after acquisition were greater for ex90 than con ( p < 0.001 ) and ex90 + 2 ( p = 0.001 ) . at r7 changes in performance scores for ex90 , ex90 + 1 , and ex90 + 2 were higher than con ( p < 0.001 , p = 0.008 , and p = 0.008 , resp . ) . changes for ex90 at r7 were greater than ex90 + 2 ( p = 0.049 ) . exercise - induced improvements in procedural memory diminish as the temporal proximity of exercise from acquisition is increased . timing of exercise following motor practice is important for motor memory consolidation .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

the consolidation phase of motor skill learning involves the transformation of memories from a trace or engram [ 16 ] , established through an encoding or acquisition phase , to a more enhanced or stabile long - term memory [ 1 , 7 ] . the interplay of factors during consolidation including rest , sleep , alternative tasks , and exercise is complicated as there also appears to be different temporal aspects of consolidation itself : synaptic and systems consolidation [ 3 , 10 , 11 ] . the different temporal phases of motor memory consolidation are therefore complex and studies are only beginning to provide evidence of how exercise may affect these phases and how the underlying cellular mechanisms are affected . it can be argued that exercise prior to motor skill acquisition mainly influences acquisition itself potentially via increased awareness [ 14 , 15 ] and/or a priming effect , but it can possibly affect consolidation , although this is hard to differentiate . an acute bout of high intensity exercise has been shown to improve motor skill learning when carried out in the period following [ 13 , 17 , 18 ] and preceding acquisition of the skill when measured with delayed retention tests . exercise placed after acquisition demonstrates the greatest effect on the consolidation and the strengthening of this type of procedural memory [ 12 , 17 , 18 ] . increases in peripheral ( and central ) availability of neurotrophic factors , energy substrates , and signaling molecules are capable of influencing memory consolidation . transient changes in corticospinal excitability ( cse ) and intracortical inhibition and facilitation in the primary motor cortex , relating to motor skill learning , have also provided evidence to suggest that these mechanisms may be enhanced or protected by exercise [ 2022 ] . the literature supports the idea that exercise intensity plays a central role for improvements in motor performance [ 15 , 24 ] , but the importance of the temporal placement of exercise in relation to motor practice has not been thoroughly investigated . muellbacher and coworkers demonstrated that repetitive transcranial magnetic stimulation ( rtms ) caused interference when placed immediately following motor learning but not 6 h later and more recently lundbye - jensen and coworkers reported a shorter period of 3 - 4 h after which rtms did not interfere with consolidation . following practice , the newly formed motor memory is susceptible to interference in a time - dependent manner from other motor tasks competing for the same neural resources and suprathreshold rtms , both resulting in impaired performance on subsequent retention test [ 11 , 25 ] . this raises the question as to whether there exists a temporal gradient relating to the effects of the exercise bout or perhaps a window of opportunity for affecting the transient plastic neural mechanisms , after which the effect disappears or is greatly reduced . in rodents , a recent study by siette and coworkers indicated that close temporal proximity of exercise was central to enhancing the acquisition , extinction , and reconsolidation of context conditioned fear compared to delayed exercise . furthermore , xu and coworkers demonstrated in mice that a rapid ( < 1 h ) synaptic reorganization occurs immediately following motor skill learning . the results from these studies provide preliminary evidence for exercise placement in close temporal proximity to acquisition in order to have a positive effect . in order to investigate the role of exercise timing on motor memory consolidation , we varied the temporal proximity of a high intensity exercise bout after motor skill acquisition and then measured performance changes with delayed retention tests . the primary hypothesis for this study was that a temporal gradient exists relating to the positive effects of exercise on procedural memory . therefore , we propose that the closer the temporal proximity of the acute exercise bout to acquisition , the larger the positive effect on the retention of the procedural memory . confirmation of this hypothesis would provide us with new important information relating to the importance of exercise timing in relation to aiding learning . subjects were required to visit the laboratory on four separate occasions with the aim of assessing the effects of a high intensity exercise bout on the consolidation and retention of a newly acquired motor skill . subjects then returned to the laboratory exactly 1 d and 7 d after the main experiment to complete the retention tests , r1 and r7 , respectively . randomization was stratified to ensure that the groups were matched for age , body mass index ( bmi ) , relative maximal oxygen consumption ( vo2 peak : ml o2minkg ) , and baseline score in the visuomotor accuracy tracking task ( vat ) . the subjects were divided between a resting control group ( con ) and three exercise groups ( ex90 , ex90 + 1 , and ex90 + 2 ) . data from subjects in the con and ex90 have been reported in a previous study focusing on the importance of exercise intensity . forty - eight able - bodied , healthy , right - handed males ( 24.0 2.5 yrs ) were recruited from the copenhagen area to participate in the study ( table 1 ) . at the time of recruitment for the study all subjects were nave to the vat used to investigate motor skill learning and procedural memory . exclusion criteria for participation in the study included the following : age below 20 or over 35 , body mass index ( bmi ) above 30 , a history of neurological , psychiatric , or medical diseases , and a current intake of medication and/or recreational drugs , which could have an impact on learning and/or the central nervous system . maximal oxygen consumption was determined when at least one of the following criteria was met : a plateau in the vo2 curve , a rer 1.1 , an inability to maintain 80 rpm , and/or volitional exhaustion . questionnaires were completed including the academic motivation scale ( ams ) , the task and ego orientation in sport questionnaire ( teosq ) , the international physical activity questionnaire- ( ipaq- ) long , flow proneness [ 38 , 39 ] , health background via a standardized general eligibility questionnaire , and positive and negative affective status ( panas ) . no differences were observed between groups for rvp ( sustained attention ) , swm ( table 2 ) , and ipaq levels at baseline ( table 1 ) . on arrival at the laboratory subjects were required to complete the panas to determine positive ( pas ) and negative ( nas ) affects before vat acquisition , r1 , and r7 , respectively . the vat involved 5 training blocks ( b1 , b2 , b3 , b4 , and b5 ) of 20 identical targets ( 4 min per block ) with feedback in the form of a performance score , separated by 2 min rest periods between blocks . similarly an intrinsic motivation inventory ( imi ) was filled out on completion of the vat . at 20 min after vat subjects in group ex90 completed a standardized acute exercise bout on a cycle ergometer in an adjacent laboratory . at 1 h and 2 h after vat subjects in groups ex90 + 1 and ex90 + 2 completed the same exercise bout , respectively . in the period following completion of the vat and start of exercise , subjects sat quietly in an adjacent office and were allowed to read nonacademic literature ( newspapers and magazines were provided ) . the exercise bout consisted of a short warm - up followed by 3 3 min intervals at 90% of peak power output ( wmax ) for all exercise groups . at 1 day after acquisition subjects were required to complete a retention test in the vat ( r1 ) , without feedback on motor performance . the removal of feedback at the retention tests was done to exclude any learning effects , which might relate to receiving feedback such as guidance and motivation . this format was also repeated at the 7 d retention test ( r7 ) and an additional training block ( r7tr ) in the vat with feedback was completed in order to check for continued learning potential . the protocol was designed to ensure high levels of peak blood lactate ( 10 mmol / l [ 17 , 50 ] ) and the total duration of the exercise was limited ( 17 min ) in order to avoid excessive fatigue and/or dehydration , which could potentially have a negative effect on memory processing [ 51 , 52 ] . subjects performed a two - minute warm - up on a cycle ergometer ( ergomedic 939e , monark , sweden ) at 100 w followed by a further 2 min at 60% of wmax after which they completed 3 blocks of high intensity cycling ( 90% of wmax ) keeping a cadence of 80 rpm interspersed by a 2 min lower intensity active rest interval ( 60% of wmax ) also with a cadence of 80 rpm . heart rate , via heart rate monitors ( polar electro , kempele , finland ) , and rating of perceived exertion ( rpe ) values ( borg scale ) were recorded during exercise and blood lactate measurements ( accutrend plus system , roche diagnostics , switzerland ) were taken at rest prior to exercise ( pre ) , at completion of each work interval ( 1 , 2 , and 3 ) then again at 5 min after exercise completion ( post ) . augmented feedback was only presented during motor skill acquisition , not during delayed retention tests . subjects performed the vat on four occasions : at the main experiment ( acquisition ) , at the 1 d retention test ( r1 ) , at the 7 d retention test ( r7 ) , and an additional motor skill training block at 7 d ( r7tr ) , which was included to check for a ceiling effect in the vat . the two retention tests ( r1 and r7 ) consisted of 1 block of 20 targets with mean scores for targets 220 used for analysis . the test at 7 d to check for a ceiling effect ( r7tr ) was 1 block of 20 targets under the same conditions as acquisition with the mean score for targets 220 used for analysis . for the vat parameters linear mixed models including group - time interactions as fixed effects and subject - specific random effects were fitted to account for the 4-by-7 two - way analysis of variance ( anova ) layout of the study ( 4 groups and 7 time points ) with repeated measurements per subjects . the same linear mixed model was also fitted to the exercise parameters for the three groups ex90 , ex90 + 1 , and ex90 + 2 with a 3-by-4 two - way anova layout . separate models were fitted for the vat acquisition phase and then subsequently the retention tests r1 and r7 . likewise , model - based t - tests were used to evaluate differences in changes between group mean scores at the time points block 5 ( b5 ) , r1 , and r7 . in particular , linear mixed models were fitted using the functionality of the package lme4 , whereas multiple comparisons and adjusted p values were calculated using the single - step method provided by the package multcomp . from b1 to b5 all groups showed significant improvement ( all p < 0.05 ) , with a mean increase of 39.4 1.2% equivalent to an increase of 20.6 0.5 in mean performance score . there were no between - group differences in performance score changes from b1 to b5 , indicating that skill improvements during acquisition and , more importantly , skill level at the end of acquisition ( b5 ) , were similar among groups ( figure 3(a ) ) . changes in mean performance score from b5 to r1 for ex90 were greater than con 3.14 0.82 ( p < 0.001 ) and this change for ex90 was also greater than the ex90 + 2 group there was a significant difference of 4.56 0.81 ( p < 0.001 ) between the ex90 and con groups as well as between the ex90 + 1 and con groups 2.54 0.81 ( p = 0.008 ) , between the con and ex90 + 2 groups 2.52 0.81 ( p = 0.008 ) , and between the ex90 + 2 and ex90 groups 2.04 0.81 ( p = 0.049 ) . there was a tendency towards a greater change between the ex90 group and the ex90 + 1 group between b5 and r7 2.03 0.81 ( p = 0.052 ) . figure 3(b ) shows the changes in mean vat scores at r1 and r7 relative to b5 . for the ex90 group there was a significant increase of 2.15 0.59 ( p < 0.001 ) in mean performance score at r1 compared to b5 and likewise from b5 to r7 3.47 0.57 ( p < 0.001 ) ( b5 70.48 1.26 ; r1 72.63 1.26 : r7 73.95 1.26 ) . there was a significant increase of 1.45 0.57 ( p = 0.011 ) in mean performance score for ex90 + 1 from b5 to r7 ( b5 72.79 1.26 : r7 74.24 1.26 ) and for ex90 + 2 1.43 0.57 ( p = 0.012 ) . at r1 a significant between - group difference of 3.69 1.79 ( p = 0.038 ) was observed between con and ex90 + 1 ( con 69.77 1.26 ; ex90 + 1 73.46 1.26 ) . at r7 significant between - group differences in mean scores of 4.29 1.78 ( p = 0.016 ) and 4.58 1.78 ( p = 0.010 ) were found between con and ex90 and con and ex90 + 1 ( con 69.66 1.26 ; ex90 73.30 1.26 ; ex90 + 1 74.24 1.26 ) , respectively , and between con and ex90 + 2 3.64 1.78 ( p = 0.041 ) . changes in performance score from r7 to r7tr for the con group were greater than the ex90 group 2.11 0.73 ( p = 0.017 ) and likewise the change for the ex90 + 2 group was greater than the ex90 group 1.96 0.73 ( p = 0.030 ) . there was a significant increase of 2.32 0.51 ( p < 0.001 ) for the con group between time points r7 and r7tr ( r7 69.66 1.22 ; r7tr 71.98 1.22 ) . likewise , for the ex90 + 1 group 1.94 0.51 ( p < 0.001 ) ( r7 74.24 1.22 ; r7tr 76.18 1.22 ) and the ex90 + 2 group 2.18 0.51 ( p < 0.001 ) ( r7 73.30 1.22 ; r7tr 75.48 1.22 ) demonstrating that learning was not saturated and that there was potential for further learning . however , this was not the case for ex90 ( r7 73.95 1.22 ; r7tr 74.17 1.22 ) . blood lactate levels for time points before exercise ( pre ) , intervals 1 , 2 , and 3 , and 5 min after exercise ( post ) are presented in figure 4 . the main finding of this study is that a temporal gradient exists in the positive effect of an acute high intensity exercise bout on the retention of a motor skill . exercise carried out 20 min after motor skill acquisition led to superior retention ( changes in performance scores ) compared to a resting control group and the delayed ( + 2 h ) exercise group at both 1 and 7 d. delaying exercise by 2 h appears to significantly diminish this effect , although changes in performance scores at 7 d were still greater than a resting control group . these findings may further our understanding of how exercise plays a role as a potent amplifier of practice - dependent plasticity and demonstrates how we may be able to advance practical guidelines in rehabilitation , sports , ergonomics , and education settings in order to aid learning processes . the present findings are in accordance with recent studies [ 13 , 17 , 19 ] and support the existence of a temporal window in which motor memory consolidation can be influenced . this behavioural intervention resulted in a broad enhancement and protection of the offline gains when performed immediately prior to a second interfering task at 2 h. the performance level at 24 h was comparable to a control group with no interference and greater than an exercise condition immediately following the initial task . whereas exercise immediately after practicing a visuomotor precision task is beneficial for motor memory consolidation the findings by rhee et al . do however support the notion of beneficial effects of exercise following learning . additionally , the current findings demonstrate that enhancing memory consolidation through intense aerobic exercise may be restricted to a narrower timeframe compared to that of negative interference . early work suggested a causal link between changes in cse and the retention of motor memory . more recently it has been suggested that the increase in cse relative to the potential for increases in cse , the so - called occlusion index , rather than the change itself relating to offline changes in motor memory [ 26 , 64 , 65 ] . these results correspond to findings by tunovic and colleagues who observed that when cse decreases were abolished via theta burst tms following an implicit version of the serial reaction time task skill improvements were observed and these improvements corresponded to the changes in cse . exercise - induced transient increases in circulating levels of brain derived neurotrophic factor ( bdnf ) , norepinephrine , epinephrine , dopamine , and lactate [ 13 , 66 , 67 ] might support or even amplify the tagging process , which in turn could potentially stimulate and signal a variety of pathways supporting long - term potentiation ( ltp ) and an upregulation of protein synthesis . many studies have reported effects on cognitive function when the exercise bout is placed before or during the task , but here we focus on motor skill learning with exercise placed after acquisition to investigate motor memory consolidation . the transient and high ( > 10 mmol / l ) increase in blood lactate level , which in turn signals an upregulation of the monoamine carboxylate transporters , a release of bdnf , and increases in cse excitability , may represent an amplification effect that could explain the increase in the postacquisition susceptibility of the neural networks to be affected by the exercise bout . the catecholamine hypothesis relating to the effects of acute exercise on cognition may also provide a neurochemical basis for the improvements in procedural memory . if the plastic mechanisms triggered by motor learning itself are transient and , as several theories suggest , are susceptible in a time frame of ~2 h after learning then it follows that the effects of exercise on consolidation would be the greatest when adding to these already upregulated processes at < 1 h after motor learning . offline gains in motor memory have been demonstrated and investigated in order to try and understand the neurobiology of consolidation [ 1 , 3 , 4 , 7073 ] . similarly , the role of sleep and the interaction between sleep and exercise is also currently poorly understood although sleep - dependent motor memory plasticity has been reported in the literature [ 8 , 9 , 14 , 56 ] . the results of this study show that the positive effects of an acute bout of high intensity exercise on the consolidation and retention of motor skill learning are greatest when placed in close temporal proximity following the acquisition of the skill with this positive effect diminishing as time to exercise increases . this temporal gradient would support the idea that in order to maximize the effects of exercise on motor memory consolidation , it should be placed immediately following acquisition of the skill / encoding of the memory ( < 1 h ) . the specific neurophysiological mechanisms , which are affected by exercise in this period , are currently not fully understood , but the systemic effect of an acute exercise bout may well amplify processes of neuroplasticity at a systemic , molecular , and cellular level .

a literature search of pubmed ( 1994april 2009 ) and scopus ( 1994april 2009 ) was conducted using combinations of relevant keywords associated with pediatric type 1 diabetes , adherence- or self - management promoting interventions , and glycemic control . the term type 1 diabetes was separately paired with each of the terms designating a pediatric population ( children , adolescents , pediatric ) . each resultant search combination was paired with an indicator of an adherence - promoting intervention ( e.g. , adherence , compliance , blood glucose monitoring interventions ) and an indicator of glycemic outcomes ( glycemic control , a1c ) . the results of these searches were then cross - checked and overlapping citations were removed . in addition , we reviewed several recent reviews and meta - analyses that included pediatric type 1 diabetes studies ( 12,13 ) . studies were included if they met the following criteria : 1 ) study was a randomized , controlled trial , 2 ) individuals in the study sample had type 1 diabetes , 3 ) study sample included youth aged < 19 years , 4 ) study reported results on adherence and glycemic control ; and 5 ) study reported using an intervention with adherence - promoting components . direct interventions were those that focused entirely ( or almost entirely ) on direct , behavioral processes . for example , an intervention that targeted an increase in the frequency of bgm and provided education or training on this topic was considered a direct intervention . combined interventions focused on one or several behavioral tasks , but the emphasis of the intervention was on promoting coping or problem - solving skills or a family process involved in diabetes management . for example , a combined intervention may have targeted an increase in bgm adherence but was also focused on identifying and resolving barriers to improving bgm adherence . for example , a study included in this meta - analysis ( 14 ) provided a description of the intervention as well as an extensive table detailing session objectives and components . , j.m.r . , and c.m.p . ) separately determined the percentage of the information described that focused on adherence promotion ( e.g. , education and skill development for adherence tasks and overcoming barriers to adherence ) . we also coded the control group as either standard care in which the control group only received their standard care in the diabetes clinic or as standard care plus controlled variable in addition , two authors ( j.m.r . and c.m.p . ) coded studies across 10 characteristics of the sample ( year of publication , mean age , percent female , percent ethnic minority , mean duration of type 1 diabetes , sample size [ total , experimental , and control ] , mean a1c at baseline and follow - up [ experimental and control ] , delivery format of the intervention [ group versus individual ] , time to follow - up , and statistics reported for adherence and a1c ) . time to follow - up was defined as the posttreatment time point designated by the authors as the primary focus of the analysis . this was either the posttreatment effect or the first follow - up effect , typically within 6 months of the end of treatment . finally , we reversed the valence of the effect size to be consistent with typical health promotion interventions . in other words , a positive effect size showed that the a1c value was reduced : positive health promotion . a negative effect size in this meta - analysis indicates a worsening of glycemic control . the double - entered data were then compared and coefficients ( for categorical variables ) or intraclass correlations ( for interval variables ) were calculated . we were primarily interested in the magnitude of the effect size for change in glycemic control from pre- to posttreatment for the intervention group versus the control group . to arrive at this , we first calculated an effect size for 1 ) pre- to posttreatment change for the intervention group and 2 ) pre- to posttreatment change for the control group . then , we calculated change scores for the pre- to posttreatment intervention versus control comparison using an approach similar to that described by lipsey and wilson ( 15 ) and winkley et al . specifically , the mean change was divided by the pooled sd of the two groups in each study . it was necessary to proceed with these steps to not only test the primary treatment effect but also to be sure the studies were adequately controlled ( i.e. , there was no change in the control groups ) . we also examined the association among study effect size , characteristics of the sample , time to follow - up , and the rated percentage of adherence promotion in the intervention . we used the analytic approach described in lipsey and wilson ( 15 ) and completed analyses in sas ( sas institute , cary , nc ) . when calculating the mean effect size across studies , we used a weighted least squares approach in which each effect size was weighted by the inverse of its variance ( 16 ) . this accounts for variability across individual observations ( i.e. , studies ) while considering the size of the sample . specifically , the mean effect size , its se , and 95% ci were calculated . homogeneity analyses ( 16 ) were conducted to determine whether the mean effect size was accurately predicted by each individual effect size . homogeneity analyses provide a fuller understanding of similarities and differences among the studies included in the meta - analysis . significant q values indicate rejection of the null hypothesis of homogeneity ( i.e. , there is heterogeneity in study effect sizes ) . a literature search of pubmed ( 1994april 2009 ) and scopus ( 1994april 2009 ) was conducted using combinations of relevant keywords associated with pediatric type 1 diabetes , adherence- or self - management promoting interventions , and glycemic control . the term type 1 diabetes was separately paired with each of the terms designating a pediatric population ( children , adolescents , pediatric ) . each resultant search combination was paired with an indicator of an adherence - promoting intervention ( e.g. , adherence , compliance , blood glucose monitoring interventions ) and an indicator of glycemic outcomes ( glycemic control , a1c ) . the results of these searches were then cross - checked and overlapping citations were removed . in addition , we reviewed several recent reviews and meta - analyses that included pediatric type 1 diabetes studies ( 12,13 ) . studies were included if they met the following criteria : 1 ) study was a randomized , controlled trial , 2 ) individuals in the study sample had type 1 diabetes , 3 ) study sample included youth aged < 19 years , 4 ) study reported results on adherence and glycemic control ; and 5 ) study reported using an intervention with adherence - promoting components . direct interventions were those that focused entirely ( or almost entirely ) on direct , behavioral processes . for example , an intervention that targeted an increase in the frequency of bgm and provided education or training on this topic was considered a direct intervention . combined interventions focused on one or several behavioral tasks , but the emphasis of the intervention was on promoting coping or problem - solving skills or a family process involved in diabetes management . for example , a combined intervention may have targeted an increase in bgm adherence but was also focused on identifying and resolving barriers to improving bgm adherence . for example , a study included in this meta - analysis ( 14 ) provided a description of the intervention as well as an extensive table detailing session objectives and components . , j.m.r . , and c.m.p . ) separately determined the percentage of the information described that focused on adherence promotion ( e.g. , education and skill development for adherence tasks and overcoming barriers to adherence ) . we also coded the control group as either standard care in which the control group only received their standard care in the diabetes clinic or as standard care plus controlled variable in addition , two authors ( j.m.r . and c.m.p . ) coded studies across 10 characteristics of the sample ( year of publication , mean age , percent female , percent ethnic minority , mean duration of type 1 diabetes , sample size [ total , experimental , and control ] , mean a1c at baseline and follow - up [ experimental and control ] , delivery format of the intervention [ group versus individual ] , time to follow - up , and statistics reported for adherence and a1c ) . time to follow - up was defined as the posttreatment time point designated by the authors as the primary focus of the analysis . this was either the posttreatment effect or the first follow - up effect , typically within 6 months of the end of treatment . finally , we reversed the valence of the effect size to be consistent with typical health promotion interventions . in other words , a positive effect size showed that the a1c value was reduced : positive health promotion . a negative effect size in this meta - analysis indicates a worsening of glycemic control . the double - entered data were then compared and coefficients ( for categorical variables ) or intraclass correlations ( for interval variables ) were calculated . we were primarily interested in the magnitude of the effect size for change in glycemic control from pre- to posttreatment for the intervention group versus the control group . to arrive at this , we first calculated an effect size for 1 ) pre- to posttreatment change for the intervention group and 2 ) pre- to posttreatment change for the control group . then , we calculated change scores for the pre- to posttreatment intervention versus control comparison using an approach similar to that described by lipsey and wilson ( 15 ) and winkley et al . specifically , the mean change was divided by the pooled sd of the two groups in each study . it was necessary to proceed with these steps to not only test the primary treatment effect but also to be sure the studies were adequately controlled ( i.e. , there was no change in the control groups ) . we also examined the association among study effect size , characteristics of the sample , time to follow - up , and the rated percentage of adherence promotion in the intervention . we used the analytic approach described in lipsey and wilson ( 15 ) and completed analyses in sas ( sas institute , cary , nc ) . when calculating the mean effect size across studies , we used a weighted least squares approach in which each effect size was weighted by the inverse of its variance ( 16 ) . this accounts for variability across individual observations ( i.e. , studies ) while considering the size of the sample . specifically , the mean effect size , its se , and 95% ci were calculated . homogeneity analyses ( 16 ) were conducted to determine whether the mean effect size was accurately predicted by each individual effect size . homogeneity analyses provide a fuller understanding of similarities and differences among the studies included in the meta - analysis . significant q values indicate rejection of the null hypothesis of homogeneity ( i.e. , there is heterogeneity in study effect sizes ) . our search identified 27 studies ; however , upon further review , 12 studies did not meet our inclusion criteria . these 12 studies were excluded because they were not randomized , controlled trials ( n = 10 ) , only a small fraction of the sample was aged < 19 years ( n = 1 ) , or no data were provided ( nor could be obtained via contact with authors ) on a1c values ( n = 1 ) . coding of the study design , intervention components , and categories of interest in each study were completed and and intraclass correlation coefficients were all 1.00 . most studies were conducted in the u.s . ; one study was conducted in the u.k . study sample sizes ranged from 22 to 127 , and the total sample size across studies was 997 . all studies reported recruiting the study sample from the larger pediatric diabetes clinic population , suggestive of a convenience sample . however , two studies reported an inclusion criterion of poor glycemic control indicated by a certain a1c threshold ( e.g. , 8.5% ) ( 11,18 ) . the average age of the pooled samples was 13.55 2.23 years . all but two studies reported the percentage of female participants ; the average across studies was 50.2% . eight studies reported the race / ethnicity of the sample ; the average proportion of nonwhite individuals in those samples was 27.0% . however , there was considerable variability in that the studies ranged from 4 to 74% inclusion of minority participants . very few studies reported indicators of socioeconomic status and family structure ( e.g. , proportion of families with two caregivers in home ) ; thus , we did not collect data on these variables . time to follow - up ranged from 12 to 52 weeks with an average length of 29.5 weeks . attrition rates were available for nine of the studies ; it ranged from 3.4 to 13.3% . characteristics of studies in meta - analysis * type of intervention is defined as combined ( direct and indirect processes targeted ) or direct ( direct processes only ) . percent intervention targeting adherence was determined by review of articles by three authors ( k.k.h . effect size is the pre- to posttreatment , intervention vs. control group effect size ; positive effect sizes reflect improvement in glycemic control ( i.e. , health promotion ) and negative effect sizes reflect deterioration in glycemic control . ( 25 ) suggest potential selection bias ; recruited individuals were from a pool of assessed individuals . diabetes - specific variables consistently reported in studies were duration of type 1 diabetes and glycemic control . across the 15 studies , mean duration of type 1 diabetes ranged from 2.7 to 8.7 years ; average across the studies was 5.89 3.60 years . the mean a1c for the experimental group was 9.61 1.83% ( range 7.811.9% ) at baseline and 9.17 1.53% ( range 7.512.3% ) at follow - up . the mean a1c for the control group at baseline was 9.75 1.83% and at follow - up was 9.70 1.76% . the mean effect size for the pre- to posttreatment change in glycemic control for the intervention group was 0.13 , with individual study effects ranging from 0.50 to 0.99 . the mean effect size for the pre- to posttreatment change in the control group was 0.01 , with individual study effects ranging from 0.24 to 0.32 . this effect size indicates that glycemic control did not change over time for the pooled control group . the mean effect size for pre- to posttreatment change for the intervention versus control group comparison was 0.11 ( 95% ci 0.01 to 0.23 ) . effects ranged from 0.55 to 0.59 , and five were negative ( i.e. , deterioration in glycemic control ) . the mean effect size of 0.11 meets conventional standards for a small effect and indicates that there was very modest improvement in glycemic control over time . 1 . the association between the mean effect size for pre- to posttreatment change for the intervention versus control group and time to follow - up was not significant ( r = 0.07 , p > 0.05 ) . the correlation between the pre- to posttreatment intervention versus control effect size and whether the intervention targeted direct , behavioral processes alone or had combined targets was significant ( spearman r = 0.56 , p < 0.03 ) . this finding indicates that multicomponent interventions ( i.e. , those that targeted direct and indirect processes together ) were associated with larger effects on glycemic control . finally , because only a small number of studies were delivered in a group format and all others were done individually , we did not conduct a comparison based on intervention delivery format . homogeneity analysis of the pre- to posttreatment intervention versus control group comparison revealed homogeneity across the 15 studies [ q(14 ) = 18.40 ; p > 0.05 ] . in other words , there was little variability in effects across these randomized , controlled trials . however , analysis for the pre- to posttreatment effects for the intervention group alone did show significant variability [ q ( 14 ) = 33.11 , p < 0.05 ] . this result illustrates that even though studies were adequately controlled , the interventions produced different magnitudes of a1c change . we hypothesized that this variability could be attributed to the percentage of the intervention targeting adherence promotion . in other words , interventions that focused almost entirely on adherence promotion should do better than those that included components other than adherence promotion . our search identified 27 studies ; however , upon further review , 12 studies did not meet our inclusion criteria . these 12 studies were excluded because they were not randomized , controlled trials ( n = 10 ) , only a small fraction of the sample was aged < 19 years ( n = 1 ) , or no data were provided ( nor could be obtained via contact with authors ) on a1c values ( n = 1 ) . coding of the study design , intervention components , and categories of interest in each study were completed and and intraclass correlation coefficients were all 1.00 . most studies were conducted in the u.s . ; one study was conducted in the u.k . study sample sizes ranged from 22 to 127 , and the total sample size across studies was 997 . all studies reported recruiting the study sample from the larger pediatric diabetes clinic population , suggestive of a convenience sample . however , two studies reported an inclusion criterion of poor glycemic control indicated by a certain a1c threshold ( e.g. , 8.5% ) ( 11,18 ) . all but two studies reported the percentage of female participants ; the average across studies was 50.2% . eight studies reported the race / ethnicity of the sample ; the average proportion of nonwhite individuals in those samples was 27.0% . however , there was considerable variability in that the studies ranged from 4 to 74% inclusion of minority participants . very few studies reported indicators of socioeconomic status and family structure ( e.g. , proportion of families with two caregivers in home ) ; thus , we did not collect data on these variables . time to follow - up ranged from 12 to 52 weeks with an average length of 29.5 weeks . attrition rates were available for nine of the studies ; it ranged from 3.4 to 13.3% . characteristics of studies in meta - analysis * type of intervention is defined as combined ( direct and indirect processes targeted ) or direct ( direct processes only ) . percent intervention targeting adherence was determined by review of articles by three authors ( k.k.h . effect size is the pre- to posttreatment , intervention vs. control group effect size ; positive effect sizes reflect improvement in glycemic control ( i.e. , health promotion ) and negative effect sizes reflect deterioration in glycemic control . ( 25 ) suggest potential selection bias ; recruited individuals were from a pool of assessed individuals . diabetes - specific variables consistently reported in studies were duration of type 1 diabetes and glycemic control . across the 15 studies , mean duration of type 1 diabetes ranged from 2.7 to 8.7 years ; average across the studies was 5.89 3.60 years . the mean a1c for the experimental group was 9.61 1.83% ( range 7.811.9% ) at baseline and 9.17 1.53% ( range 7.512.3% ) at follow - up . the mean a1c for the control group at baseline was 9.75 1.83% and at follow - up was 9.70 1.76% . the mean effect size for the pre- to posttreatment change in glycemic control for the intervention group was 0.13 , with individual study effects ranging from 0.50 to 0.99 . the mean effect size for the pre- to posttreatment change in the control group was 0.01 , with individual study effects ranging from 0.24 to 0.32 . this effect size indicates that glycemic control did not change over time for the pooled control group . the mean effect size for pre- to posttreatment change for the intervention versus control group comparison was 0.11 ( 95% ci 0.01 to 0.23 ) . effects ranged from 0.55 to 0.59 , and five were negative ( i.e. , deterioration in glycemic control ) . the mean effect size of 0.11 meets conventional standards for a small effect and indicates that there was very modest improvement in glycemic control over time . the association between the mean effect size for pre- to posttreatment change for the intervention versus control group and time to follow - up was not significant ( r = 0.07 , p > 0.05 ) . the correlation between the pre- to posttreatment intervention versus control effect size and whether the intervention targeted direct , behavioral processes alone or had combined targets was significant ( spearman r = 0.56 , p < 0.03 ) . this finding indicates that multicomponent interventions ( i.e. , those that targeted direct and indirect processes together ) were associated with larger effects on glycemic control . finally , because only a small number of studies were delivered in a group format and all others were done individually , we did not conduct a comparison based on intervention delivery format . homogeneity analysis of the pre- to posttreatment intervention versus control group comparison revealed homogeneity across the 15 studies [ q(14 ) = 18.40 ; p > 0.05 ] . in other words , there was little variability in effects across these randomized , controlled trials . however , analysis for the pre- to posttreatment effects for the intervention group alone did show significant variability [ q ( 14 ) = 33.11 , p < 0.05 ] . this result illustrates that even though studies were adequately controlled , the interventions produced different magnitudes of a1c change . we hypothesized that this variability could be attributed to the percentage of the intervention targeting adherence promotion . in other words , interventions that focused almost entirely on adherence promotion should do better than those that included components other than adherence promotion . results of this meta - analysis show that the mean effect size for the pre- to posttreatment change between intervention and control groups was 0.11 . whereas the mean change of a1c values from baseline to follow - up for youth in the intervention groups of these studies was 9.6 to 9.2% , variability in effects and the weighting of effects based on sample size translates to just a modest 0.20% reduction in a1c values . overall , this meta - analysis shows that the pooled effect on glycemic control of these 15 interventions was very modest . before identifying the implications of these meta - analytic results , we want to highlight the context of these findings as well as the positive aspects of these results . this meta - analysis included 14 studies not reviewed in the meta - analysis of winkley et al . ( 12 ) ; the only overlapping study was by wysocki , harris , and colleagues ( 26 ) . thus , this meta - analysis extends the current evidence base for interventions deployed in pediatric type 1 diabetes . interestingly , the interventions that we described as adherence - promoting interventions fared worse overall than the psychological interventions in the prior meta - analysis . specifically , the mean effect size in this meta - analysis was 3 times smaller than that reported by winkley et al . one reason for this may be our inclusion of interventions targeting direct , behavioral processes because these studies conferred little benefit on glycemic control . in our meta - analysis , the interventions that fared the best were those that were multicomponent and addressed both the direct and indirect processes involved in diabetes management . this program trains groups of youth with type 1 diabetes to increase the[ir ] ability to cope with problems that they face on a day - to - day basis and to be more effective in achieving therapeutic goals ( 8) . their inclusion of diabetes - specific social scenarios may have served to buttress the development of general coping skills . at least three other studies ( 20,22,25 ) were similarly multicomponent and had effect sizes of 0.40 . two other studies ( 24,26 ) also contained similar components and had effect sizes of 0.20 . overall , these interventions seem to be exemplars for the type of interventions that carry considerable benefit for improvement in glycemic control . on the other hand , the mean effect size of this meta - analysis provides a sobering look at the current state of interventions with adherence - promoting components if improvement in glycemic control is the measuring stick . why is the mean effect size so small and why do only a few interventions produce considerable benefit on glycemic control ? first , the percentage of the intervention directly targeting adherence promotion does not seem to be a factor . what appears important is whether or not the indirect processes are targeted in the intervention . as noted nearly two decades ago by johnson and her colleagues ( 2,27 ) , adherence is just one of the multiple factors that influence glycemic control . perhaps the primary focus on adherence behaviors and the neglect of other areas related to glycemic control ( social support , family relationships , coping , and problem - solving skills ) is what drove the present findings . second , and related to the first point , the dose of the intervention does not appear to be a factor . a sizable effect on glycemic control was observed in the study by laffel , anderson , and colleagues ( 20 ) , which included approximately four clinic - based intervention sessions across the year plus support from the care ambassador in scheduling appointments and working with clinic staff . the dose in this study was considerably less than that in other time- and resource - heavy interventions ( 11,26 ) , both with more modest effects on glycemic control , as well as that in an earlier iteration of the anderson and laffel family - based intervention ( 10 ) . admittedly , the unmeasured competing needs of the participants across these studies may not make this a fair comparison . nevertheless , dose appears to be less important than the inclusion of multiple components with the aim of facilitating adherence promotion . third , there was no association between time to follow - up and effect sizes . because these studies were controlled and there was little change in glycemic control for the control participants , simply stopping the deterioration of glycemic control that is so commonly seen across late childhood and adolescence ( 2830 ) was not enough to demonstrate statistical significance . although there is certainly clinical significance in preventing further deterioration , the interventions that reduced a1c values stood out and the length of time to the follow - up assessment did not matter . the primary implication of these meta - analytic results is that there is considerable work left to be done to make a significant impact on glycemic outcomes . however , after a thorough review of the most potent interventions , we conclude there is no need to start over . for example , we should learn from the interventions that produce reductions in a1c values approaching 1.0% ( e.g. , from 9.5 to 8.5% ) . we should also obtain a better understanding of how much cost - savings these interventions provide . for example , to what extent do they reduce acute and long - term medical complications that show up in extra medical and emergency department visits ? further , what are the direct costs to the clinics in terms of staffing and supplies and will insurance companies reimburse for these services ? likewise , an evaluation of psychosocial functioning ( e.g. , health - related quality of life ) will also provide critical information about the broader impact of these interventions . at the same time efforts are made to document effectiveness , the interventions demonstrating modest effects on glycemic control may benefit from the addition of other components and then a reexamination of their efficacy . for example , the most efficacious interventions target processes other than direct , behavioral processes ; they target the critical emotional , social , and family factors that youth with type 1 diabetes face on a daily basis . by focusing more so on these facilitators of adherence considered together , the collection of effectiveness data on the most potent interventions and adapting and refining less potent interventions will push the field closer to providing evidence - based care in pediatric diabetes clinics . the results of this meta - analysis and our recommendations should be considered in the context of several limitations . first , a meta - analysis pools studies and can not account for differing methodologies and sampling strategies ( 31 ) . even with randomization and control conditions , the heterogeneity in individual effect sizes among the interventions included highlights that fact that there is considerable variability in the potency of these interventions . the extent to which that variability is related to the sampling methodology and features of study design can not be ascertained through meta - analysis . second , the teams of investigators from these 15 studies may not have viewed adherence promotion as the primary mechanism to change glycemic control . the primary targets of the interventions ( i.e. , hypothesized mechanism to promote change in a1c ) may have been broader than adherence promotion and encompassed family , system , or emotional variables . we simply identified interventions with adherence - promoting components and may not be fully characterizing the whole treatment package . third , and related to the last point , our team made decisions about the coding of multiple variables ( time to follow - up , treatment components , and percentage of adherence promotion ) . although we were guided by past research and clinically guided assumptions , others may have coded these variables in a different manner . fourth , other variables that influence glycemic control ( e.g. , growth , puberty , and insulin regimen ) were not examined in this study , yet there is compelling evidence for their contribution to suboptimal glycemic outcomes ( 29,32,33 ) . further , there are inconsistent reports of sociodemographic variables such as socioeconomic status and family structure across these studies . in fact , some do not provide any data on these characteristics . given the links between these variables and glycemic outcomes ( 3436 ) , future reports should include indicators of socioeconomic status such as family income , educational achievement , occupational status , or full socioeconomic scales . a related limitation is that few of the studies targeted high - risk youth , those who consistently demonstrate high a1c values and have fewer resources and support . these youth and their families may respond better to higher doses of treatment and attention to the multiple systems in which they attempt to manage diabetes . indeed , there are compelling data on the effectiveness of these types of programs with these high - risk youth ( 37,38 ) . finally , we did not include an examination of changes in adherence in the included studies . given the data that adherence is linked to glycemic control ( 1 ) and a focus on optimizing glycemic control in practice guidelines ( 3 ) , we were primarily concerned with understanding whether these interventions alter glycemic outcomes . although we do not suspect this to be the case , it is possible that some interventions included in this meta - analysis improved adherence significantly while not altering glycemic control . in sum , this review and meta - analysis of interventions focused on adherence promotion in pediatric type 1 diabetes emphasizes two points . first , it is likely that glycemic control will not improve if interventions focus on direct , behavioral processes involved in diabetes management and neglect emotional , social , and family processes . second , we need to implement the effective programs in pediatric diabetes clinics and obtain effectiveness data . it is important that we better understand the costs and savings from a monetary perspective and also from the perspective of health outcomes of the pediatric patients cared for in these clinics .

objectiveto review interventions with adherence - promoting components and document their impact on glycemic control via meta-analysis.research design and methodsdata from 15 studies that met the following criteria were subjected to meta - analysis : 1 ) randomized , controlled trial , 2 ) study sample included youth aged < 19 years , 3 ) youth had type 1 diabetes , 4 ) study reported results on glycemic control ; and 5 ) study reported use of adherence- or self - management promoting components.resultsthe 15 studies included 997 youth with type 1 diabetes . the mean effect size for pre- to posttreatment change for the intervention versus control group comparison was 0.11 ( 95% ci 0.01 to 0.23 ) . this is a small effect , demonstrating very modest improvements in glycemic control . however , analysis for the pre- to posttreatment effects for the intervention group alone did show significant variability [ q(14 ) = 33.11 ; p < 0.05 ] . multicomponent interventions , those that targeted emotional , social , or family processes that facilitate diabetes management , were more potent than interventions just targeting a direct , behavioral process ( e.g. , increase in blood glucose monitoring frequency).conclusionsinterventions that focus on direct , behavioral processes and neglect emotional , social , and family processes are unlikely to have an impact on glycemic control ; multicomponent interventions showed more robust effects on a1c . future clinical research should focus on refining interventions and gathering more efficacy and effectiveness data on health outcomes of the pediatric patients treated with these interventions .

RESEARCH DESIGN AND METHODS Search strategy and study selection Study coding and analysis of reliability Primary and secondary analyses Statistical analysis RESULTS Search results and coding reliability Study characteristics Mean effect sizes Factors associated with mean effect size CONCLUSIONS

a literature search of pubmed ( 1994april 2009 ) and scopus ( 1994april 2009 ) was conducted using combinations of relevant keywords associated with pediatric type 1 diabetes , adherence- or self - management promoting interventions , and glycemic control . the term type 1 diabetes was separately paired with each of the terms designating a pediatric population ( children , adolescents , pediatric ) . each resultant search combination was paired with an indicator of an adherence - promoting intervention ( e.g. , adherence , compliance , blood glucose monitoring interventions ) and an indicator of glycemic outcomes ( glycemic control , a1c ) . in addition , we reviewed several recent reviews and meta - analyses that included pediatric type 1 diabetes studies ( 12,13 ) . studies were included if they met the following criteria : 1 ) study was a randomized , controlled trial , 2 ) individuals in the study sample had type 1 diabetes , 3 ) study sample included youth aged < 19 years , 4 ) study reported results on adherence and glycemic control ; and 5 ) study reported using an intervention with adherence - promoting components . direct interventions were those that focused entirely ( or almost entirely ) on direct , behavioral processes . for example , an intervention that targeted an increase in the frequency of bgm and provided education or training on this topic was considered a direct intervention . combined interventions focused on one or several behavioral tasks , but the emphasis of the intervention was on promoting coping or problem - solving skills or a family process involved in diabetes management . for example , a study included in this meta - analysis ( 14 ) provided a description of the intervention as well as an extensive table detailing session objectives and components . separately determined the percentage of the information described that focused on adherence promotion ( e.g. coded studies across 10 characteristics of the sample ( year of publication , mean age , percent female , percent ethnic minority , mean duration of type 1 diabetes , sample size [ total , experimental , and control ] , mean a1c at baseline and follow - up [ experimental and control ] , delivery format of the intervention [ group versus individual ] , time to follow - up , and statistics reported for adherence and a1c ) . finally , we reversed the valence of the effect size to be consistent with typical health promotion interventions . a negative effect size in this meta - analysis indicates a worsening of glycemic control . we were primarily interested in the magnitude of the effect size for change in glycemic control from pre- to posttreatment for the intervention group versus the control group . to arrive at this , we first calculated an effect size for 1 ) pre- to posttreatment change for the intervention group and 2 ) pre- to posttreatment change for the control group . then , we calculated change scores for the pre- to posttreatment intervention versus control comparison using an approach similar to that described by lipsey and wilson ( 15 ) and winkley et al . we also examined the association among study effect size , characteristics of the sample , time to follow - up , and the rated percentage of adherence promotion in the intervention . when calculating the mean effect size across studies , we used a weighted least squares approach in which each effect size was weighted by the inverse of its variance ( 16 ) . specifically , the mean effect size , its se , and 95% ci were calculated . homogeneity analyses ( 16 ) were conducted to determine whether the mean effect size was accurately predicted by each individual effect size . homogeneity analyses provide a fuller understanding of similarities and differences among the studies included in the meta - analysis . a literature search of pubmed ( 1994april 2009 ) and scopus ( 1994april 2009 ) was conducted using combinations of relevant keywords associated with pediatric type 1 diabetes , adherence- or self - management promoting interventions , and glycemic control . the term type 1 diabetes was separately paired with each of the terms designating a pediatric population ( children , adolescents , pediatric ) . each resultant search combination was paired with an indicator of an adherence - promoting intervention ( e.g. , adherence , compliance , blood glucose monitoring interventions ) and an indicator of glycemic outcomes ( glycemic control , a1c ) . in addition , we reviewed several recent reviews and meta - analyses that included pediatric type 1 diabetes studies ( 12,13 ) . studies were included if they met the following criteria : 1 ) study was a randomized , controlled trial , 2 ) individuals in the study sample had type 1 diabetes , 3 ) study sample included youth aged < 19 years , 4 ) study reported results on adherence and glycemic control ; and 5 ) study reported using an intervention with adherence - promoting components . direct interventions were those that focused entirely ( or almost entirely ) on direct , behavioral processes . for example , an intervention that targeted an increase in the frequency of bgm and provided education or training on this topic was considered a direct intervention . combined interventions focused on one or several behavioral tasks , but the emphasis of the intervention was on promoting coping or problem - solving skills or a family process involved in diabetes management . for example , a study included in this meta - analysis ( 14 ) provided a description of the intervention as well as an extensive table detailing session objectives and components . separately determined the percentage of the information described that focused on adherence promotion ( e.g. coded studies across 10 characteristics of the sample ( year of publication , mean age , percent female , percent ethnic minority , mean duration of type 1 diabetes , sample size [ total , experimental , and control ] , mean a1c at baseline and follow - up [ experimental and control ] , delivery format of the intervention [ group versus individual ] , time to follow - up , and statistics reported for adherence and a1c ) . this was either the posttreatment effect or the first follow - up effect , typically within 6 months of the end of treatment . finally , we reversed the valence of the effect size to be consistent with typical health promotion interventions . a negative effect size in this meta - analysis indicates a worsening of glycemic control . we were primarily interested in the magnitude of the effect size for change in glycemic control from pre- to posttreatment for the intervention group versus the control group . to arrive at this , we first calculated an effect size for 1 ) pre- to posttreatment change for the intervention group and 2 ) pre- to posttreatment change for the control group . then , we calculated change scores for the pre- to posttreatment intervention versus control comparison using an approach similar to that described by lipsey and wilson ( 15 ) and winkley et al . specifically , the mean change was divided by the pooled sd of the two groups in each study . we also examined the association among study effect size , characteristics of the sample , time to follow - up , and the rated percentage of adherence promotion in the intervention . when calculating the mean effect size across studies , we used a weighted least squares approach in which each effect size was weighted by the inverse of its variance ( 16 ) . specifically , the mean effect size , its se , and 95% ci were calculated . homogeneity analyses ( 16 ) were conducted to determine whether the mean effect size was accurately predicted by each individual effect size . homogeneity analyses provide a fuller understanding of similarities and differences among the studies included in the meta - analysis . these 12 studies were excluded because they were not randomized , controlled trials ( n = 10 ) , only a small fraction of the sample was aged < 19 years ( n = 1 ) , or no data were provided ( nor could be obtained via contact with authors ) on a1c values ( n = 1 ) . coding of the study design , intervention components , and categories of interest in each study were completed and and intraclass correlation coefficients were all 1.00 . study sample sizes ranged from 22 to 127 , and the total sample size across studies was 997 . however , two studies reported an inclusion criterion of poor glycemic control indicated by a certain a1c threshold ( e.g. very few studies reported indicators of socioeconomic status and family structure ( e.g. characteristics of studies in meta - analysis * type of intervention is defined as combined ( direct and indirect processes targeted ) or direct ( direct processes only ) . effect size is the pre- to posttreatment , intervention vs. control group effect size ; positive effect sizes reflect improvement in glycemic control ( i.e. , health promotion ) and negative effect sizes reflect deterioration in glycemic control . diabetes - specific variables consistently reported in studies were duration of type 1 diabetes and glycemic control . across the 15 studies , mean duration of type 1 diabetes ranged from 2.7 to 8.7 years ; average across the studies was 5.89 3.60 years . the mean a1c for the control group at baseline was 9.75 1.83% and at follow - up was 9.70 1.76% . the mean effect size for the pre- to posttreatment change in glycemic control for the intervention group was 0.13 , with individual study effects ranging from 0.50 to 0.99 . the mean effect size for the pre- to posttreatment change in the control group was 0.01 , with individual study effects ranging from 0.24 to 0.32 . this effect size indicates that glycemic control did not change over time for the pooled control group . the mean effect size for pre- to posttreatment change for the intervention versus control group comparison was 0.11 ( 95% ci 0.01 to 0.23 ) . effects ranged from 0.55 to 0.59 , and five were negative ( i.e. , deterioration in glycemic control ) . the mean effect size of 0.11 meets conventional standards for a small effect and indicates that there was very modest improvement in glycemic control over time . the association between the mean effect size for pre- to posttreatment change for the intervention versus control group and time to follow - up was not significant ( r = 0.07 , p > 0.05 ) . the correlation between the pre- to posttreatment intervention versus control effect size and whether the intervention targeted direct , behavioral processes alone or had combined targets was significant ( spearman r = 0.56 , p < 0.03 ) . , those that targeted direct and indirect processes together ) were associated with larger effects on glycemic control . homogeneity analysis of the pre- to posttreatment intervention versus control group comparison revealed homogeneity across the 15 studies [ q(14 ) = 18.40 ; p > 0.05 ] . however , analysis for the pre- to posttreatment effects for the intervention group alone did show significant variability [ q ( 14 ) = 33.11 , p < 0.05 ] . these 12 studies were excluded because they were not randomized , controlled trials ( n = 10 ) , only a small fraction of the sample was aged < 19 years ( n = 1 ) , or no data were provided ( nor could be obtained via contact with authors ) on a1c values ( n = 1 ) . coding of the study design , intervention components , and categories of interest in each study were completed and and intraclass correlation coefficients were all 1.00 . however , two studies reported an inclusion criterion of poor glycemic control indicated by a certain a1c threshold ( e.g. very few studies reported indicators of socioeconomic status and family structure ( e.g. effect size is the pre- to posttreatment , intervention vs. control group effect size ; positive effect sizes reflect improvement in glycemic control ( i.e. diabetes - specific variables consistently reported in studies were duration of type 1 diabetes and glycemic control . across the 15 studies , mean duration of type 1 diabetes ranged from 2.7 to 8.7 years ; average across the studies was 5.89 3.60 years . the mean a1c for the experimental group was 9.61 1.83% ( range 7.811.9% ) at baseline and 9.17 1.53% ( range 7.512.3% ) at follow - up . the mean a1c for the control group at baseline was 9.75 1.83% and at follow - up was 9.70 1.76% . the mean effect size for the pre- to posttreatment change in glycemic control for the intervention group was 0.13 , with individual study effects ranging from 0.50 to 0.99 . the mean effect size for the pre- to posttreatment change in the control group was 0.01 , with individual study effects ranging from 0.24 to 0.32 . this effect size indicates that glycemic control did not change over time for the pooled control group . the mean effect size for pre- to posttreatment change for the intervention versus control group comparison was 0.11 ( 95% ci 0.01 to 0.23 ) . effects ranged from 0.55 to 0.59 , and five were negative ( i.e. the mean effect size of 0.11 meets conventional standards for a small effect and indicates that there was very modest improvement in glycemic control over time . the association between the mean effect size for pre- to posttreatment change for the intervention versus control group and time to follow - up was not significant ( r = 0.07 , p > 0.05 ) . the correlation between the pre- to posttreatment intervention versus control effect size and whether the intervention targeted direct , behavioral processes alone or had combined targets was significant ( spearman r = 0.56 , p < 0.03 ) . , those that targeted direct and indirect processes together ) were associated with larger effects on glycemic control . homogeneity analysis of the pre- to posttreatment intervention versus control group comparison revealed homogeneity across the 15 studies [ q(14 ) = 18.40 ; p > 0.05 ] . however , analysis for the pre- to posttreatment effects for the intervention group alone did show significant variability [ q ( 14 ) = 33.11 , p < 0.05 ] . we hypothesized that this variability could be attributed to the percentage of the intervention targeting adherence promotion . results of this meta - analysis show that the mean effect size for the pre- to posttreatment change between intervention and control groups was 0.11 . whereas the mean change of a1c values from baseline to follow - up for youth in the intervention groups of these studies was 9.6 to 9.2% , variability in effects and the weighting of effects based on sample size translates to just a modest 0.20% reduction in a1c values . overall , this meta - analysis shows that the pooled effect on glycemic control of these 15 interventions was very modest . thus , this meta - analysis extends the current evidence base for interventions deployed in pediatric type 1 diabetes . interestingly , the interventions that we described as adherence - promoting interventions fared worse overall than the psychological interventions in the prior meta - analysis . specifically , the mean effect size in this meta - analysis was 3 times smaller than that reported by winkley et al . one reason for this may be our inclusion of interventions targeting direct , behavioral processes because these studies conferred little benefit on glycemic control . in our meta - analysis , the interventions that fared the best were those that were multicomponent and addressed both the direct and indirect processes involved in diabetes management . this program trains groups of youth with type 1 diabetes to increase the[ir ] ability to cope with problems that they face on a day - to - day basis and to be more effective in achieving therapeutic goals ( 8) . overall , these interventions seem to be exemplars for the type of interventions that carry considerable benefit for improvement in glycemic control . on the other hand , the mean effect size of this meta - analysis provides a sobering look at the current state of interventions with adherence - promoting components if improvement in glycemic control is the measuring stick . why is the mean effect size so small and why do only a few interventions produce considerable benefit on glycemic control ? first , the percentage of the intervention directly targeting adherence promotion does not seem to be a factor . what appears important is whether or not the indirect processes are targeted in the intervention . perhaps the primary focus on adherence behaviors and the neglect of other areas related to glycemic control ( social support , family relationships , coping , and problem - solving skills ) is what drove the present findings . second , and related to the first point , the dose of the intervention does not appear to be a factor . a sizable effect on glycemic control was observed in the study by laffel , anderson , and colleagues ( 20 ) , which included approximately four clinic - based intervention sessions across the year plus support from the care ambassador in scheduling appointments and working with clinic staff . the dose in this study was considerably less than that in other time- and resource - heavy interventions ( 11,26 ) , both with more modest effects on glycemic control , as well as that in an earlier iteration of the anderson and laffel family - based intervention ( 10 ) . because these studies were controlled and there was little change in glycemic control for the control participants , simply stopping the deterioration of glycemic control that is so commonly seen across late childhood and adolescence ( 2830 ) was not enough to demonstrate statistical significance . the primary implication of these meta - analytic results is that there is considerable work left to be done to make a significant impact on glycemic outcomes . however , after a thorough review of the most potent interventions , we conclude there is no need to start over . for example , we should learn from the interventions that produce reductions in a1c values approaching 1.0% ( e.g. likewise , an evaluation of psychosocial functioning ( e.g. at the same time efforts are made to document effectiveness , the interventions demonstrating modest effects on glycemic control may benefit from the addition of other components and then a reexamination of their efficacy . for example , the most efficacious interventions target processes other than direct , behavioral processes ; they target the critical emotional , social , and family factors that youth with type 1 diabetes face on a daily basis . by focusing more so on these facilitators of adherence considered together , the collection of effectiveness data on the most potent interventions and adapting and refining less potent interventions will push the field closer to providing evidence - based care in pediatric diabetes clinics . the results of this meta - analysis and our recommendations should be considered in the context of several limitations . first , a meta - analysis pools studies and can not account for differing methodologies and sampling strategies ( 31 ) . the extent to which that variability is related to the sampling methodology and features of study design can not be ascertained through meta - analysis . second , the teams of investigators from these 15 studies may not have viewed adherence promotion as the primary mechanism to change glycemic control . we simply identified interventions with adherence - promoting components and may not be fully characterizing the whole treatment package . fourth , other variables that influence glycemic control ( e.g. a related limitation is that few of the studies targeted high - risk youth , those who consistently demonstrate high a1c values and have fewer resources and support . indeed , there are compelling data on the effectiveness of these types of programs with these high - risk youth ( 37,38 ) . given the data that adherence is linked to glycemic control ( 1 ) and a focus on optimizing glycemic control in practice guidelines ( 3 ) , we were primarily concerned with understanding whether these interventions alter glycemic outcomes . although we do not suspect this to be the case , it is possible that some interventions included in this meta - analysis improved adherence significantly while not altering glycemic control . in sum , this review and meta - analysis of interventions focused on adherence promotion in pediatric type 1 diabetes emphasizes two points . first , it is likely that glycemic control will not improve if interventions focus on direct , behavioral processes involved in diabetes management and neglect emotional , social , and family processes . it is important that we better understand the costs and savings from a monetary perspective and also from the perspective of health outcomes of the pediatric patients cared for in these clinics .

gastric cancer is the fourth most common malignancy worldwide , and most common malignancy in korea . although survival time has been lengthened , the life expectancy of advanced disease still remains within one year . for patients with advanced gastric carcinoma or recurrent cancer , the response rate ( rr ) for first - line chemotherapy ranges from 33 - 55% , and the survival time following chemotherapy is superior to that of patients given best supportive care . despite poorer survival benefit and tolerance of second - line chemotherapy , about 20% of patients with failure of first - line chemotherapy receive second - line chemotherapy . asian studies have reported a rr of second - line chemotherapy as 5 - 25% and progression - free survival ( pfs ) of 5.2 - 8.8 months . many clinicians consider second - line chemotherapy after failure of first - line chemotherapy for patients with advanced gastric carcinoma . there is a controversy about the benefit of sequential salvage therapy further to second - line . however , with prolonged survival time of patients and tolerable toxicity of drugs , there has been renewed interest in chemotherapy after failure of second - line treatments . it has been our empiric observation that some patients maintain good performance after failure of second - line chemotherapy , and live longer with salvage chemotherapy than other patients with supportive care . the aim of the present study was to clarify the relationship between the sequence of second- and third - line chemotherapy regimen including taxane and irinotecan with survival of patients , and to identify clinical characteristics of patients with better response to salvage chemotherapy than others . we retrospectively evaluated the medical records of advanced or recurred gastric carcinoma patients who were diagnosed and treated at gangnam severance hospital between november 2004 and july 2010 . patients who met the following criteria were eligible : histologically proven advanced gastric adenocarcinoma with metastatic or recurrent disease ; received third - line chemotherapy at gangnam severance hospital using folinic acid , 5-fluorouracil ( 5-fu ) and oxaliplatin ( folfox ) or 5-fu+cisplatin ( fp ) , folinic acid , 5-fu and irinotecan ( folfiri ) , paclitaxel or docetaxel with 5-fu , with or without epirubicin ; measurable lesion according to the response evaluation criteria in solid tumors ( recist ) or a non - measurable lesion , assessed using either computed tomography ( ct ) or magnetic resonance imaging ; 18 - 75 years of age ; eastern cooperative oncology group ( ecog ) performance status2 ; life expectancy of at least 3 months ; and adequate hematologic function , evident as absolute neutrophil count1500/l , platelet count100,000/l , hepatic function ( total bilirubin2 times the upper normal limit [ unl ] , serum transaminase level2 times the unl ) and renal function ( serum creatine1.5 times the unl ) . exclusion criteria were the presence of other severe medical illness , central nervous system metastasis , another active malignancy , or history of anaphylaxis to drugs . clinical information included gender , age at diagnosis , ecog performance status , pathologic differentiation , lauren classification , coexisting peritoneal or hepatic metastases , date of chemotherapy , and number of cycles . in addition , laboratory data including hemoglobin , serum albumin , carcinoembryonic antigen ( cea ) , and cancer antigen ( ca ) 19 - 9 were also reviewed . sequential chemotherapy was defined as chemotherapy using more than two different chemotherapeutic regimens performed consecutively . patients were assigned to either arm a or arm b , according to the sequence of three chemotherapeutic regimens . arm a comprised folfox or fp for first - line chemotherapy ; folfiri for second - line chemotherapy ; and docetaxel or paclitaxel , leucovorin , and 5-fu ( with or without epirubicin ) for third - line chemotherapy . in arm b , patients were treated with folfox or fp for first - line chemotherapy ; docetaxel or paclitaxel , leucovorin , and fu with or without epirubicin for second - line chemotherapy ; and folfiri for third - line chemotherapy . patients received oxaliplatin ( 100 mg / m in 500 ml normal saline ) or 5% dextrose water over 2 hours followed by leucovorin ( 100 mg / m ) over 2 hours on day 1 and continuous infusion of 5-fu 1,000 mg / m / day for 2 days . cisplatin ( 70 mg / m in 500 ml normal saline ) or 5% dextrose water was intravenously administered over 2 hours on day 1 followed by continuous infusion of 5-fu ( 1,000 mg / m / day ) for 2 days . irinotecan ( 150 mg / m in 500 ml normal saline ) was intravenously administered over 2 hours on followed by leucovorin ( 100 mg / m over 2 hours ) on day 1 and 5-fu ( 1,000 docetaxel ( 75 mg / m in 500 ml normal saline ) or paclitaxel ( 175 mg / m in 500 ml normal saline ) was intravenously administered over 2 hours followed by leucovorin ( 100 mg / m over 2 hours ) on day 1 and 5-fu ( 1,000 for some patients , epirubicin ( 50 mg / m in 100 ml normal saline ) was intravenously administered over 30 minutes on day 2 . response was assessed after two cycles of chemotherapy and when disease progression was clinically suspected . a measurable lesion was defined as a lesion with the longest diameter 10 mm in any dimension assessed by spiral ct imaging . tumor response was evaluated according to the recist guidelines as follows : complete response ( cr ) if all target lesions disappeared , partial response ( pr ) if the sum of diameters of the target lesions decreased by at least 30% , progressive disease ( pd ) if the sum of the longest diameters of the target lesions increased by at least 20% and one or more new lesions appeared , and stable disease ( sd ) for responses that were neither pr nor pd . the rr was defined as proportion of the patients with responses of cr or pr out of all patients . overall survival ( os ) was defined as the interval from the first date of chemotherapy to death or the last follow - up date . time to progression ( ttp ) was defined as the time from the first date of chemotherapy to the time of progression of the disease or the last follow - up date . the kaplan - meier method was used for survival analysis , and survival curves were compared assessed using the log - rank test . following variables were included in univariate analysis : age at diagnosis , gender , performance status by ecog criteria , pathologic differentiation , coexisting peritoneal or hepatic metastases , type of chemotherapeutic regimen , hemoglobin , serum albumin , cea , ca19 - 9 , and ttp of first- or second - line chemotherapy . for variables with a probability p - value0.3 in univariate analysis , multivariate analysis was also performed using the cox 's proportional hazard regression model ( two - sided ) . we retrospectively evaluated the medical records of advanced or recurred gastric carcinoma patients who were diagnosed and treated at gangnam severance hospital between november 2004 and july 2010 . patients who met the following criteria were eligible : histologically proven advanced gastric adenocarcinoma with metastatic or recurrent disease ; received third - line chemotherapy at gangnam severance hospital using folinic acid , 5-fluorouracil ( 5-fu ) and oxaliplatin ( folfox ) or 5-fu+cisplatin ( fp ) , folinic acid , 5-fu and irinotecan ( folfiri ) , paclitaxel or docetaxel with 5-fu , with or without epirubicin ; measurable lesion according to the response evaluation criteria in solid tumors ( recist ) or a non - measurable lesion , assessed using either computed tomography ( ct ) or magnetic resonance imaging ; 18 - 75 years of age ; eastern cooperative oncology group ( ecog ) performance status2 ; life expectancy of at least 3 months ; and adequate hematologic function , evident as absolute neutrophil count1500/l , platelet count100,000/l , hepatic function ( total bilirubin2 times the upper normal limit [ unl ] , serum transaminase level2 times the unl ) and renal function ( serum creatine1.5 times the unl ) . exclusion criteria were the presence of other severe medical illness , central nervous system metastasis , another active malignancy , or history of anaphylaxis to drugs . clinical information included gender , age at diagnosis , ecog performance status , pathologic differentiation , lauren classification , coexisting peritoneal or hepatic metastases , date of chemotherapy , and number of cycles . in addition , laboratory data including hemoglobin , serum albumin , carcinoembryonic antigen ( cea ) , and cancer antigen ( ca ) 19 - 9 were also reviewed . sequential chemotherapy was defined as chemotherapy using more than two different chemotherapeutic regimens performed consecutively . patients were assigned to either arm a or arm b , according to the sequence of three chemotherapeutic regimens . arm a comprised folfox or fp for first - line chemotherapy ; folfiri for second - line chemotherapy ; and docetaxel or paclitaxel , leucovorin , and 5-fu ( with or without epirubicin ) for third - line chemotherapy . in arm b , patients were treated with folfox or fp for first - line chemotherapy ; docetaxel or paclitaxel , leucovorin , and fu with or without epirubicin for second - line chemotherapy ; and folfiri for third - line chemotherapy . patients received oxaliplatin ( 100 mg / m in 500 ml normal saline ) or 5% dextrose water over 2 hours followed by leucovorin ( 100 mg / m ) over 2 hours on day 1 and continuous infusion of 5-fu 1,000 mg / m / day for 2 days . cisplatin ( 70 mg / m in 500 ml normal saline ) or 5% dextrose water was intravenously administered over 2 hours on day 1 followed by continuous infusion of 5-fu ( 1,000 irinotecan ( 150 mg / m in 500 ml normal saline ) was intravenously administered over 2 hours on followed by leucovorin ( 100 mg / m over 2 hours ) on day 1 and 5-fu ( 1,000 docetaxel ( 75 mg / m in 500 ml normal saline ) or paclitaxel ( 175 mg / m in 500 ml normal saline ) was intravenously administered over 2 hours followed by leucovorin ( 100 mg / m over 2 hours ) on day 1 and 5-fu ( 1,000 for some patients , epirubicin ( 50 mg / m in 100 ml normal saline ) was intravenously administered over 30 minutes on day 2 . response was assessed after two cycles of chemotherapy and when disease progression was clinically suspected . a measurable lesion was defined as a lesion with the longest diameter 10 mm in any dimension assessed by spiral ct imaging . tumor response was evaluated according to the recist guidelines as follows : complete response ( cr ) if all target lesions disappeared , partial response ( pr ) if the sum of diameters of the target lesions decreased by at least 30% , progressive disease ( pd ) if the sum of the longest diameters of the target lesions increased by at least 20% and one or more new lesions appeared , and stable disease ( sd ) for responses that were neither pr nor pd . the rr was defined as proportion of the patients with responses of cr or pr out of all patients . patients received oxaliplatin ( 100 mg / m in 500 ml normal saline ) or 5% dextrose water over 2 hours followed by leucovorin ( 100 mg / m ) over 2 hours on day 1 and continuous infusion of 5-fu 1,000 mg / m / day for 2 days . cisplatin ( 70 mg / m in 500 ml normal saline ) or 5% dextrose water was intravenously administered over 2 hours on day 1 followed by continuous infusion of 5-fu ( 1,000 mg / m / day ) for 2 days . irinotecan ( 150 mg / m in 500 ml normal saline ) was intravenously administered over 2 hours on followed by leucovorin ( 100 mg / m over 2 hours ) on day 1 and 5-fu ( 1,000 docetaxel ( 75 mg / m in 500 ml normal saline ) or paclitaxel ( 175 mg / m in 500 ml normal saline ) was intravenously administered over 2 hours followed by leucovorin ( 100 mg / m over 2 hours ) on day 1 and 5-fu ( 1,000 for some patients , epirubicin ( 50 mg / m in 100 ml normal saline ) was intravenously administered over 30 minutes on day 2 . response was assessed after two cycles of chemotherapy and when disease progression was clinically suspected . a measurable lesion was defined as a lesion with the longest diameter 10 mm in any dimension assessed by spiral ct imaging . tumor response was evaluated according to the recist guidelines as follows : complete response ( cr ) if all target lesions disappeared , partial response ( pr ) if the sum of diameters of the target lesions decreased by at least 30% , progressive disease ( pd ) if the sum of the longest diameters of the target lesions increased by at least 20% and one or more new lesions appeared , and stable disease ( sd ) for responses that were neither pr nor pd . the rr was defined as proportion of the patients with responses of cr or pr out of all patients . overall survival ( os ) was defined as the interval from the first date of chemotherapy to death or the last follow - up date . time to progression ( ttp ) was defined as the time from the first date of chemotherapy to the time of progression of the disease or the last follow - up date . the kaplan - meier method was used for survival analysis , and survival curves were compared assessed using the log - rank test . following variables were included in univariate analysis : age at diagnosis , gender , performance status by ecog criteria , pathologic differentiation , coexisting peritoneal or hepatic metastases , type of chemotherapeutic regimen , hemoglobin , serum albumin , cea , ca19 - 9 , and ttp of first- or second - line chemotherapy . for variables with a probability p - value0.3 in univariate analysis , multivariate analysis was also performed using the cox 's proportional hazard regression model ( two - sided ) . between november 2004 and july 2010 , 50 patients with gastric carcinoma were treated with a sequential chemotherapy protocol , based only on folfox or fp , folfiri , and paclitaxel or docetaxel , at gangnam severance hospital . the median age was 52.5 years ( range , 29 to 70 years ) , and 34 patients ( 68% ) were male . forty subjects ( 80% ) had good performance status ( ecog 0 - 1 ) . seven patients had diffuse type , 13 patients had interstitial type , and the other two patients had mixed type adenocarcinoma . no significant difference except for peritoneal carcinomatosis was observed . fifty patients received median fourth - line chemotherapy ( range , 3 to 10 ) , and a median of 18 cycles ( range , 4 to 60 cycles ) were administered . median os of all patients was 16.0 months ( 95% confidence interval [ ci ] , 13.6 to 18.3 months ) ( fig . 1 ) , and median ttp was 5.0 months ( 95% ci , 3.9 to 6.1 months ) in first - line chemotherapy , 2.4 months ( 95% ci , 1.6 to 3.2 months ) in second - line chemotherapy , and 2.5 months ( 95% ci , 1.8 to 3.3 months ) in third - line chemotherapy . the median os time of arm a and arm b was 17.1 months ( 95% ci , 10.7 to 23.6 months ) , and 15.4 months ( range , 13.8 to 17.1 months ) , respectively . 1 ) . ttp of arm a and arm b with first - line chemotherapy was 5.5 months ( 95% ci , 1.2 to 9.8 months ) and 4.8 months ( 95% ci , 3.8 to 5.8 months ) , respectively ( p=0.064 ) . for the second - line chemotherapy , ttp of arm a and arm b was 2.7 months and 2.2 months , respectively ( p=0.850 ) . each ttp of the two groups were 2.2 months in arm a and 2.7 months in arm b ( p=0.297 ) . in addition , we analyzed clinicopathologic characteristics of the patients . patients with poorly - differentiated adenocarcinoma or signet ring cell feature showed poorer prognosis than others . furthermore , elevated cea level and shorter ttp of first - line chemotherapy were significantly related with shorter survival times ( table 2 ) . the rr was 50% for first - line chemotherapy , 8% for second - line chemotherapy , and 8.5% for third - line chemotherapy . in second - line chemotherapy , rr was 11.1% in arm a and 6.3% in arm b. in third - line chemotherapy , rr was 6.3% in arm a and 9.7% in arm b. the disease control rate ( dcr ) which was determined by the disease status ( cr , pr , or sd ) controlled by treatment , of first - line chemotherapy was 88.9% in arm a and 78.1% in arm b ( p=0.342 ) . in second - line chemotherapy , dcr of arm a was 38.9% , and that of arm b was 53.1% ( p=0.333 ) . in third - line chemotherapy , dcr was 25% in arm a and 41.9% in arm b ( p=0.252 ) . twenty - one patients did not show any response ( pr or cr ) to any of the three sequential chemotherapies . the median os was 20.0 months ( 95% ci , 2.4 to 37.5 months ) in the good - responders and 13.9 months ( 95% ci , 12.0 to 15.8 months ) in the poor - responders . between november 2004 and july 2010 , 50 patients with gastric carcinoma were treated with a sequential chemotherapy protocol , based only on folfox or fp , folfiri , and paclitaxel or docetaxel , at gangnam severance hospital . the median age was 52.5 years ( range , 29 to 70 years ) , and 34 patients ( 68% ) were male . forty subjects ( 80% ) had good performance status ( ecog 0 - 1 ) . seven patients had diffuse type , 13 patients had interstitial type , and the other two patients had mixed type adenocarcinoma . no significant difference except for peritoneal carcinomatosis was observed . fifty patients received median fourth - line chemotherapy ( range , 3 to 10 ) , and a median of 18 cycles ( range , 4 to 60 cycles ) were administered . median os of all patients was 16.0 months ( 95% confidence interval [ ci ] , 13.6 to 18.3 months ) ( fig . 1 ) , and median ttp was 5.0 months ( 95% ci , 3.9 to 6.1 months ) in first - line chemotherapy , 2.4 months ( 95% ci , 1.6 to 3.2 months ) in second - line chemotherapy , and 2.5 months ( 95% ci , 1.8 to 3.3 months ) in third - line chemotherapy . the median os time of arm a and arm b was 17.1 months ( 95% ci , 10.7 to 23.6 months ) , and 15.4 months ( range , 13.8 to 17.1 months ) , respectively . 1 ) . ttp of arm a and arm b with first - line chemotherapy was 5.5 months ( 95% ci , 1.2 to 9.8 months ) and 4.8 months ( 95% ci , 3.8 to 5.8 months ) , respectively ( p=0.064 ) . for the second - line chemotherapy , ttp of arm a and arm b was 2.7 months and 2.2 months , respectively ( p=0.850 ) . each ttp of the two groups were 2.2 months in arm a and 2.7 months in arm b ( p=0.297 ) . patients with poorly - differentiated adenocarcinoma or signet ring cell feature showed poorer prognosis than others . furthermore , elevated cea level and shorter ttp of first - line chemotherapy were significantly related with shorter survival times ( table 2 ) . the rr was 50% for first - line chemotherapy , 8% for second - line chemotherapy , and 8.5% for third - line chemotherapy . in second - line chemotherapy , rr was 11.1% in arm a and 6.3% in arm b. in third - line chemotherapy , rr was 6.3% in arm a and 9.7% in arm b. the disease control rate ( dcr ) which was determined by the disease status ( cr , pr , or sd ) controlled by treatment , of first - line chemotherapy was 88.9% in arm a and 78.1% in arm b ( p=0.342 ) . in second - line chemotherapy , dcr of arm in third - line chemotherapy , dcr was 25% in arm a and 41.9% in arm b ( p=0.252 ) . twenty - one patients did not show any response ( pr or cr ) to any of the three sequential chemotherapies . the median os was 20.0 months ( 95% ci , 2.4 to 37.5 months ) in the good - responders and 13.9 months ( 95% ci , 12.0 to 15.8 months ) in the poor - responders . it has generally been accepted that palliative chemotherapy can significantly prolong survival of patients with advanced gastric carcinoma , longer than best supportive care . platinum - based chemotherapy is widely - used as first - line chemotherapy for advanced gastric cancer . given the reported tolerance and survival benefits of second - line chemotherapy , studies have sought to identify the benefit and adequate regimen of second - line chemotherapy . the current study evaluated two groups of gastric cancer patients who received third - line chemotherapy . previous studies reported the median pfs of 2.5 - 3.3 months and the median os of 5.3 - 8.7 months in gastric cancer patients who received second - line chemotherapy [ 6,8 - 10 ] . in this study , the median pfs was 2.4 months ( 95% ci , 1.6 to 3.2 months ) for patients treated with second - line chemotherapy , similar to the previous report . the median os was 16.0 months ( 95% ci , 13.6 to 18.3 months ) , and 32.0% of patients survived after 2 years . these times were slightly longer than that of historical control . we suggest that this is due to sequential treatment of the patients . this study included a restricted population with a performance status that permitted the use of third - line chemotherapy after failure of second - line chemotherapy . therefore , this result was subject to limitation in comparison with other survival data of patients without third - line chemotherapy . nevertheless , based on 2.5 months of median ttp in third - line chemotherapy , the authors suggest that third - line chemotherapy would provide a survival benefit for patients with favorable performance status . in many phase ii clinical trials of second - line chemotherapy for advanced gastric cancer , rr was about 20.8% , and dcr was about 51% . in the present study , rr for second - line chemotherapy was 8% and dcr was 48% . there is still little information to support the provision of a second - line chemotherapeutic regimen after failure of platinum - based first - line chemotherapy . reported that sequential chemotherapy appears to be both a feasible and effective treatment for advanced gastric cancer . their sequential protocol was s-1 based chemotherapy for first - line chemotherapy , a low dose irinotecan / cisplatin for second - line chemotherapy , and paclitaxel for third - line chemotherapy . thirty - two gastric cancer patients showed relatively long median survival ( 17.4 months ; 95% ci , 323 to 723 days ) . this result shows that a consecutive use of chemotherapy has a substantial impact on os of gastric cancer patients . these facts also demonstrate that second- and third - line chemotherapy is necessary to treat advanced gastric cancer . many clinicians consider the folfiri regimen and paclitaxel or docetaxel with 5-fu regimen as second - line chemotherapy after failure of platinum - based first line chemotherapy . numerous physicians suggested that folfiri is tolerable and has a modest effect as a second - line chemotherapy for gastric carcinoma . in an asian study , the median pfs was 3.2 months , and the median os was 9.1 months . in another study , rr was 18.2% , median os was 5.1 months , and median ttp was 2.3 months . on the other hand , the tolerance and effectiveness of taxane - based chemotherapy apparent in several studies ( median ttp , 2.6 - 3.9 months ; median os , 7.2 to 10.1 months ) warrants its use as second - line chemotherapy in advanced gastric carcinoma . kim et al . reported that overall rr and dcr for patients with docetaxel / cisplatin followed by folfiri and patients with folfiri followed by a docetaxel / cisplatin regimen showed no significant difference . however , to the best of our knowledge , survival benefits according to sequence of regimens of second - line and third - line chemotherapy after failure of first line chemotherapy have not been reported . in this study , we analyzed 50 patients treated with either folfox or fp chemotherapy followed by folfiri , and paclitaxel or docetaxel , or folfox or fp chemotherapy followed by paclitaxel or docetaxel , and folfiri regimen . however , we found some patients responded better to chemotherapy , and their os was longer than the others . previous investigators suggested that low hemoglobin level , low serum albumin level , high cea level , poor performance status [ 8 - 10,16,17 ] , shorter ttp of first - line chemotherapy , shorter ttp of second - line chemotherapy , and poor differentiation of tumor are poor prognostic factors for patients with gastric carcinoma under sequential chemotherapy . we analyzed the previously reported factors and found some differences in survival according to histologic differentiation , elevated cea level , and ttp of first line chemotherapy in this study . there has been a controversy regarding the influence of the histological feature of signet ring cell on the prognosis of gastric carcinoma . some researchers reported poorer prognosis of advanced gastric carcinoma with signet ring cell histology , while better prognosis of early gastric carcinoma with signet ring cell histology has been reported . in this study , gastric adenocarcinoma with poorly differentiated or signet ring cell features had poorer response to sequential chemotherapy , and had shorter median os than well or moderately differentiated ones . concerning cea level , some investigators reported the relationship of serum cea level with tumor invasion , lymph node metastasis , and higher recurrence rate . herein , we suggest that differences in gene and protein expressions are other prognostic values that should be taken into account . in a recent study , we suppose that there are some genetic differences in diffuse gastric adenocarcinoma and that the different genetic defect influences a poorer response to sequential chemotherapy . furthermore , cea is a widely used tumor marker , and the cea gene has been widely - used as a tumor - specific promoter and target of treatment over the last decade . we propose that the difference in response could also be attributed to the differences in gene expression profiles between the two groups . in recent years , it has become clear that a pharmacogenic approach is a potential tool for optimizing treatment for several human tumors . the ability of genetic polymorphisms to influence pharmacogenetics of 5-fu , platinum derivatives , anthracyclines , irinotecan , and docetaxel in gastric cancer has been reported . the use of multiple gene analyses in the development of individualized gastric cancer chemotherapies could be helpful in selecting patients who are more likely to benefit better to a particular therapy . the possible prediction of cancer outcome from gene expression classifiers , sets of genes , or signatures associated with prognosis together with classification rules has been suggested . limitations of this study include the retrospective study design and small patient pool . nevertheless , the results of this study provides evidence that third - line chemotherapy , including folfox or fp , folfiri , paclitaxel or docetaxel regimen , helps to improve os of patients , regardless of the sequence of use of taxane and irinotecan . in addition , os seems to be poorly affected by poorly differentiated or signet ring cell feature of adenocarcinoma , elevated cea level , and shorter ttp of first - line chemotherapy . different chemotherapy - responses to locally advanced or metastatic gastric carcinoma treated with second and third - line chemotherapy using irinotecan or taxane could be attributed not to sequence of chemotherapeutic regimen , but to patients ' different clinical features underlying different gene expression profiles . future studies will be required to investigate the impact of genetic difference on response to sequential chemotherapy .

purposethe aims of this study are to find out whether the sequence of chemotherapeutic regimens including second- and third - line taxane and irinotecan influences the survival of patients with unresectable gastric carcinoma and to identify clinical characteristics of patients with improved response.materials and methodsfifty gastric carcinoma patients who were treated by third - line sequential chemotherapy between november 2004 and july 2010 were enrolled in this study . their overall survival ( os ) and time to progression ( ttp ) were set up as primary and secondary end points . for the sequence of chemotherapy regimen , two arms were used . arm a was defined as 5-fluorouracil ( 5-fu)+cisplatin ( fp ) or folinic acid , 5-fu and oxaliplati ( folfox ) , followed by folinic acid , 5-fu and irinotecan ( folfiri ) , and paclitaxel or docetaxel plus 5-fu , with or without epirubicin . arm b was defined as fp or folfox , followed by paclitaxel or docetaxel plus 5-fu , and folfiri.resultsthe median os of all patients was 16.0 months ( 95% confidence interval , 13.6 to 18.3 months ) , which is longer than historical control of patients who did not receive third - line chemotherapy . the sequence of second and third - line regimen , including irinotecan and taxane , did not present significant difference in os or ttp after failure of 5-fu with platinum chemotherapy . in survival analysis of patients ' clinicopathologic characteristics , poor prognosis was shown in patients with poorly differentiated histologic features , elevated serum carcinoembryonic level , and shorter ttp of first line chemotherapy.conclusionit is possible for patients to respond differently to chemotherapy due to differences in clinical features and underlying gene expression profiles . development of individualized chemotherapy regimens based on gene expression profiles is warranted .

Introduction Materials and Methods 1. Patients 2. Treatment 1) FOLFOX chemotherapy 2) FP chemotherapy 3) FOLFIRI chemotherapy 4) Docetaxel or paclitaxel, leucovorin, and 5-FU chemotherapy, with or without epirubicin 5) Efficacy 3. Statistical analyses Results 1. Baseline characteristics 2. Treatment outcomes 3. Efficacy Discussion Conclusion

for patients with advanced gastric carcinoma or recurrent cancer , the response rate ( rr ) for first - line chemotherapy ranges from 33 - 55% , and the survival time following chemotherapy is superior to that of patients given best supportive care . despite poorer survival benefit and tolerance of second - line chemotherapy , about 20% of patients with failure of first - line chemotherapy receive second - line chemotherapy . asian studies have reported a rr of second - line chemotherapy as 5 - 25% and progression - free survival ( pfs ) of 5.2 - 8.8 months . many clinicians consider second - line chemotherapy after failure of first - line chemotherapy for patients with advanced gastric carcinoma . however , with prolonged survival time of patients and tolerable toxicity of drugs , there has been renewed interest in chemotherapy after failure of second - line treatments . it has been our empiric observation that some patients maintain good performance after failure of second - line chemotherapy , and live longer with salvage chemotherapy than other patients with supportive care . the aim of the present study was to clarify the relationship between the sequence of second- and third - line chemotherapy regimen including taxane and irinotecan with survival of patients , and to identify clinical characteristics of patients with better response to salvage chemotherapy than others . we retrospectively evaluated the medical records of advanced or recurred gastric carcinoma patients who were diagnosed and treated at gangnam severance hospital between november 2004 and july 2010 . patients who met the following criteria were eligible : histologically proven advanced gastric adenocarcinoma with metastatic or recurrent disease ; received third - line chemotherapy at gangnam severance hospital using folinic acid , 5-fluorouracil ( 5-fu ) and oxaliplatin ( folfox ) or 5-fu+cisplatin ( fp ) , folinic acid , 5-fu and irinotecan ( folfiri ) , paclitaxel or docetaxel with 5-fu , with or without epirubicin ; measurable lesion according to the response evaluation criteria in solid tumors ( recist ) or a non - measurable lesion , assessed using either computed tomography ( ct ) or magnetic resonance imaging ; 18 - 75 years of age ; eastern cooperative oncology group ( ecog ) performance status2 ; life expectancy of at least 3 months ; and adequate hematologic function , evident as absolute neutrophil count1500/l , platelet count100,000/l , hepatic function ( total bilirubin2 times the upper normal limit [ unl ] , serum transaminase level2 times the unl ) and renal function ( serum creatine1.5 times the unl ) . sequential chemotherapy was defined as chemotherapy using more than two different chemotherapeutic regimens performed consecutively . patients were assigned to either arm a or arm b , according to the sequence of three chemotherapeutic regimens . arm a comprised folfox or fp for first - line chemotherapy ; folfiri for second - line chemotherapy ; and docetaxel or paclitaxel , leucovorin , and 5-fu ( with or without epirubicin ) for third - line chemotherapy . in arm b , patients were treated with folfox or fp for first - line chemotherapy ; docetaxel or paclitaxel , leucovorin , and fu with or without epirubicin for second - line chemotherapy ; and folfiri for third - line chemotherapy . irinotecan ( 150 mg / m in 500 ml normal saline ) was intravenously administered over 2 hours on followed by leucovorin ( 100 mg / m over 2 hours ) on day 1 and 5-fu ( 1,000 docetaxel ( 75 mg / m in 500 ml normal saline ) or paclitaxel ( 175 mg / m in 500 ml normal saline ) was intravenously administered over 2 hours followed by leucovorin ( 100 mg / m over 2 hours ) on day 1 and 5-fu ( 1,000 for some patients , epirubicin ( 50 mg / m in 100 ml normal saline ) was intravenously administered over 30 minutes on day 2 . the rr was defined as proportion of the patients with responses of cr or pr out of all patients . overall survival ( os ) was defined as the interval from the first date of chemotherapy to death or the last follow - up date . time to progression ( ttp ) was defined as the time from the first date of chemotherapy to the time of progression of the disease or the last follow - up date . following variables were included in univariate analysis : age at diagnosis , gender , performance status by ecog criteria , pathologic differentiation , coexisting peritoneal or hepatic metastases , type of chemotherapeutic regimen , hemoglobin , serum albumin , cea , ca19 - 9 , and ttp of first- or second - line chemotherapy . we retrospectively evaluated the medical records of advanced or recurred gastric carcinoma patients who were diagnosed and treated at gangnam severance hospital between november 2004 and july 2010 . patients who met the following criteria were eligible : histologically proven advanced gastric adenocarcinoma with metastatic or recurrent disease ; received third - line chemotherapy at gangnam severance hospital using folinic acid , 5-fluorouracil ( 5-fu ) and oxaliplatin ( folfox ) or 5-fu+cisplatin ( fp ) , folinic acid , 5-fu and irinotecan ( folfiri ) , paclitaxel or docetaxel with 5-fu , with or without epirubicin ; measurable lesion according to the response evaluation criteria in solid tumors ( recist ) or a non - measurable lesion , assessed using either computed tomography ( ct ) or magnetic resonance imaging ; 18 - 75 years of age ; eastern cooperative oncology group ( ecog ) performance status2 ; life expectancy of at least 3 months ; and adequate hematologic function , evident as absolute neutrophil count1500/l , platelet count100,000/l , hepatic function ( total bilirubin2 times the upper normal limit [ unl ] , serum transaminase level2 times the unl ) and renal function ( serum creatine1.5 times the unl ) . sequential chemotherapy was defined as chemotherapy using more than two different chemotherapeutic regimens performed consecutively . patients were assigned to either arm a or arm b , according to the sequence of three chemotherapeutic regimens . arm a comprised folfox or fp for first - line chemotherapy ; folfiri for second - line chemotherapy ; and docetaxel or paclitaxel , leucovorin , and 5-fu ( with or without epirubicin ) for third - line chemotherapy . in arm b , patients were treated with folfox or fp for first - line chemotherapy ; docetaxel or paclitaxel , leucovorin , and fu with or without epirubicin for second - line chemotherapy ; and folfiri for third - line chemotherapy . cisplatin ( 70 mg / m in 500 ml normal saline ) or 5% dextrose water was intravenously administered over 2 hours on day 1 followed by continuous infusion of 5-fu ( 1,000 irinotecan ( 150 mg / m in 500 ml normal saline ) was intravenously administered over 2 hours on followed by leucovorin ( 100 mg / m over 2 hours ) on day 1 and 5-fu ( 1,000 docetaxel ( 75 mg / m in 500 ml normal saline ) or paclitaxel ( 175 mg / m in 500 ml normal saline ) was intravenously administered over 2 hours followed by leucovorin ( 100 mg / m over 2 hours ) on day 1 and 5-fu ( 1,000 for some patients , epirubicin ( 50 mg / m in 100 ml normal saline ) was intravenously administered over 30 minutes on day 2 . the rr was defined as proportion of the patients with responses of cr or pr out of all patients . the rr was defined as proportion of the patients with responses of cr or pr out of all patients . overall survival ( os ) was defined as the interval from the first date of chemotherapy to death or the last follow - up date . time to progression ( ttp ) was defined as the time from the first date of chemotherapy to the time of progression of the disease or the last follow - up date . following variables were included in univariate analysis : age at diagnosis , gender , performance status by ecog criteria , pathologic differentiation , coexisting peritoneal or hepatic metastases , type of chemotherapeutic regimen , hemoglobin , serum albumin , cea , ca19 - 9 , and ttp of first- or second - line chemotherapy . between november 2004 and july 2010 , 50 patients with gastric carcinoma were treated with a sequential chemotherapy protocol , based only on folfox or fp , folfiri , and paclitaxel or docetaxel , at gangnam severance hospital . fifty patients received median fourth - line chemotherapy ( range , 3 to 10 ) , and a median of 18 cycles ( range , 4 to 60 cycles ) were administered . median os of all patients was 16.0 months ( 95% confidence interval [ ci ] , 13.6 to 18.3 months ) ( fig . 1 ) , and median ttp was 5.0 months ( 95% ci , 3.9 to 6.1 months ) in first - line chemotherapy , 2.4 months ( 95% ci , 1.6 to 3.2 months ) in second - line chemotherapy , and 2.5 months ( 95% ci , 1.8 to 3.3 months ) in third - line chemotherapy . the median os time of arm a and arm b was 17.1 months ( 95% ci , 10.7 to 23.6 months ) , and 15.4 months ( range , 13.8 to 17.1 months ) , respectively . ttp of arm a and arm b with first - line chemotherapy was 5.5 months ( 95% ci , 1.2 to 9.8 months ) and 4.8 months ( 95% ci , 3.8 to 5.8 months ) , respectively ( p=0.064 ) . for the second - line chemotherapy , ttp of arm a and arm b was 2.7 months and 2.2 months , respectively ( p=0.850 ) . furthermore , elevated cea level and shorter ttp of first - line chemotherapy were significantly related with shorter survival times ( table 2 ) . the rr was 50% for first - line chemotherapy , 8% for second - line chemotherapy , and 8.5% for third - line chemotherapy . in second - line chemotherapy , rr was 11.1% in arm a and 6.3% in arm b. in third - line chemotherapy , rr was 6.3% in arm a and 9.7% in arm b. the disease control rate ( dcr ) which was determined by the disease status ( cr , pr , or sd ) controlled by treatment , of first - line chemotherapy was 88.9% in arm a and 78.1% in arm b ( p=0.342 ) . in second - line chemotherapy , dcr of arm a was 38.9% , and that of arm b was 53.1% ( p=0.333 ) . in third - line chemotherapy , dcr was 25% in arm a and 41.9% in arm b ( p=0.252 ) . the median os was 20.0 months ( 95% ci , 2.4 to 37.5 months ) in the good - responders and 13.9 months ( 95% ci , 12.0 to 15.8 months ) in the poor - responders . between november 2004 and july 2010 , 50 patients with gastric carcinoma were treated with a sequential chemotherapy protocol , based only on folfox or fp , folfiri , and paclitaxel or docetaxel , at gangnam severance hospital . fifty patients received median fourth - line chemotherapy ( range , 3 to 10 ) , and a median of 18 cycles ( range , 4 to 60 cycles ) were administered . median os of all patients was 16.0 months ( 95% confidence interval [ ci ] , 13.6 to 18.3 months ) ( fig . 1 ) , and median ttp was 5.0 months ( 95% ci , 3.9 to 6.1 months ) in first - line chemotherapy , 2.4 months ( 95% ci , 1.6 to 3.2 months ) in second - line chemotherapy , and 2.5 months ( 95% ci , 1.8 to 3.3 months ) in third - line chemotherapy . the median os time of arm a and arm b was 17.1 months ( 95% ci , 10.7 to 23.6 months ) , and 15.4 months ( range , 13.8 to 17.1 months ) , respectively . ttp of arm a and arm b with first - line chemotherapy was 5.5 months ( 95% ci , 1.2 to 9.8 months ) and 4.8 months ( 95% ci , 3.8 to 5.8 months ) , respectively ( p=0.064 ) . for the second - line chemotherapy , ttp of arm a and arm b was 2.7 months and 2.2 months , respectively ( p=0.850 ) . furthermore , elevated cea level and shorter ttp of first - line chemotherapy were significantly related with shorter survival times ( table 2 ) . the rr was 50% for first - line chemotherapy , 8% for second - line chemotherapy , and 8.5% for third - line chemotherapy . in second - line chemotherapy , rr was 11.1% in arm a and 6.3% in arm b. in third - line chemotherapy , rr was 6.3% in arm a and 9.7% in arm b. the disease control rate ( dcr ) which was determined by the disease status ( cr , pr , or sd ) controlled by treatment , of first - line chemotherapy was 88.9% in arm a and 78.1% in arm b ( p=0.342 ) . in second - line chemotherapy , dcr of arm in third - line chemotherapy , dcr was 25% in arm a and 41.9% in arm b ( p=0.252 ) . the median os was 20.0 months ( 95% ci , 2.4 to 37.5 months ) in the good - responders and 13.9 months ( 95% ci , 12.0 to 15.8 months ) in the poor - responders . it has generally been accepted that palliative chemotherapy can significantly prolong survival of patients with advanced gastric carcinoma , longer than best supportive care . given the reported tolerance and survival benefits of second - line chemotherapy , studies have sought to identify the benefit and adequate regimen of second - line chemotherapy . the current study evaluated two groups of gastric cancer patients who received third - line chemotherapy . previous studies reported the median pfs of 2.5 - 3.3 months and the median os of 5.3 - 8.7 months in gastric cancer patients who received second - line chemotherapy [ 6,8 - 10 ] . in this study , the median pfs was 2.4 months ( 95% ci , 1.6 to 3.2 months ) for patients treated with second - line chemotherapy , similar to the previous report . the median os was 16.0 months ( 95% ci , 13.6 to 18.3 months ) , and 32.0% of patients survived after 2 years . this study included a restricted population with a performance status that permitted the use of third - line chemotherapy after failure of second - line chemotherapy . therefore , this result was subject to limitation in comparison with other survival data of patients without third - line chemotherapy . nevertheless , based on 2.5 months of median ttp in third - line chemotherapy , the authors suggest that third - line chemotherapy would provide a survival benefit for patients with favorable performance status . in many phase ii clinical trials of second - line chemotherapy for advanced gastric cancer , rr was about 20.8% , and dcr was about 51% . there is still little information to support the provision of a second - line chemotherapeutic regimen after failure of platinum - based first - line chemotherapy . their sequential protocol was s-1 based chemotherapy for first - line chemotherapy , a low dose irinotecan / cisplatin for second - line chemotherapy , and paclitaxel for third - line chemotherapy . these facts also demonstrate that second- and third - line chemotherapy is necessary to treat advanced gastric cancer . many clinicians consider the folfiri regimen and paclitaxel or docetaxel with 5-fu regimen as second - line chemotherapy after failure of platinum - based first line chemotherapy . on the other hand , the tolerance and effectiveness of taxane - based chemotherapy apparent in several studies ( median ttp , 2.6 - 3.9 months ; median os , 7.2 to 10.1 months ) warrants its use as second - line chemotherapy in advanced gastric carcinoma . reported that overall rr and dcr for patients with docetaxel / cisplatin followed by folfiri and patients with folfiri followed by a docetaxel / cisplatin regimen showed no significant difference . however , to the best of our knowledge , survival benefits according to sequence of regimens of second - line and third - line chemotherapy after failure of first line chemotherapy have not been reported . in this study , we analyzed 50 patients treated with either folfox or fp chemotherapy followed by folfiri , and paclitaxel or docetaxel , or folfox or fp chemotherapy followed by paclitaxel or docetaxel , and folfiri regimen . previous investigators suggested that low hemoglobin level , low serum albumin level , high cea level , poor performance status [ 8 - 10,16,17 ] , shorter ttp of first - line chemotherapy , shorter ttp of second - line chemotherapy , and poor differentiation of tumor are poor prognostic factors for patients with gastric carcinoma under sequential chemotherapy . we analyzed the previously reported factors and found some differences in survival according to histologic differentiation , elevated cea level , and ttp of first line chemotherapy in this study . in this study , gastric adenocarcinoma with poorly differentiated or signet ring cell features had poorer response to sequential chemotherapy , and had shorter median os than well or moderately differentiated ones . we propose that the difference in response could also be attributed to the differences in gene expression profiles between the two groups . nevertheless , the results of this study provides evidence that third - line chemotherapy , including folfox or fp , folfiri , paclitaxel or docetaxel regimen , helps to improve os of patients , regardless of the sequence of use of taxane and irinotecan . in addition , os seems to be poorly affected by poorly differentiated or signet ring cell feature of adenocarcinoma , elevated cea level , and shorter ttp of first - line chemotherapy . different chemotherapy - responses to locally advanced or metastatic gastric carcinoma treated with second and third - line chemotherapy using irinotecan or taxane could be attributed not to sequence of chemotherapeutic regimen , but to patients ' different clinical features underlying different gene expression profiles .

it is well known that oxidative stress plays a major role in the neurodegenerative process that underlies parkinson s disease ( pd ) ( 1 , 2 ) . various experimental studies have also shown that levodopa ( ld ) , the most effective dopaminergic agent for pd , may paradoxically contribute to neuronal damage through formation of free radicals and reactive oxygen species ( ros ) ( 1 , 3 , 4 ) . in this regard , a previous report indicated increased hydroxyl radical formation in blood cells of pd patients under treatment with ld when compared with both untreated pd patients and healthy subjects ( 5 ) . homocysteine ( hcy ) is a neuro and vascular toxic sulfur - containing intermediate product . because the adverse effects of hcy are most likely related to its prooxidant properties ( 6 ) , a direct involvement of the amino acid in this phenomenon was hypothesized moreover , it has been shown that the elevated plasma hcy levels found in pd patients treated with ld are associated with a nearly two - fold increased prevalence of coronary artery diseases ( 7 , 8) . previous reports also suggest that elevated plasma hcy levels may be a risk factor for neuropsychiatric disorders such as stroke , dementia , depression , and pd ( 6 , 8) . although the brain has defenses against ros including dietary free radical scavengers ( ascorbate , -tocopherol ) , the endogenous tripeptide glutathione ( gsh ) , and antioxidant enzymes such as glutathione peroxidase ( gpx ) , superoxide dismutase ( sod ) , and catalase ( cat ) , there is considerable evidence that oxidative damage directly or indirectly , due to free radical production and ros , can lead to brain injury ( 6 , 9 ) . hcy passes the blood - brain - barrier ( bbb ) ( 10 , 11 ) , additional functional disturbance of the bbb leads to an unprotected exposure of the brain to hcy . hcy has various consequences for neural cells : oxidative stress , activation of caspases , mitochondrial dysfunction and increase of cytosolic calcium , which contribute to apoptosis ( 6 , 12 , 13 ) . hcy also inhibits the expression of antioxidant enzymes , which might potentiate the toxic effects of ros ( 6 , 14 - 16 ) . in addition , autooxidation of hcy is known to generate ros which overload oxidative stress in neurodegene - rative disorders ( 6 ) . in this sense , hcy is considered a predictor for dementia and alzheimer s disease ( 11 , 17 , 18 ) . therefore , the high occurrence of hyperhomocysteinemia in brain disorders and its easy treatment , make hcy an interesting amino acid in the prevention of neurodegenerative disorders ( 6 ) . ld is metabolized by four major metabolic pathways as follows : decarboxylation to dopamine , o - methylation to 3-o - methyl dopa , transamination , and oxidation ( 19 ) . as hcy synthesis represents a secondary reaction product of o - methylation of ld to 3-o - methyldopa ( 3-omd ) , one may hypothesize that catechol - o - methyltransferase ( comt ) inhibitors and/or vitamin supplementation may exert a certain preventive effect on the onset of axonal polyneuropathy during ld treatment ( 20 ) . a previous report demonstrated elevated total homocysteine ( thcy ) levels with concomitant tolcapone ( as a comt inhibitor ) and/or vitamin intake ( 21 ) . from this point of view , it is concluded that comt inhibition and vitamin supplementation only provide a limited impact on thcy accumulation . in recent years , ld has been used in combination with a dopa decarboxylase inhibitor ( ddi ) , such as benserazide to reduce its peripheral metabolism so as to avoid peripheral toxicity and to enhance its brain penetration in pd ( 22 , 23 ) . however , hyperhomocysteinemia has been considered one of the side effects of this treatment protocol in pd . in the present study we used betaine ( trimethyl - glycine ) , as a vital methylating agent for prevention of hyperhomocysteinemia and oxidative stress according to our previous reports ( 24 - 26 ) . betaine transfers a methyl group via the enzyme betaine homocysteine methyl transferase ( bhmt ) to become dimethylglycine ( 6 ) . it is well - known that bhmt is the only known enzyme that utilizes betaine as a substrate and transfers a methyl group from betaine to hcy ; thereafter hcy converts to methionine ( supplementary file ) ( 24 , 25 ) . animal studies have shown that both pathways are equally important and that betaine is a vital methylating agent ( 26 ) . although betaine can cross the bbb , hcy remethylation to methionine catalyzed by bhmt occurs mainly in liver ( 6 ) . herein , we investigated possible neuroprotective effects of betaine on cerebellum in an animal model . to achieve this goal , we measured lipid peroxidation marker ( tbars ) , gpx activity and gsh content , which are indicators of oxidative stress , in cerebellum of rats chronically treated by ld and benserazide . dopamine hydrochloride , methanol , thiobarbi - turic acid ( tba ) , and glutathione ( gsh ) were supplied by the merck chemical company ( kgaa , darmstadt , germany ) . betaine ( betafin 96% ) was prepared from biochem company ( brinkstrasse 55 , d-49393 lohne , germany ) . the hcy enzymatic kit was prepared by axis homocysteine ( axis - shield as , uk ) . a total of 42 adult male sprague - dawley rats ( weighing 150170 g ) were housed in temperature - controlled conditions under a 12:12 light / dark photoperiod with food and tap water supplied ad libitum . all rats were treated humanely and in compliance with the recommendations of the animal care committee for lorestan university of medical sciences ( khorramabad , iran ) . all experimental procedures were carried out between 8:00 am and 5.00 pm for prevention of circadian rhythm changes . the rats were divided into six identical groups ( n=7 per group ) , weight gain and food consumption were determined at 5 day intervals , and they were treated daily for 10 consecutive days orally by gavage in the following order : the control group received 1 ml distilled water , the levodopa ( ld ) group was treated with ld ( 3 100 mg / kg po at 8:30 am , 12:30 pm , and 4:30 pm ) , the betaine ( bet ) group received betaine ( 1.5% w / w of the total diet at 8.00 am ) , the levodopa plus betaine ( ld / bet ) group were treated by ld ( 3 100 mg / kg po at 4 hr intervals ) plus betaine ( 1.5% w / w of the total diet at 8.00 am ) , the levodopa plus benserazide ( ld / ben ) group received levodopa+benserazide ( 3 ( ld 100 mg+ben 25 mg ) at 4 hr intervals ) , and the final group , levodopa plus betaine - benserazide , ( ld / bet - ben ) was treated via levodopa+benserazide and betaine . ld , betaine , and benserazide were dissolved in distilled water before administration , daily . doses of ld and benserazide were determined according to a previous report ( 8) , and betaine was found in our previous works ( 6 , 24 , 25 ) . 2 hr after the last gavage , the rats were sacrificed upon light diethyl ether anesthesia ( dagenham , uk ) . blood samples were collected via cardiac puncture , in order to provide serum and plasma , separately . the cerebellum was removed and carefully cleaned by cold phosphate buffer ( 0.1 mol / l , ph 7.4 ) . dopamine concentration in serum was measured by the hplc method as described previously ( 19 ) . in brief , serum ( 250 l ) was extracted with 125 l of 2 mol / l hclo4 and the extract was centrifuged for 10 min at 500 g ( centrifuge 5415 r ; rotofix 32a , germany ) , the supernatant ( 20 l ) injected into the hplc system . analytical reversed phase high performance liquid chromatography ( shimadzu , lc-10ad vp , japan ) with a uv - vis detector ( spd-10avp ) and reverse phase column : rp-18 , 250 4.6 mm , 5 m , was used in this study . a mixture of phosphate buffer ( kh2po4 ) at ph = 2.5 and methanol with the ratio of 50/50 ( v / v ) , and flow rate of 1 ml / min was used as mobile phase . the temperature of the column was maintained at 30 c by a column oven ( cto-10as vp ) . dopamine concentration was expressed as millimol per milliliter ( mmol / ml ) of serum . total hcy of plasma which refers to the sum of protein - bound , free - oxidized , and reduced species of hcy was determined by the axis homocysteine enzymatic kit according to the manufacturer s instructions , by enzyme immunoassay method ( 6 , 27 , 28 ) . absorbance was measured at a wavelength of 450 nm using an elisa reader ( stat fax 2100 , usa ) . all estimations were performed in duplicate and the intra assay coefficient of variation was < 10% . thcy concentration was expressed as micromoles per liter ( mol / l ) of plasma . rat cerebellums were thawed and manually homogenized in cold phosphate buffer ( 0.1 mol / l , ph 7.4 ) , containing 5 mmol / l edta , and debris were removed by centrifugation at 2000 g for 10 min . supernatants were recovered and used for gpx assay , tbars and gsh concentrations and protein measurement . protein content of tissue homogenates was determined using the colorimetric method of lowry with bovine serum albumin as a standard ( 29 ) . the amount of lipid peroxidation was indicated by the content of thiobarbituric acid reactive substances ( tbars ) in the cerebellum . tissue tbars was determined by following its production as described previously ( 30 ) and reported in our previous works ( 6 , 24 , 25 ) . in short , 40 l of homogenate was added to 40 l of 0.9% nacl and 40 l of deionized h2o , resulting in a total reaction volume of 120 l . the reaction was incubated at 37 c for 20 min and stopped by the addition of 600 l of cold 0.8 mol / l hydrochloride acid , containing 12.5% trichloroacetic acid . following the addition of 780 l of 1% tba , the reaction was boiled for 20 min and then cooled at 4c for 1 hr . in order to measure the amount of tbars produced by the homogenate , the cooled reaction was spun at 1500 g in a microcentrifuge for 20 min and the absorbance of the supernatant was spectrophotometrically ( s2000 uv model ; wpa , cambridge , uk ) read at 532 nm , using and extinction coefficient of 1.5610/m.cm . the blanks for all of the tbars assays contained an additional 40 l of 0.9% nacl instead of homogenate as just described . tbars results were expressed as nanomoles per mg of tissue protein ( nmol / mg protein ) . the activity of gpx was evaluated using randox gpx detection kit according to the manufacturer s instructions , as reported in our previous works ( 6 , 24 , 25 ) . gpx catalyzes the oxidation of gsh by cumene hydroperoxide . in the presence of glutathione reductase ( gr ) and nadph , the oxidized glutathione ( gssg ) is immediately converted to the reduced form with a concomitant oxidation of nadph to nadp . one unit ( u ) of gpx was defined as l mol of oxidized nadph per min per mg of tissue protein . the gpx activity was expressed as unit per mg of tissue protein ( u / mg protein ) . total gsh was measured by the model as described previously ( 31 ) , which was reported in our previous work ( 32 ) . in brief , 20 l of tissue homogenates was prepared in 20 mmol / l edta , ph 4.7 , and 100 l of the homogenate or pure gsh was added to 0.2 mol / l tris edta ( 1.0 ml , ph 8.2 ) buffer ( fluka , switzerland ) and 20 mmol / l edta , ph 4.7 ( 0.9 ml ) , followed by 20 l of ellman s reagent ( 10 mmol / l dtnb in methanol ) . after 30 min of incubation at room temperature , absorbance was read at 412 nm . the blank was prepared with the same method ; however , instead of 100 l of the tissue homogenates , 100 l of distilled water was applied . gsh concentration was calculated on the basis of millimolar extinction coefficient of 13.6/m.cm and molecular weight of 307 g. results for gsh content were expressed as micromoles per mg of tissue protein ( mol / mg protein ) . statistical analysis was performed using the statistical package graphpad prism version 5 ( graphpad software inc . , san diego , ca , usa ) ( 24 , 25 ) . the statistical differences were tested among all groups by one - way analysis of variance ( anova ) with tukey s post hoc analysis . dopamine hydrochloride , methanol , thiobarbi - turic acid ( tba ) , and glutathione ( gsh ) were supplied by the merck chemical company ( kgaa , darmstadt , germany ) . betaine ( betafin 96% ) was prepared from biochem company ( brinkstrasse 55 , d-49393 lohne , germany ) . the hcy enzymatic kit was prepared by axis homocysteine ( axis - shield as , uk ) . a total of 42 adult male sprague - dawley rats ( weighing 150170 g ) were housed in temperature - controlled conditions under a 12:12 light / dark photoperiod with food and tap water supplied ad libitum . all rats were treated humanely and in compliance with the recommendations of the animal care committee for lorestan university of medical sciences ( khorramabad , iran ) . all experimental procedures were carried out between 8:00 am and 5.00 pm for prevention of circadian rhythm changes . the rats were divided into six identical groups ( n=7 per group ) , weight gain and food consumption were determined at 5 day intervals , and they were treated daily for 10 consecutive days orally by gavage in the following order : the control group received 1 ml distilled water , the levodopa ( ld ) group was treated with ld ( 3 100 mg / kg po at 8:30 am , 12:30 pm , and 4:30 pm ) , the betaine ( bet ) group received betaine ( 1.5% w / w of the total diet at 8.00 am ) , the levodopa plus betaine ( ld / bet ) group were treated by ld ( 3 100 mg / kg po at 4 hr intervals ) plus betaine ( 1.5% w / w of the total diet at 8.00 am ) , the levodopa plus benserazide ( ld / ben ) group received levodopa+benserazide ( 3 ( ld 100 mg+ben 25 mg ) at 4 hr intervals ) , and the final group , levodopa plus betaine - benserazide , ( ld / bet - ben ) was treated via levodopa+benserazide and betaine . ld , betaine , and benserazide were dissolved in distilled water before administration , daily . doses of ld and benserazide were determined according to a previous report ( 8) , and betaine was found in our previous works ( 6 , 24 , 25 ) . 2 hr after the last gavage , the rats were sacrificed upon light diethyl ether anesthesia ( dagenham , uk ) . blood samples were collected via cardiac puncture , in order to provide serum and plasma , separately . the cerebellum was removed and carefully cleaned by cold phosphate buffer ( 0.1 mol / l , ph 7.4 ) . dopamine concentration in serum was measured by the hplc method as described previously ( 19 ) . in brief , serum ( 250 l ) was extracted with 125 l of 2 mol / l hclo4 and the extract was centrifuged for 10 min at 500 g ( centrifuge 5415 r ; rotofix 32a , germany ) , the supernatant ( 20 l ) injected into the hplc system . analytical reversed phase high performance liquid chromatography ( shimadzu , lc-10ad vp , japan ) with a uv - vis detector ( spd-10avp ) and reverse phase column : rp-18 , 250 4.6 mm , 5 m , was used in this study . a mixture of phosphate buffer ( kh2po4 ) at ph = 2.5 and methanol with the ratio of 50/50 ( v / v ) , and flow rate of 1 ml / min was used as mobile phase . the temperature of the column was maintained at 30 c by a column oven ( cto-10as vp ) . dopamine concentration was expressed as millimol per milliliter ( mmol / ml ) of serum . total hcy of plasma which refers to the sum of protein - bound , free - oxidized , and reduced species of hcy was determined by the axis homocysteine enzymatic kit according to the manufacturer s instructions , by enzyme immunoassay method ( 6 , 27 , 28 ) . absorbance was measured at a wavelength of 450 nm using an elisa reader ( stat fax 2100 , usa ) . all estimations were performed in duplicate and the intra assay coefficient of variation was < 10% . thcy concentration was expressed as micromoles per liter ( mol / l ) of plasma . rat cerebellums were thawed and manually homogenized in cold phosphate buffer ( 0.1 mol / l , ph 7.4 ) , containing 5 mmol / l edta , and debris were removed by centrifugation at 2000 g for 10 min . supernatants were recovered and used for gpx assay , tbars and gsh concentrations and protein measurement . protein content of tissue homogenates was determined using the colorimetric method of lowry with bovine serum albumin as a standard ( 29 ) . the amount of lipid peroxidation was indicated by the content of thiobarbituric acid reactive substances ( tbars ) in the cerebellum . tissue tbars was determined by following its production as described previously ( 30 ) and reported in our previous works ( 6 , 24 , 25 ) . in short , 40 l of homogenate was added to 40 l of 0.9% nacl and 40 l of deionized h2o , resulting in a total reaction volume of 120 l . the reaction was incubated at 37 c for 20 min and stopped by the addition of 600 l of cold 0.8 mol / l hydrochloride acid , containing 12.5% trichloroacetic acid . following the addition of 780 l of 1% tba , the reaction was boiled for 20 min and then cooled at 4c for 1 hr . in order to measure the amount of tbars produced by the homogenate , the cooled reaction was spun at 1500 g in a microcentrifuge for 20 min and the absorbance of the supernatant was spectrophotometrically ( s2000 uv model ; wpa , cambridge , uk ) read at 532 nm , using and extinction coefficient of 1.5610/m.cm . the blanks for all of the tbars assays contained an additional 40 l of 0.9% nacl instead of homogenate as just described . tbars results were expressed as nanomoles per mg of tissue protein ( nmol / mg protein ) . the activity of gpx was evaluated using randox gpx detection kit according to the manufacturer s instructions , as reported in our previous works ( 6 , 24 , 25 ) . gpx catalyzes the oxidation of gsh by cumene hydroperoxide . in the presence of glutathione reductase ( gr ) and nadph , the oxidized glutathione ( gssg ) is immediately converted to the reduced form with a concomitant oxidation of nadph to nadp . one unit ( u ) of gpx was defined as l mol of oxidized nadph per min per mg of tissue protein . the gpx activity was expressed as unit per mg of tissue protein ( u / mg protein ) . total gsh was measured by the model as described previously ( 31 ) , which was reported in our previous work ( 32 ) . in brief , 20 l of tissue homogenates was prepared in 20 mmol / l edta , ph 4.7 , and 100 l of the homogenate or pure gsh was added to 0.2 mol / l tris edta ( 1.0 ml , ph 8.2 ) buffer ( fluka , switzerland ) and 20 mmol / l edta , ph 4.7 ( 0.9 ml ) , followed by 20 l of ellman s reagent ( 10 mmol / l dtnb in methanol ) . after 30 min of incubation at room temperature , absorbance was read at 412 nm . the blank was prepared with the same method ; however , instead of 100 l of the tissue homogenates , 100 l of distilled water was applied . gsh concentration was calculated on the basis of millimolar extinction coefficient of 13.6/m.cm and molecular weight of 307 g. results for gsh content were expressed as micromoles per mg of tissue protein ( mol / mg protein ) . statistical analysis was performed using the statistical package graphpad prism version 5 ( graphpad software inc . , san diego , ca , usa ) ( 24 , 25 ) . the statistical differences were tested among all groups by one - way analysis of variance ( anova ) with tukey s post hoc analysis . treatment of rats with levodopa plus dopa - decarboxylase inhibitor ( ld+benserazide ) significantly increased thcy of the ld / ben group compared to the other groups , while administration of betaine to the ld / bet group could suppress thcy increase ( p<0.05 ) . moreover , thcy in the ld / bet - ben group was significantly lower compared to the ld / ben - treated rats ( p<0.05 ; figure 1 ) . the effects of levodopa , betaine and benserazide treatments on plasma total homocysteine ( thcy ) concentration in the control and treated rats ( n=7 per group ) . # indicates statistical difference between ld / ben and the other groups ( p<0.05 ) . * ; denotes a significant difference ( p<0.05 ) versus levodopa group . ld ; levodopa , bet ; betaine , ben ; benserazide lipid peroxidation ( tbars concentration ) increased significantly in ld - treated rats when compared to the other groups ( p<0.05 ) . however , the concentrations of tbars in the ld / bet - ben , control and bet groups were significantly lower when compared to the ld / bet - treated rats ( p<0.05 ; figure 2 ) . the effects of levodopa , betaine and benserazide treatments on thiobarbituric acid reactive substances ( tbars ) concentration in control and treated rats ( n=7 per group ) . values represent meansem of tbars ( nanomoles per milligram protein of cerebellum tissue ) . # indicates statistical difference between ld and the other groups ( p<0.05 ) . * ; denotes a significant difference ( p<0.05 ) versus control , betaine , and ld / bet - ben groups . ld ; levodopa , bet ; betaine , ben ; benserazide the mean valuessem of the glutathione peroxidase ( gpx ) activity and gsh content of the cerebellum tissue from control and experimental groups are presented in figures 3 and 4 . gpx activity was significantly higher in the betaine - treated rats when compared to the ld and ld / ben groups ( p<0.05 ) . while betaine treatment could increase gpx activity as significantly in the ld / bet group in comparison with ld - treated rats ( p<0.05 ) , the enhancement of gpx activity in the ld / bet - ben group in comparison with ld / ben - treated rats was not significant ( p>0.05 ) . regarding gsh molecule as a cofactor for gpx activity , gsh content increased significantly in bet and ld / bet groups in comparison with ld- , and ld / ben - treated rats ( p<0.05 ) . although betaine treatment could increase gsh content in the ld / bet - ben group in comparison with ld / ben - treated rats , the enhancement was not significant ( p>0.05 ; figures 3 and 4 ) . the effects of levodopa , betaine and benserazide treatments on glutathione peroxidase ( gpx ) activity in control and treated rats ( n=7 per group ) . values represent meansem of enzyme activity ( unit / mg protein of cerebellum tissue ) . * ; denotes a significant difference ( p<0.05 ) versus ld and ld / ben groups . * * ; denotes a significant difference ( p<0.05 ) versus levodopa and ld / ben groups . ld ; levodopa , bet ; betaine , ben ; benserazide the effects of levodopa , betaine and benserazide treatments on glutathione content ( gsh ) in control and treated rats ( n=7 per group ) . values represent meansem of gsh ( micromoles per milligram protein of cerebellum tissue ) . * ; denotes a significant difference ( p<0.05 ) versus levodopa and ld / ben groups . * * ; denotes a significant difference ( p<0.05 ) versus levodopa and ld / ben groups . ld ; levodopa , bet ; betaine , ben ; benserazide regarding ld metabolism , dopamine concentration increased significantly in ld - treated rats in comparison with the ld / ben group ( p<0.05 ) . moreover , ld / bet treatment elevated dopamine level significantly when compared to the ld / ben treatment ( p<0.05 ; figure 5 ) . the effects of levodopa , betaine and benserazide treatments on dopamine levels in control and treated rats ( n=7 per group ) . values represent meansem of dopamine ( mmol / ml serum ) . * ; denotes a significant difference ( p<0.05 ) versus levodopa and ld / bet groups . there is abundant evidence for oxidative stress in substantia nigra of brain in pd ( 1 ) . nevertheless , it remains unknown whether an increased oxidative load produced by ld would induce oxidative stress in cerebellum tissue of pd patients . the results demonstrated , for the first time , the antioxidant and methyl donor effects of betaine against ld / ben - induced oxidative stress in cerebellum of rats . the significant increase of thcy levels in ld / ben treated rats and the significant decrease of gpx activity and gsh content in ld and ld / ben groups in comparison with betaine - treated rats , support the ld / ben - mediated hyperhomocysteinemia and ld - induced oxidative stress in cerebellum of rats . in addition , in line with the previous reports benserazide administration ( as a ddi ) was associated with a reduction of dopamine level in serum . based on the present data ld / ben , as a new drug for pd , induces hyperhomocysteinemia and this effect was more evident than in ld treatment . in contrast , betaine is not only a methyl donor , but it is also an antioxidant agent versus oxidative stress mediated by ld and ld / ben in the rat cerebellum . therefore , it appears that the methyl donor and antioxidant properties of betaine are promising particularly in management of plasma thcy and oxidative stress in cerebellum tissue . ld , the most effective drug known in the treatment of pd , has been observed to induce elevations in plasma thcy concentrations ( 8) . the processes of methyl group transfer are involved in the metabolism of ld ( 4 , 33 ) . the reaction involves the enzyme comt , with s - adenosyl methionine ( sam ) forms s - adenosyl homocysteine ( sah ) , which is hydrolyzed to hcy . hcy is then metabolized via a remethylation cycle , which leads back to methionine , or a transsulfuration pathway , forming cystathionine ( supplementary file ) . it is well known that catabolism of ld interferes , at various steps , with hcy metabolism . indeed , there is experimental evidence that ld administration increases thcy levels in plasma and is able to increase cerebellar sah ( 34 , 35 ) . in the present study , betaine as a methyl donor agent that continuously generates sam could decrease thcy level as significantly in the ld / bet group when compared to ld - treated rats . this effect is in agreement with our previous reports ( 6 , 24 - 26 ) . putative therapeutic approaches for reduction of thcy levels include vitamin ( b12 and folic acid ) supplementation because folic acid and cyanocobalamin catalyze and enhance metabolism of hcy into methionine ( 26 ) . a further hypothetical therapeutic alternative would be application of peripheral acting comt inhibitors as adjunctives to levodopa / dopa decarboxylase inhibitor ( ld / ddi ) treatment . the comt inhibitors increase the peripheral bioavailability of ld . the combination of ld with a ddi such as benserazide also reduces decarboxylation of ld in peripheral tissues and increases bioavailability of ld for central nervous system ( cns ) ( 22 ) . however , a previous report indicated an elevation of thcy levels with concomitant tolcapone ( as a comt inhibitor ) ( 21 ) , it is concluded that comt inhibitors only provide a limited impact on thcy increasing . in the present study , comt is the essential enzyme for this o - methylation of ld , which demands for a methyl group transfer from the donor sam ( 20 ) . as one consequence , sam is transformed into sah and then to hcy . in this setting , thcy level in ld / ben - treated rats ( figure 1 ) well indicates the bioavailability of ld and its conversion to hcy in comparison with other groups . thus , we assume that a certain balance between ld and betaine is developed during this treatment protocol . indeed , betaine supplementation exerted a certain preventive effect on the onset of hyperhomocysteinemia in ld / bet and ld / bet - ben groups . during the past decade , sam was used as a co - adjuvant in depression and has a potential neuroprotective effect in animal models ( 36 ) . sam application also increases brain gsh levels via intracellular biochemical pathways of transsulfura - tion and decreases membrane lipid peroxidation caused by free radical damage , acting as a potential antioxidant drug ( 6 , 36 , 37 ) . in theory , good titration and carefully monitoring sam supplementation with normalizing of reduced sam levels in pd patients may cause an effective reduction of hcy levels , while elevated sam dosage may also worsen motor symptoms or even accelerate neuronal cell death via the apoptotic pathway ( 22 , 38 ) . betaine is a methylating agent like sam and it also stabilizes sam levels via remethylation . in this sense , betaine indicated similar methyl donor effects against ethanol - induced hyperhomocysteinemia in rats and rabbits ( 6 , 26 ) . betaine treatment may also have some advantages over endogenous sam application ; because sam application enhances the levels of hcy , which is undesirable due to the toxicity of this amino acid , whereas betaine treatment decreases hcy levels by directly inducing the remethylating process , which transforms hcy into methionine ( 6 , 37 , 39 ) . furthermore , since humans produce little betaine from choline due to lack of choline oxidase ( 40 ) , betaine is practical for investigations in hyperhomocysteinemia conditions such as neurodegenerative disorders . the brain is more vulnerable to oxidative stress than other organs due to its low antioxidant protection system and increased exposure of target molecules to ros ( 6 ) . the nervous tissue has a high content of polyunsaturated fatty acids , which are easy targets to oxidative damage by free radicals ( 6 , 41 ) . in our study , ld consumption caused significantly increased tbars concentration ( as a lipid peroxidation marker ) in the ld - treated rats , and betaine treatment restored this elevated tbars concentration in the ld / bet - treated rats to near the ld / ben grouplevel . on the other hand , there were significant differences between ld and the other groups in tbars , indicating occurrence of oxidative stress in ld - treated rats . ld / ben group also indicated increased tbars level , but this elevation was not significant when compared to the control group . the results of lipid peroxidation were well consistent with our previous report on ethanol - induced oxidative stress in cerebellum of rats ( 6 ) . betaine plays a significant role in maintaining the structural and functional integrity of cell membranes . betaine through its participation in sequential methylation within the cellular membranes maintains a proper balance between phosphatidylethanolamine and phosphatidylcholine , thus sustaining proper membranes and prevents lipid peroxidation ( 6 , 42 , 43 ) . animal models have shown that a number of antioxidants prevent oxidative brain injury through a variety of cellular mechanisms which have described oxidative damage on the cns ( 6 , 9 , 44 , 45 ) . the cellular tripeptide gsh thwarts peroxidative damage by neutralizing the free radicals ( 42 ) . in the present study , gpx activity and gsh content were increased as significantly in betaine- and ld / bet - treated rats when compared to ld and ld / ben groups . it is well known that gpx and cat are two key antioxidant enzymes that can decompose hydrogen peroxide to water ( 6 ) . although , h2o2 is not a particularly reactive product , it can be reduced to the highly reactive metabolites hydroxyl radicals ( 6 , 24 , 25 ) . the increase in cerebellar gpx activity in betaine - treated rats correlates well with the increase of gsh content as a cofactor for gpx activity in cerebellum . the protective effect of betaine against ld - induced oxidative stress observed in this study may be associated with the restoration of sam . the increase in sam concentration contributes through an increase in the supply of substrate needed for the synthesis of gsh ( 6 , 42 ) . the principal path is decarboxylation , whereby ld converts to dopamine via l - dopa decarboxylase ( 19 ) . in the present study , the co - administration of benserazide with ld results in increased metabolism of ld to 3-o - md via the enzyme comt in peripheral tissues , thus , dopamine concentration in ld / ben - treated rats in comparison with ld and ld / bet groups is decreased ( figure 5 ) . we measured dopamine concentration in blood to evaluate decarboxylation pathway of ld in peripheral tissues . because dopamine does not go through bbb , the serum dopamine level after application of ld , betaine and benserazide shall not reflect dopamine concentration in the cns . in this setting , benserazide treatment prevented ld metabolism to dopamine in the peripheral tissues and is able to enhance dopamine in cns . however , dopamine measurement in the cns is needed to clarify the drug s effects in a future experimental study . the present study demonstrates that betaine may have a potential as a neuroprotective agent for prevention of ld - induced oxidative damage in cerebellum and benserazide - mediated hyper - homocysteinemia in rats . however , further studies including physiological parameters and histochemical techniques should be performed to validate this hypothesis . all rats were treated humanely and in compliance with the recommendations of animal care committee of lorestan university of medical sciences ( khorramabad , iran ) .

objective(s):the aim of the present study was to evaluate antioxidant and methyl donor effects of betaine in cerebellum following levodopa and benserazide administration in rats.materials and methods : sprague - dawley male rats were treated with levodopa ( ld ) , betaine ( bet ) , levodopa plus betaine ( ld / bet ) , levodopa plus benserazide ( ld / ben ) , levodopa plus betaine - benserazide ( ld / bet - ben ) , and the controls with vehicle for 10 consecutive days , orally.results:treatment of rats with ld and benserazide significantly increased total homocysteine in plasma of the ld / ben group when compared to the other groups . lipid peroxidation of cerebellum increased significantly in ld - treated rats when compared to the other groups . in contrast , glutathione peroxidase activity and glutathione content in cerebellum were significantly higher in the betaine - treated rats when compared to the ld and ld / ben groups . serum dopamine concentration increased significantly in ld - treated rats in comparison with the ld / ben group . ld / bet - treated rats also demonstrated significantly higher dopamine levels when compared to the ld / ben group.conclusion:we observed valuable effects of bet in combination with ld and benserazide , which routinely were used for parkinson s disease ( pd ) treatment , in experimentally - induced oxidative stress and hyperhomocysteinemia in rats . therefore , it seems that bet is a vital and promising agent regarding pd for future clinical trials in humans .

Introduction Materials and Methods Materials Experimental design Measurement of dopamine concentration Measurement of tHcy concentration Tissue preparation for protein measurement, TBARS and GSH content and GPx assay Measurement of lipid peroxidation Measurement of GPx activity Measurement of total glutathione (GSH content) Statistical analysis Results Discussion Conclusion Animal studies

it is well known that oxidative stress plays a major role in the neurodegenerative process that underlies parkinson s disease ( pd ) ( 1 , 2 ) . various experimental studies have also shown that levodopa ( ld ) , the most effective dopaminergic agent for pd , may paradoxically contribute to neuronal damage through formation of free radicals and reactive oxygen species ( ros ) ( 1 , 3 , 4 ) . because the adverse effects of hcy are most likely related to its prooxidant properties ( 6 ) , a direct involvement of the amino acid in this phenomenon was hypothesized moreover , it has been shown that the elevated plasma hcy levels found in pd patients treated with ld are associated with a nearly two - fold increased prevalence of coronary artery diseases ( 7 , 8) . although the brain has defenses against ros including dietary free radical scavengers ( ascorbate , -tocopherol ) , the endogenous tripeptide glutathione ( gsh ) , and antioxidant enzymes such as glutathione peroxidase ( gpx ) , superoxide dismutase ( sod ) , and catalase ( cat ) , there is considerable evidence that oxidative damage directly or indirectly , due to free radical production and ros , can lead to brain injury ( 6 , 9 ) . hcy passes the blood - brain - barrier ( bbb ) ( 10 , 11 ) , additional functional disturbance of the bbb leads to an unprotected exposure of the brain to hcy . hcy has various consequences for neural cells : oxidative stress , activation of caspases , mitochondrial dysfunction and increase of cytosolic calcium , which contribute to apoptosis ( 6 , 12 , 13 ) . hcy also inhibits the expression of antioxidant enzymes , which might potentiate the toxic effects of ros ( 6 , 14 - 16 ) . therefore , the high occurrence of hyperhomocysteinemia in brain disorders and its easy treatment , make hcy an interesting amino acid in the prevention of neurodegenerative disorders ( 6 ) . in recent years , ld has been used in combination with a dopa decarboxylase inhibitor ( ddi ) , such as benserazide to reduce its peripheral metabolism so as to avoid peripheral toxicity and to enhance its brain penetration in pd ( 22 , 23 ) . however , hyperhomocysteinemia has been considered one of the side effects of this treatment protocol in pd . in the present study we used betaine ( trimethyl - glycine ) , as a vital methylating agent for prevention of hyperhomocysteinemia and oxidative stress according to our previous reports ( 24 - 26 ) . herein , we investigated possible neuroprotective effects of betaine on cerebellum in an animal model . to achieve this goal , we measured lipid peroxidation marker ( tbars ) , gpx activity and gsh content , which are indicators of oxidative stress , in cerebellum of rats chronically treated by ld and benserazide . dopamine hydrochloride , methanol , thiobarbi - turic acid ( tba ) , and glutathione ( gsh ) were supplied by the merck chemical company ( kgaa , darmstadt , germany ) . all rats were treated humanely and in compliance with the recommendations of the animal care committee for lorestan university of medical sciences ( khorramabad , iran ) . the rats were divided into six identical groups ( n=7 per group ) , weight gain and food consumption were determined at 5 day intervals , and they were treated daily for 10 consecutive days orally by gavage in the following order : the control group received 1 ml distilled water , the levodopa ( ld ) group was treated with ld ( 3 100 mg / kg po at 8:30 am , 12:30 pm , and 4:30 pm ) , the betaine ( bet ) group received betaine ( 1.5% w / w of the total diet at 8.00 am ) , the levodopa plus betaine ( ld / bet ) group were treated by ld ( 3 100 mg / kg po at 4 hr intervals ) plus betaine ( 1.5% w / w of the total diet at 8.00 am ) , the levodopa plus benserazide ( ld / ben ) group received levodopa+benserazide ( 3 ( ld 100 mg+ben 25 mg ) at 4 hr intervals ) , and the final group , levodopa plus betaine - benserazide , ( ld / bet - ben ) was treated via levodopa+benserazide and betaine . ld , betaine , and benserazide were dissolved in distilled water before administration , daily . doses of ld and benserazide were determined according to a previous report ( 8) , and betaine was found in our previous works ( 6 , 24 , 25 ) . in brief , serum ( 250 l ) was extracted with 125 l of 2 mol / l hclo4 and the extract was centrifuged for 10 min at 500 g ( centrifuge 5415 r ; rotofix 32a , germany ) , the supernatant ( 20 l ) injected into the hplc system . a mixture of phosphate buffer ( kh2po4 ) at ph = 2.5 and methanol with the ratio of 50/50 ( v / v ) , and flow rate of 1 ml / min was used as mobile phase . total hcy of plasma which refers to the sum of protein - bound , free - oxidized , and reduced species of hcy was determined by the axis homocysteine enzymatic kit according to the manufacturer s instructions , by enzyme immunoassay method ( 6 , 27 , 28 ) . rat cerebellums were thawed and manually homogenized in cold phosphate buffer ( 0.1 mol / l , ph 7.4 ) , containing 5 mmol / l edta , and debris were removed by centrifugation at 2000 g for 10 min . in brief , 20 l of tissue homogenates was prepared in 20 mmol / l edta , ph 4.7 , and 100 l of the homogenate or pure gsh was added to 0.2 mol / l tris edta ( 1.0 ml , ph 8.2 ) buffer ( fluka , switzerland ) and 20 mmol / l edta , ph 4.7 ( 0.9 ml ) , followed by 20 l of ellman s reagent ( 10 mmol / l dtnb in methanol ) . dopamine hydrochloride , methanol , thiobarbi - turic acid ( tba ) , and glutathione ( gsh ) were supplied by the merck chemical company ( kgaa , darmstadt , germany ) . all rats were treated humanely and in compliance with the recommendations of the animal care committee for lorestan university of medical sciences ( khorramabad , iran ) . the rats were divided into six identical groups ( n=7 per group ) , weight gain and food consumption were determined at 5 day intervals , and they were treated daily for 10 consecutive days orally by gavage in the following order : the control group received 1 ml distilled water , the levodopa ( ld ) group was treated with ld ( 3 100 mg / kg po at 8:30 am , 12:30 pm , and 4:30 pm ) , the betaine ( bet ) group received betaine ( 1.5% w / w of the total diet at 8.00 am ) , the levodopa plus betaine ( ld / bet ) group were treated by ld ( 3 100 mg / kg po at 4 hr intervals ) plus betaine ( 1.5% w / w of the total diet at 8.00 am ) , the levodopa plus benserazide ( ld / ben ) group received levodopa+benserazide ( 3 ( ld 100 mg+ben 25 mg ) at 4 hr intervals ) , and the final group , levodopa plus betaine - benserazide , ( ld / bet - ben ) was treated via levodopa+benserazide and betaine . ld , betaine , and benserazide were dissolved in distilled water before administration , daily . doses of ld and benserazide were determined according to a previous report ( 8) , and betaine was found in our previous works ( 6 , 24 , 25 ) . in brief , serum ( 250 l ) was extracted with 125 l of 2 mol / l hclo4 and the extract was centrifuged for 10 min at 500 g ( centrifuge 5415 r ; rotofix 32a , germany ) , the supernatant ( 20 l ) injected into the hplc system . a mixture of phosphate buffer ( kh2po4 ) at ph = 2.5 and methanol with the ratio of 50/50 ( v / v ) , and flow rate of 1 ml / min was used as mobile phase . rat cerebellums were thawed and manually homogenized in cold phosphate buffer ( 0.1 mol / l , ph 7.4 ) , containing 5 mmol / l edta , and debris were removed by centrifugation at 2000 g for 10 min . the amount of lipid peroxidation was indicated by the content of thiobarbituric acid reactive substances ( tbars ) in the cerebellum . in brief , 20 l of tissue homogenates was prepared in 20 mmol / l edta , ph 4.7 , and 100 l of the homogenate or pure gsh was added to 0.2 mol / l tris edta ( 1.0 ml , ph 8.2 ) buffer ( fluka , switzerland ) and 20 mmol / l edta , ph 4.7 ( 0.9 ml ) , followed by 20 l of ellman s reagent ( 10 mmol / l dtnb in methanol ) . the blank was prepared with the same method ; however , instead of 100 l of the tissue homogenates , 100 l of distilled water was applied . treatment of rats with levodopa plus dopa - decarboxylase inhibitor ( ld+benserazide ) significantly increased thcy of the ld / ben group compared to the other groups , while administration of betaine to the ld / bet group could suppress thcy increase ( p<0.05 ) . moreover , thcy in the ld / bet - ben group was significantly lower compared to the ld / ben - treated rats ( p<0.05 ; figure 1 ) . the effects of levodopa , betaine and benserazide treatments on plasma total homocysteine ( thcy ) concentration in the control and treated rats ( n=7 per group ) . # indicates statistical difference between ld / ben and the other groups ( p<0.05 ) . ld ; levodopa , bet ; betaine , ben ; benserazide lipid peroxidation ( tbars concentration ) increased significantly in ld - treated rats when compared to the other groups ( p<0.05 ) . however , the concentrations of tbars in the ld / bet - ben , control and bet groups were significantly lower when compared to the ld / bet - treated rats ( p<0.05 ; figure 2 ) . the effects of levodopa , betaine and benserazide treatments on thiobarbituric acid reactive substances ( tbars ) concentration in control and treated rats ( n=7 per group ) . # indicates statistical difference between ld and the other groups ( p<0.05 ) . * ; denotes a significant difference ( p<0.05 ) versus control , betaine , and ld / bet - ben groups . ld ; levodopa , bet ; betaine , ben ; benserazide the mean valuessem of the glutathione peroxidase ( gpx ) activity and gsh content of the cerebellum tissue from control and experimental groups are presented in figures 3 and 4 . gpx activity was significantly higher in the betaine - treated rats when compared to the ld and ld / ben groups ( p<0.05 ) . while betaine treatment could increase gpx activity as significantly in the ld / bet group in comparison with ld - treated rats ( p<0.05 ) , the enhancement of gpx activity in the ld / bet - ben group in comparison with ld / ben - treated rats was not significant ( p>0.05 ) . regarding gsh molecule as a cofactor for gpx activity , gsh content increased significantly in bet and ld / bet groups in comparison with ld- , and ld / ben - treated rats ( p<0.05 ) . although betaine treatment could increase gsh content in the ld / bet - ben group in comparison with ld / ben - treated rats , the enhancement was not significant ( p>0.05 ; figures 3 and 4 ) . the effects of levodopa , betaine and benserazide treatments on glutathione peroxidase ( gpx ) activity in control and treated rats ( n=7 per group ) . * ; denotes a significant difference ( p<0.05 ) versus ld and ld / ben groups . * * ; denotes a significant difference ( p<0.05 ) versus levodopa and ld / ben groups . ld ; levodopa , bet ; betaine , ben ; benserazide the effects of levodopa , betaine and benserazide treatments on glutathione content ( gsh ) in control and treated rats ( n=7 per group ) . * ; denotes a significant difference ( p<0.05 ) versus levodopa and ld / ben groups . * * ; denotes a significant difference ( p<0.05 ) versus levodopa and ld / ben groups . ld ; levodopa , bet ; betaine , ben ; benserazide regarding ld metabolism , dopamine concentration increased significantly in ld - treated rats in comparison with the ld / ben group ( p<0.05 ) . moreover , ld / bet treatment elevated dopamine level significantly when compared to the ld / ben treatment ( p<0.05 ; figure 5 ) . the effects of levodopa , betaine and benserazide treatments on dopamine levels in control and treated rats ( n=7 per group ) . * ; denotes a significant difference ( p<0.05 ) versus levodopa and ld / bet groups . nevertheless , it remains unknown whether an increased oxidative load produced by ld would induce oxidative stress in cerebellum tissue of pd patients . the results demonstrated , for the first time , the antioxidant and methyl donor effects of betaine against ld / ben - induced oxidative stress in cerebellum of rats . the significant increase of thcy levels in ld / ben treated rats and the significant decrease of gpx activity and gsh content in ld and ld / ben groups in comparison with betaine - treated rats , support the ld / ben - mediated hyperhomocysteinemia and ld - induced oxidative stress in cerebellum of rats . in addition , in line with the previous reports benserazide administration ( as a ddi ) was associated with a reduction of dopamine level in serum . based on the present data ld / ben , as a new drug for pd , induces hyperhomocysteinemia and this effect was more evident than in ld treatment . in contrast , betaine is not only a methyl donor , but it is also an antioxidant agent versus oxidative stress mediated by ld and ld / ben in the rat cerebellum . therefore , it appears that the methyl donor and antioxidant properties of betaine are promising particularly in management of plasma thcy and oxidative stress in cerebellum tissue . ld , the most effective drug known in the treatment of pd , has been observed to induce elevations in plasma thcy concentrations ( 8) . in the present study , betaine as a methyl donor agent that continuously generates sam could decrease thcy level as significantly in the ld / bet group when compared to ld - treated rats . a further hypothetical therapeutic alternative would be application of peripheral acting comt inhibitors as adjunctives to levodopa / dopa decarboxylase inhibitor ( ld / ddi ) treatment . in the present study , comt is the essential enzyme for this o - methylation of ld , which demands for a methyl group transfer from the donor sam ( 20 ) . in this setting , thcy level in ld / ben - treated rats ( figure 1 ) well indicates the bioavailability of ld and its conversion to hcy in comparison with other groups . indeed , betaine supplementation exerted a certain preventive effect on the onset of hyperhomocysteinemia in ld / bet and ld / bet - ben groups . in this sense , betaine indicated similar methyl donor effects against ethanol - induced hyperhomocysteinemia in rats and rabbits ( 6 , 26 ) . betaine treatment may also have some advantages over endogenous sam application ; because sam application enhances the levels of hcy , which is undesirable due to the toxicity of this amino acid , whereas betaine treatment decreases hcy levels by directly inducing the remethylating process , which transforms hcy into methionine ( 6 , 37 , 39 ) . in our study , ld consumption caused significantly increased tbars concentration ( as a lipid peroxidation marker ) in the ld - treated rats , and betaine treatment restored this elevated tbars concentration in the ld / bet - treated rats to near the ld / ben grouplevel . on the other hand , there were significant differences between ld and the other groups in tbars , indicating occurrence of oxidative stress in ld - treated rats . ld / ben group also indicated increased tbars level , but this elevation was not significant when compared to the control group . the results of lipid peroxidation were well consistent with our previous report on ethanol - induced oxidative stress in cerebellum of rats ( 6 ) . in the present study , gpx activity and gsh content were increased as significantly in betaine- and ld / bet - treated rats when compared to ld and ld / ben groups . although , h2o2 is not a particularly reactive product , it can be reduced to the highly reactive metabolites hydroxyl radicals ( 6 , 24 , 25 ) . the increase in cerebellar gpx activity in betaine - treated rats correlates well with the increase of gsh content as a cofactor for gpx activity in cerebellum . the protective effect of betaine against ld - induced oxidative stress observed in this study may be associated with the restoration of sam . in the present study , the co - administration of benserazide with ld results in increased metabolism of ld to 3-o - md via the enzyme comt in peripheral tissues , thus , dopamine concentration in ld / ben - treated rats in comparison with ld and ld / bet groups is decreased ( figure 5 ) . because dopamine does not go through bbb , the serum dopamine level after application of ld , betaine and benserazide shall not reflect dopamine concentration in the cns . the present study demonstrates that betaine may have a potential as a neuroprotective agent for prevention of ld - induced oxidative damage in cerebellum and benserazide - mediated hyper - homocysteinemia in rats . all rats were treated humanely and in compliance with the recommendations of animal care committee of lorestan university of medical sciences ( khorramabad , iran ) .

how should patient categories , or case - mix groups , be defined if they are to be used for prospective payment for inpatient care ? two criteria are important for evaluating the effectiveness of a case - mix grouping system : medical meaningfulness of the grouping system and homogeneity of resource consumption within each of the case - mix groups . in addition , if a case - mix grouping system is to be used for prospective payment , two additional criteria must be considered : independence from the medical treatment process , and administrative feasibility . with these criteria in mind , a severity of illness index was developed by a team of researchers , physicians , and nurses at the johns hopkins university . it was designed to be a medically meaningful generic classification system that could differentiate the severity of illness of hospital inpatients . because discharge abstract data contain only labels of principal and secondary diagnoses , procedures performed , age , etc . , the conventional discharge abstract data base is not rich enough to describe patient severity of illness accurately . using the patient 's total medical record , the severity of illness index assigns to each patient at discharge an overall severity score that is determined from the scores of each of seven medically meaningful dimensions chosen to reflect burden of illness . these seven dimensions , as shown in figure 1 , are : stage of the principal diagnosis the severity of illness index is described in detail in various reports ( horn , sharkey , and bertram , 1983 ; horn , 1983a ; horn and sharkey , 1983 ; horn , chachich , and clopton , 1983 ; horn , 1983b ) . to determine the severity of illness score for an individual case , a rater scores each of the seven dimensions into one of four levels of increasing severity by examining data in the patient 's medical record following discharge . definitions of each of the four levels for each dimension are provided , and raters are intensively trained to distinguish the reasons for selecting the level of each dimension . the rater then assigns an overall severity score for the patient on a four - point scale by implicitly integrating the values of the seven dimensions . the reliability and validity of the severity of illness instrument has been studied in several ways . more than 95 percent of the individual raters achieve greater than 90 percent agreement on blind re - rating of a sample of their charts after using the severity of illness index for 2 months . all the hospital data reported here had greater than 90 percent agreement rates on blind re - rating of samples of charts . the actual agreement percent , rather than agreement corrected for chance agreement , has been reported here because of the very different distributions of overall severity of illness in the samples of charts selected for blind re - rating . we did not feel that each rater 's reliability assessment should be affected because the random sample selected for that review happened to contain charts from mostly one of the severity of illness levels . however , over 90 percent of the time , the agreement percent and the agreement corrected for chance agreement statistic were within 2 percentage points of each other . thus , we found that severity of illness data can be collected in a reproducible manner . with respect to the validity of the severity of illness instrument , we used the following procedures to assure that the instrument would come as close as possible to accurately measuring patient severity of illness . content validity was assessed by presenting the criteria for classification ( definitions of severity levels ) to a panel of medical experts who systematically examined the definitions and came to a consensus . the clinical experts felt that the criteria were representative of patient severity of illness at each identified severity level . face validity was examined by asking the developers and users of the instrument to make subjective judgments as to whether or not the instrument seemed reasonable and had the ability to obtain reasonable data . empirical investigation suggests that the index is a good measure of severity , but the process of validation is constantly being examined . the severity of illness index can not be compared with another validated measure of severity because none is available at this time . the research and statistical analyses discussed below , however , present evidence that the instrument leads to an accurate measurement of illness severity . the four levels of severity are an ordinal scale from the least severe ( level 1 ) to the most severe ( level 4 ) ; the distances between successive levels have no significance and are not necessarily equal . the severity of illness index is a generic measure , referring to the patients themselves . thus , any grouping system for patient classification can be subdivided into severity of illness levels . in particular , the severity of illness index can be used within drg 's or within any other case - mix system . another feature of the severity of illness index , which it shares with other case - mix grouping systems such as drg 's and disease staging , is that it does not explicitly take into account the quality of care received by the patient . the severity of illness index asks how sick the patient is and what burden of illness the patient exhibits while in the hospital . it does not ask whether the patient 's burden of illness was naturally caused or iatrogenically caused , ( i.e. , was exacerbated by accident or infection during hospitalization ) . thus , a patient may become sicker as a result of poor quality of care . severity of illness data are being or have been collected in more than 30 hospitals in the united states and canada . these hospitals include university teaching hospitals , community teaching hospitals , and community nonteaching hospitals . the results presented here are based on those hospitals for which we had at least six months of data . these results are preliminary and will need to be studied more thoroughly when additional data become available . because of the controversies about what trim points should be used , the data have not been trimmed . this permits generalization to an entire patient population , and thus application of a prospective payment system to all patients . severity of illness data are usually collected at discharge by medical records personnel concurrent with the coding of discharge abstract data , or by utilization review personnel when a final utilization review on a patient is completed . for analysis , severity of illness data are merged with discharge abstract data and financial data on each patient . at this point , a computer algorithm takes the presence of an operating room procedure into account . the data are subdivided into three subgroups depending on whether the patient had no operating room procedure , a moderate operating room procedure , or a major operating room procedure . the designation of moderate versus major operating room procedure is based on a list developed by a surgeon panel . a major operating room procedure either requires very special skills and education or takes a long time from which to recover . the subdivision into procedure type subgroups is done for analytical purposes , but does not affect the patient 's severity rating . within each severity of illness group , we examined resource consumption as expressed by total charges , length of stay , and ancillary service charges such as laboratory , radiology , pharmacy , and routine charges . we studied severity of illness within drg 's and within major diagnostic categories ( mdc 's ) , of which the drg 's are a finer subdivision . the results are similar for all types of resource use , so we shall demonstrate them using total charges . the necessary data are being collected to take charges back to costs , but the results are not yet available . in figure 2 , we show a sample of the reduction in variance ( riv ) results obtained when patients are grouped within an mdc . the patients are placed either into drg 's ( from 5 to 48 subgroups depending on the mdc ) or into the four severity levels divided into procedure type subgroups ( up to 12 subgroups ) . is represented by the formula : the data come from a total sample of over 19,000 cases in one university teaching hospital . these results are representative of the kinds of riv results that have been seen for all the mdc 's in the hospitals in the data set . the data show that even though the severity of illness index may place patients into fewer groups ( at most 12 ) than the drg 's , the variability in resource use that is explained by the severity of illness groups is greater than the variability that is explained by drg 's . the weighted ( by sample size ) coefficient of variation statistics for the same mdc 's are shown in figure 3 . the coefficient of variation of a data set is the standard deviation divided by the mean . we see that the severity of illness groups have lower weighted coefficients of variation , and hence , by this measure , have greater homogeneity than the drg 's . analysis of variance f test statistics for the severity of illness index and for drg 's are shown in figure 4 . the analysis of variance f test indicates how different the means are between the various groups , compared with the variability within the groups . a higher f value indicates a grouping system with greater differentiation among the groups as well as better homogeneity within the groups . we see that the severity of illness groups have much higher f values than the drg 's . thus , by three common measures of homogeneity , the severity of illness groups , even though almost always fewer in number , are found to be more homogeneous with respect to resource use than drg 's . these results are representative and typical of those seen from all the hospitals collecting severity of illness data on an ongoing basis . homogeneity of resource use is a desirable feature of a case - mix grouping system for prospective payment . it implies that most cases in a group will actually have resource - use levels which are approximately that of the typical ( average , median , or whatever ) case in the group the norm used to determine the amount of prospective payment for all patients in the group . even if a particular hospital 's patient distribution is atypical , it will be reimbursed appropriately at levels that fairly reflect the resource consumption of its patients . if groups are heterogeneous , the possibility exists that a hospital 's patients in a group will cluster far from the typical a grouping system with more groups might be expected , in principle , to be able to produce groups that are more homogeneous than those produced by a system with fewer groups . for the drg 's and severity of illness case - mix systems thus , it is not just the number of groups that has a decisive influence on homogeneity , but also the fundamental conception of what is being quantified by the system . the data indicate that just the labels of principal and secondary diagnoses , age , procedures , etc . , on which drg 's are based , do not describe sufficiently well how severely ill the patients are . it was found , however , that dividing patients into severity of illness groups yields much better explanatory power . an example of these results for one hospital with more than 19,000 cases in 1 year is shown in figure 5 . patients were placed into drg 's and also classified by severity of illness level and procedure type alone , severity and procedure type within mdc 's , and severity and procedure type within drg 's . for each of these three ways to use severity of illness , the result is that the severity of illness groups are more homogeneous than the drg 's alone , as indicated by greater reductions in variance , larger f values , and lower coefficients of variation . the number of groups into which the patients were classified in this hospital for each of the case - mix grouping systems is given in parentheses in the legend . in tables 1 - 3 , examples are shown of three drg 's , each illustrating a different aspect of variability . table 1 is for drg 75 ( major chest procedures ) , which contained 47 patients whose total charges ranged from $ 1,117 to more than $ 205,000 . a patient was placed in the major operating room procedure category if any of the patient 's operations was on the major operating room procedures list . otherwise , the patient was placed into the moderate operating room procedures category . in particular , the patient with charges of $ 1,117 was classified by the drg grouper into drg 75 because of a procedure coded 33.27 ( other lung biopsy ) . our refined definitions of procedure codes do not list this procedure in the major category , nor does this patient 's resource use reflect such a designation . however , some differences are noted in resource use by severity of illness level even among those patients who have only a moderate operating room procedure . the same phenomenon is observed for patients with major operating room procedures . large differences can be found between the average resource use for patients in the same severity level who have a moderate operating room procedure versus a major operating room procedure . the coefficients of variation of each of the severity of illness groups are much smaller than the coefficient of variation for drg 75 overall . by dividing the patients into severity of illness and procedure type groups within this drg , table 2 is for drg 108 ( cardiothoracic procedures , except valve and coronary bypass , with pump ) , which contained 60 patients with total charges ranging from a little more than $ 5,000 to more than $ 289,000 . although all these patients had a major operating room procedure , some differences in resource use by severity of illness level were found . table 3 is for drg 296 ( nutritional and miscellaneous metabolic disorders , age over 70 and/or with a complicating or comorbid secondary diagnosis ) , which contained 52 patients and had a smaller range of total charges , from $ 374 to more than $ 57,000 . however , differences were still observed in resource use as severity of illness increased . in this case , however , the severity level 1 group had a rather large coefficient of variation . this is attributable to one patient who had difficulty in placement , and whose resource use was therefore much higher than would be expected for a patient in this level . one of the main reasons for adopting a prospective payment system is to encourage an efficient level of operation within the health care delivery system . within a hospital , a prospective payment system should provide specific incentives for reducing inefficiencies of hospital services and should have groups that are meaningful to the medical community . if it does , then physicians may be led to modify their behavior , if appropriate . it is also important that a case - mix system for prospective payment produce groups that are homogeneous ( by various measures ) with respect to patient resource use . because of the often great variability in resource use found within drg 's , a question arises about their effectiveness in inducing desirable modifications in physician practice patterns . severity of illness within drg 's produced the most homogeneous groups ( figure 5 ) . how much of this variability might be explained by differences in physician practice patterns , the drg and severity of illness groups were subdivided further into groups of patients treated by each individual physician . we could , by this means , compare how much of the variability of charges within drg 's could be explained by physician practice patterns , compared with how much of the variability of charges within severity - of - illness - adjusted drg 's was explained by physician practice patterns . the results of these comparisons are presented in figure 6 for several hospitals . in general , the results show that drg 's explain 30 - 40 percent of the variability in resource use . physician subgrouping within drg 's explains another 20 - 40 percent of the variability of resource use . taking severity of illness into account as well , raises the explanatory power to between 90 percent and 96 percent of the variability in charges for each of the hospitals . these results suggest that most of the variability in resource use within a hospital can be explained by : the case mix that is going to the hospital ( drg ) . the coefficients of variation for these same hospitals grouped in the same ways are shown in figure 7 . these results show that physicians do explain some of the variability in resource use . however , it is not clear if the variability results from different physicians treating the same type of patient differently ( efficiency ) or differences in patients within a drg , severity , procedure type group treated by the same physician ( classification ) . these results , as well as the contribution of poor quality of care and its effect on the definitions of severity of illness and drg 's , need further study . if a hospital 's ( or a physician 's ) resource use within a drg is higher than a typical level , it could be that the institution ( or physician ) is inefficient . it could also mean that the institution or physician is treating patients who are more severely ill . because severity - adjusted drg 's produced the most homogeneous groups in this study , patients from the whole institution were placed into the appropriate drg group and further subclassified by severity of illness level and procedure type ( drg , severity , procedure ) . as the norms of practice in each institution , we used the average resource use expressed in terms of total charges , length of stay , laboratory charges , radiology charges , routine charges , and pharmacy charges for patients in each drg , severity , procedure group . subsequently , we compared the resource use of each physician 's patients with the norms in the appropriate categories . for each physician , we then accumulated the differences between each of his patient 's resource use and the norms , controlling for drg , severity , and procedure type . the results are explained in more detail in another article ( horn , horn , and moses , 1984 ) . it was found that some physicians treated most of their patients with less resource use than the norms , and some with more . the same comparisons were also made when the patients were grouped only by drg 's , and not adjusted for severity or procedure type ( figure 8) . the two different methods of assessing an individual physician 's efficiency often led to different conclusions . these disparate results signal to a hospital administrator that the underlying causes of the differences should be investigated . only a more detailed review will show whether the differences are because of quality of care , efficiency , or treatment of more severe cases . however , the implications of these differences between drg 's and severity- and procedure - adjusted drg 's are great . where will medical practice be in the future if the wrong physicians are criticized ( or praised ) for atypical practice patterns ? the results in the previous sections have indicated that severity of illness refinements within drg 's can produce resource - use groups that are more homogeneous . however , the fact that there is a spread of severity within a drg is not necessarily a fatal problem . if all hospitals treat patient populations with the same distribution of severity of illness within each of the drg 's , one might expect variations in resource use within a drg to average out . exactly such a reliance on a law of large numbers was explicitly stated in secretary richard schweiker 's report to congress ( department of health and human services , 1982 ) in which he proposed the present prospective payment system for medicare . in figure 9 , the distribution of severity of illness within drg 's in several different types of hospitals is shown . each hospital the various shaded sub - bars represent the percent of drg 's having one severity of illness level ( homogeneous with respect to severity of illness ) , having two severity of illness levels , etc . in hospital ut1 , on the other hand , more than 60 percent of the drg 's in hospital c1 had one level of severity , and fewer than 10 percent of the drg 's had three and four levels of severity . thus , different hospitals have different numbers of drg 's with a spread of severity of illness . figure 10 shows the distribution of patients according to the number of severity levels that their respective drg 's contained . in hospital ut1 , these drg 's contained 63 percent of the hospital 's patients , compared with 35 percent or less of the patients in the other hospitals in drg 's with three or four levels of severity . only 18 percent of the drg 's in hospital ut1 were homogeneous with respect to severity , and these drg 's contained only 3 percent of the patients in the institution . in the other hospitals , no more than 31 percent of the patients were in drg 's that were homogeneous with respect to severity of illness . thus , the drg 's that are homogeneous with respect to severity contained only a minority of the patients in the study hospitals , whereas those drg 's that were heterogeneous with respect to severity encompassed the majority of patients in all five institutions . however , the distribution of those patients varied greatly by institution . the financial impact of patients in the drg 's that were heterogeneous with respect to severity of illness is shown in figure 11 . the drg 's that contain three or four levels of severity of illness in hospital ut1 ( 37 percent of all drg 's present in ut1 ) accounted for 81 percent of the total charges in hospital ut1 , compared with 52 percent or less of the charges in the other hospitals . in this respect , thus , different percentages of a hospital 's revenue are at risk in the drg 's that are heterogeneous . these findings indicate that there can be large inter - hospital differences in the distribution of severity of illness of patients treated within a drg . furthermore , although teaching hospitals frequently treat a greater proportion of more severely ill patients , the severity distributions of the patients in the study hospitals are not predicted reliably by a simple classification by hospital type ( teaching versus community ) or by number of residents available per bed . the prospective payment system based on drg 's is an average - cost - formula system . claims data in the health care financing administration medicare provider analysis and review file supplemented by discharge records from maryland and michigan were used to place patients into their designated drg 's , and cost weights per drg were constructed . these weights reflect the relative costs of treating patients in a particular drg across all hospitals . the actual drg payments are computed by multiplying the drg cost weight by standardized amounts with adjustments for region of the country , differences in wage rates , and differences in number of residents per bed . collection of severity of illness data could be accomplished simultaneously with the collection of discharge abstract data on which the drg system is based . professional review organizations , who have been designated to monitor the reliability of discharge abstract data , could also monitor the reliability of severity of illness data . implementation of a prospective payment system involves administrative issues of equity , cost and regulatory burden . an equitable distribution of payments is essential to pay hospitals fairly for the efficient production of services performed . definition of the hospital product as accurately and precisely as possible is of primary importance . unreliable discharge abstract and severity data from coder errors , incomplete records , and potential gaming of the system definitions can be audited and corrected . this problem will exist and will require expense and regulatory efforts to monitor it . to alleviate part of this problem , we are developing a computerized severity of illness information data base that is based on a 6th digit enrichment of the current coding system , the international classification of diseases , ninth revision , clinical modification . this new , richer discharge abstract data set will be the basis for obtaining severity of illness levels from computer algorithms . it will also permit researchers to define and study other case - mix grouping systems from the new discharge data . as mentioned earlier , a patient may become sicker , and hence may receive a higher severity of illness rating , either because of poor quality of care that allows the patient to develop complications , or a poor clinical choice of therapy , to which the patient does not respond promptly . the consequence of this for a prospective payment system would be a higher payment for iatrogenically caused , as well as naturally , caused , illness . this problem exists , however , in the current drg prospective payment system , with or without a severity of illness adjustment . what is needed is a good measure of quality of care to correct this problem in any prospective payment system . a per - case prospective payment system has many of the right incentives to deal with the financial crisis in the health care field . with more accurate descriptions of their products , hospitals should be able to manage themselves in a more businesslike manner , and accurate cross - hospital comparisons can be made . each individual hospital patient is different , but there are sufficient similarities among patients to allow them to be grouped into medically meaningful homogeneous resource - use groups . trade - offs between the number of groups in such a grouping system , their medical meaningfulness , their homogeneity , and the robustness of the system against manipulation by hospitals and physicians need to be investigated thoroughly to determine which of the various possible case - mix systems is most suitable for prospective payment purposes . although our results , which used the severity of illness index for case - mix purposes , are consistent within all the hospitals analyses to date , these results could be biased by the self - selection factor of the hospitals that have elected to gather severity data . more data from a greater cross - section of hospital types are needed to determine if these results will continue to hold for all hospitals .

this article discusses the severity of illness case - mix groups , and suggests a refinement to diagnosis - related groups ( drg 's ) designed to accommodate the important element of patient severity . an application of the suggested refinement is presented in a discussion of the efficient production of hospital services.the following areas are addressed . a brief summary of the goals and development of the severity of illness index , and the methodology used to collect severity of illness data on hospital inpatients.comparative analyses of the resulting case - mix groups within hospitals , and an application of severity - adjusted diagnosis - related groups case - mix definitions.the contribution of the variation in physician practice patterns to the variation in resource use per patient within a hospital.cross-hospital comparisons.some of the consequences of incorporating a patient severity refinement into the prospective payment system .

Introduction Measuring patient severity of illness Results within hospitals Evaluating variations in physician practice patterns Inter-hospital differences in patient severity Severity adjustments to the prospective payment system Conclusions

how should patient categories , or case - mix groups , be defined if they are to be used for prospective payment for inpatient care ? two criteria are important for evaluating the effectiveness of a case - mix grouping system : medical meaningfulness of the grouping system and homogeneity of resource consumption within each of the case - mix groups . in addition , if a case - mix grouping system is to be used for prospective payment , two additional criteria must be considered : independence from the medical treatment process , and administrative feasibility . with these criteria in mind , a severity of illness index was developed by a team of researchers , physicians , and nurses at the johns hopkins university . it was designed to be a medically meaningful generic classification system that could differentiate the severity of illness of hospital inpatients . , the conventional discharge abstract data base is not rich enough to describe patient severity of illness accurately . using the patient 's total medical record , the severity of illness index assigns to each patient at discharge an overall severity score that is determined from the scores of each of seven medically meaningful dimensions chosen to reflect burden of illness . these seven dimensions , as shown in figure 1 , are : stage of the principal diagnosis the severity of illness index is described in detail in various reports ( horn , sharkey , and bertram , 1983 ; horn , 1983a ; horn and sharkey , 1983 ; horn , chachich , and clopton , 1983 ; horn , 1983b ) . to determine the severity of illness score for an individual case , a rater scores each of the seven dimensions into one of four levels of increasing severity by examining data in the patient 's medical record following discharge . definitions of each of the four levels for each dimension are provided , and raters are intensively trained to distinguish the reasons for selecting the level of each dimension . the reliability and validity of the severity of illness instrument has been studied in several ways . more than 95 percent of the individual raters achieve greater than 90 percent agreement on blind re - rating of a sample of their charts after using the severity of illness index for 2 months . the actual agreement percent , rather than agreement corrected for chance agreement , has been reported here because of the very different distributions of overall severity of illness in the samples of charts selected for blind re - rating . we did not feel that each rater 's reliability assessment should be affected because the random sample selected for that review happened to contain charts from mostly one of the severity of illness levels . however , over 90 percent of the time , the agreement percent and the agreement corrected for chance agreement statistic were within 2 percentage points of each other . thus , we found that severity of illness data can be collected in a reproducible manner . with respect to the validity of the severity of illness instrument , we used the following procedures to assure that the instrument would come as close as possible to accurately measuring patient severity of illness . content validity was assessed by presenting the criteria for classification ( definitions of severity levels ) to a panel of medical experts who systematically examined the definitions and came to a consensus . the clinical experts felt that the criteria were representative of patient severity of illness at each identified severity level . empirical investigation suggests that the index is a good measure of severity , but the process of validation is constantly being examined . the severity of illness index can not be compared with another validated measure of severity because none is available at this time . the research and statistical analyses discussed below , however , present evidence that the instrument leads to an accurate measurement of illness severity . the four levels of severity are an ordinal scale from the least severe ( level 1 ) to the most severe ( level 4 ) ; the distances between successive levels have no significance and are not necessarily equal . the severity of illness index is a generic measure , referring to the patients themselves . thus , any grouping system for patient classification can be subdivided into severity of illness levels . in particular , the severity of illness index can be used within drg 's or within any other case - mix system . another feature of the severity of illness index , which it shares with other case - mix grouping systems such as drg 's and disease staging , is that it does not explicitly take into account the quality of care received by the patient . the severity of illness index asks how sick the patient is and what burden of illness the patient exhibits while in the hospital . thus , a patient may become sicker as a result of poor quality of care . severity of illness data are being or have been collected in more than 30 hospitals in the united states and canada . these hospitals include university teaching hospitals , community teaching hospitals , and community nonteaching hospitals . because of the controversies about what trim points should be used , the data have not been trimmed . this permits generalization to an entire patient population , and thus application of a prospective payment system to all patients . severity of illness data are usually collected at discharge by medical records personnel concurrent with the coding of discharge abstract data , or by utilization review personnel when a final utilization review on a patient is completed . for analysis , severity of illness data are merged with discharge abstract data and financial data on each patient . within each severity of illness group , we examined resource consumption as expressed by total charges , length of stay , and ancillary service charges such as laboratory , radiology , pharmacy , and routine charges . we studied severity of illness within drg 's and within major diagnostic categories ( mdc 's ) , of which the drg 's are a finer subdivision . the patients are placed either into drg 's ( from 5 to 48 subgroups depending on the mdc ) or into the four severity levels divided into procedure type subgroups ( up to 12 subgroups ) . the data show that even though the severity of illness index may place patients into fewer groups ( at most 12 ) than the drg 's , the variability in resource use that is explained by the severity of illness groups is greater than the variability that is explained by drg 's . we see that the severity of illness groups have lower weighted coefficients of variation , and hence , by this measure , have greater homogeneity than the drg 's . analysis of variance f test statistics for the severity of illness index and for drg 's are shown in figure 4 . the analysis of variance f test indicates how different the means are between the various groups , compared with the variability within the groups . we see that the severity of illness groups have much higher f values than the drg 's . thus , by three common measures of homogeneity , the severity of illness groups , even though almost always fewer in number , are found to be more homogeneous with respect to resource use than drg 's . these results are representative and typical of those seen from all the hospitals collecting severity of illness data on an ongoing basis . homogeneity of resource use is a desirable feature of a case - mix grouping system for prospective payment . it implies that most cases in a group will actually have resource - use levels which are approximately that of the typical ( average , median , or whatever ) case in the group the norm used to determine the amount of prospective payment for all patients in the group . if groups are heterogeneous , the possibility exists that a hospital 's patients in a group will cluster far from the typical a grouping system with more groups might be expected , in principle , to be able to produce groups that are more homogeneous than those produced by a system with fewer groups . for the drg 's and severity of illness case - mix systems thus , it is not just the number of groups that has a decisive influence on homogeneity , but also the fundamental conception of what is being quantified by the system . it was found , however , that dividing patients into severity of illness groups yields much better explanatory power . patients were placed into drg 's and also classified by severity of illness level and procedure type alone , severity and procedure type within mdc 's , and severity and procedure type within drg 's . for each of these three ways to use severity of illness , the result is that the severity of illness groups are more homogeneous than the drg 's alone , as indicated by greater reductions in variance , larger f values , and lower coefficients of variation . the number of groups into which the patients were classified in this hospital for each of the case - mix grouping systems is given in parentheses in the legend . a patient was placed in the major operating room procedure category if any of the patient 's operations was on the major operating room procedures list . otherwise , the patient was placed into the moderate operating room procedures category . our refined definitions of procedure codes do not list this procedure in the major category , nor does this patient 's resource use reflect such a designation . however , some differences are noted in resource use by severity of illness level even among those patients who have only a moderate operating room procedure . large differences can be found between the average resource use for patients in the same severity level who have a moderate operating room procedure versus a major operating room procedure . the coefficients of variation of each of the severity of illness groups are much smaller than the coefficient of variation for drg 75 overall . by dividing the patients into severity of illness and procedure type groups within this drg , table 2 is for drg 108 ( cardiothoracic procedures , except valve and coronary bypass , with pump ) , which contained 60 patients with total charges ranging from a little more than $ 5,000 to more than $ 289,000 . although all these patients had a major operating room procedure , some differences in resource use by severity of illness level were found . however , differences were still observed in resource use as severity of illness increased . in this case , however , the severity level 1 group had a rather large coefficient of variation . this is attributable to one patient who had difficulty in placement , and whose resource use was therefore much higher than would be expected for a patient in this level . one of the main reasons for adopting a prospective payment system is to encourage an efficient level of operation within the health care delivery system . within a hospital , a prospective payment system should provide specific incentives for reducing inefficiencies of hospital services and should have groups that are meaningful to the medical community . it is also important that a case - mix system for prospective payment produce groups that are homogeneous ( by various measures ) with respect to patient resource use . because of the often great variability in resource use found within drg 's , a question arises about their effectiveness in inducing desirable modifications in physician practice patterns . severity of illness within drg 's produced the most homogeneous groups ( figure 5 ) . how much of this variability might be explained by differences in physician practice patterns , the drg and severity of illness groups were subdivided further into groups of patients treated by each individual physician . we could , by this means , compare how much of the variability of charges within drg 's could be explained by physician practice patterns , compared with how much of the variability of charges within severity - of - illness - adjusted drg 's was explained by physician practice patterns . in general , the results show that drg 's explain 30 - 40 percent of the variability in resource use . physician subgrouping within drg 's explains another 20 - 40 percent of the variability of resource use . taking severity of illness into account as well , raises the explanatory power to between 90 percent and 96 percent of the variability in charges for each of the hospitals . these results suggest that most of the variability in resource use within a hospital can be explained by : the case mix that is going to the hospital ( drg ) . these results show that physicians do explain some of the variability in resource use . however , it is not clear if the variability results from different physicians treating the same type of patient differently ( efficiency ) or differences in patients within a drg , severity , procedure type group treated by the same physician ( classification ) . these results , as well as the contribution of poor quality of care and its effect on the definitions of severity of illness and drg 's , need further study . if a hospital 's ( or a physician 's ) resource use within a drg is higher than a typical level , it could be that the institution ( or physician ) is inefficient . because severity - adjusted drg 's produced the most homogeneous groups in this study , patients from the whole institution were placed into the appropriate drg group and further subclassified by severity of illness level and procedure type ( drg , severity , procedure ) . as the norms of practice in each institution , we used the average resource use expressed in terms of total charges , length of stay , laboratory charges , radiology charges , routine charges , and pharmacy charges for patients in each drg , severity , procedure group . subsequently , we compared the resource use of each physician 's patients with the norms in the appropriate categories . for each physician , we then accumulated the differences between each of his patient 's resource use and the norms , controlling for drg , severity , and procedure type . the results are explained in more detail in another article ( horn , horn , and moses , 1984 ) . it was found that some physicians treated most of their patients with less resource use than the norms , and some with more . the same comparisons were also made when the patients were grouped only by drg 's , and not adjusted for severity or procedure type ( figure 8) . these disparate results signal to a hospital administrator that the underlying causes of the differences should be investigated . however , the implications of these differences between drg 's and severity- and procedure - adjusted drg 's are great . the results in the previous sections have indicated that severity of illness refinements within drg 's can produce resource - use groups that are more homogeneous . however , the fact that there is a spread of severity within a drg is not necessarily a fatal problem . if all hospitals treat patient populations with the same distribution of severity of illness within each of the drg 's , one might expect variations in resource use within a drg to average out . exactly such a reliance on a law of large numbers was explicitly stated in secretary richard schweiker 's report to congress ( department of health and human services , 1982 ) in which he proposed the present prospective payment system for medicare . in figure 9 , the distribution of severity of illness within drg 's in several different types of hospitals is shown . each hospital the various shaded sub - bars represent the percent of drg 's having one severity of illness level ( homogeneous with respect to severity of illness ) , having two severity of illness levels , etc . in hospital ut1 , on the other hand , more than 60 percent of the drg 's in hospital c1 had one level of severity , and fewer than 10 percent of the drg 's had three and four levels of severity . thus , different hospitals have different numbers of drg 's with a spread of severity of illness . figure 10 shows the distribution of patients according to the number of severity levels that their respective drg 's contained . in hospital ut1 , these drg 's contained 63 percent of the hospital 's patients , compared with 35 percent or less of the patients in the other hospitals in drg 's with three or four levels of severity . only 18 percent of the drg 's in hospital ut1 were homogeneous with respect to severity , and these drg 's contained only 3 percent of the patients in the institution . in the other hospitals , no more than 31 percent of the patients were in drg 's that were homogeneous with respect to severity of illness . thus , the drg 's that are homogeneous with respect to severity contained only a minority of the patients in the study hospitals , whereas those drg 's that were heterogeneous with respect to severity encompassed the majority of patients in all five institutions . the financial impact of patients in the drg 's that were heterogeneous with respect to severity of illness is shown in figure 11 . the drg 's that contain three or four levels of severity of illness in hospital ut1 ( 37 percent of all drg 's present in ut1 ) accounted for 81 percent of the total charges in hospital ut1 , compared with 52 percent or less of the charges in the other hospitals . in this respect , thus , different percentages of a hospital 's revenue are at risk in the drg 's that are heterogeneous . these findings indicate that there can be large inter - hospital differences in the distribution of severity of illness of patients treated within a drg . furthermore , although teaching hospitals frequently treat a greater proportion of more severely ill patients , the severity distributions of the patients in the study hospitals are not predicted reliably by a simple classification by hospital type ( teaching versus community ) or by number of residents available per bed . the prospective payment system based on drg 's is an average - cost - formula system . claims data in the health care financing administration medicare provider analysis and review file supplemented by discharge records from maryland and michigan were used to place patients into their designated drg 's , and cost weights per drg were constructed . the actual drg payments are computed by multiplying the drg cost weight by standardized amounts with adjustments for region of the country , differences in wage rates , and differences in number of residents per bed . collection of severity of illness data could be accomplished simultaneously with the collection of discharge abstract data on which the drg system is based . professional review organizations , who have been designated to monitor the reliability of discharge abstract data , could also monitor the reliability of severity of illness data . implementation of a prospective payment system involves administrative issues of equity , cost and regulatory burden . an equitable distribution of payments is essential to pay hospitals fairly for the efficient production of services performed . unreliable discharge abstract and severity data from coder errors , incomplete records , and potential gaming of the system definitions can be audited and corrected . to alleviate part of this problem , we are developing a computerized severity of illness information data base that is based on a 6th digit enrichment of the current coding system , the international classification of diseases , ninth revision , clinical modification . this new , richer discharge abstract data set will be the basis for obtaining severity of illness levels from computer algorithms . it will also permit researchers to define and study other case - mix grouping systems from the new discharge data . as mentioned earlier , a patient may become sicker , and hence may receive a higher severity of illness rating , either because of poor quality of care that allows the patient to develop complications , or a poor clinical choice of therapy , to which the patient does not respond promptly . the consequence of this for a prospective payment system would be a higher payment for iatrogenically caused , as well as naturally , caused , illness . this problem exists , however , in the current drg prospective payment system , with or without a severity of illness adjustment . what is needed is a good measure of quality of care to correct this problem in any prospective payment system . a per - case prospective payment system has many of the right incentives to deal with the financial crisis in the health care field . with more accurate descriptions of their products , hospitals should be able to manage themselves in a more businesslike manner , and accurate cross - hospital comparisons can be made . trade - offs between the number of groups in such a grouping system , their medical meaningfulness , their homogeneity , and the robustness of the system against manipulation by hospitals and physicians need to be investigated thoroughly to determine which of the various possible case - mix systems is most suitable for prospective payment purposes . although our results , which used the severity of illness index for case - mix purposes , are consistent within all the hospitals analyses to date , these results could be biased by the self - selection factor of the hospitals that have elected to gather severity data . more data from a greater cross - section of hospital types are needed to determine if these results will continue to hold for all hospitals .

ribonucleotide reductases ( rnrs ) catalyze the conversion of four nucleotides ( cdp , udp , adp , gdp ; ndps ) into the corresponding deoxynucleotides ( dndps ) . class ia rnrs are found in nearly all eukaryotic and some prokaryotic organisms and are composed of two homodimeric subunits , 2 and 2 , which form an active , transient 22 complex . subunit 2 houses the catalytic substrate binding site and the binding sites for the allosteric effectors that govern specificity and activity of nucleotide diphosphate ( ndp ) reduction . subunit 2 houses the diferric - tyrosyl radical cofactor ( the fe2-y ) essential for initiating ndp reduction . during each turnover , the fe2-y-2 oxidizes c439 in the active site of 2 where dndp is produced , and then it is subsequently reoxidized . the oxidation occurs via a radical hopping mechanism over 35 between the two subunits along a specific pathway comprised of redox active amino acids ( y122 [ w48 ? ] y356 in 2 y731 y730 c439 in 2 ) . reversible oxidation and reduction of y during turnover requires release of the phenolic proton to an acceptor concomitant with the oxidation ( figure 1a ) , a mechanistic strategy to avoid formation of high - energy intermediates . while the long - range oxidation through aromatic amino acid residues in ribonucleotide reductase ( rnr ) is unprecedented in biology , proton - coupled electron transfer ( pcet ) mechanisms are involved in many fundamental processes in biology including photosynthesis , respiration and nitrogen fixation . rnrs can thus serve as a paradigm for understanding pcet in a complex biological machine and , in comparison with other systems , offer the opportunity to identify common principles that control this basic transformation . since pcet is intrinsically of quantum mechanical nature because of both electron and proton tunnels , the large difference in mass ( factor 2000 ) causes the proton translocation to be limited to very short distances ( < 1 ) , while the electron may transfer over very long distances . thus , in pcet the electron and the proton might be transferred to different acceptors ( orthogonal or bidirectional ) or they might move between the same donor / acceptor pairs ( colinear ) . description of the coordination of these events has been theoretically challenging and is summarized in recent reviews . investigation of the electron and proton pathways has required rnrs with site - specifically incorporated unnatural amino acids and electron paramagnetic resonance ( epr ) combined with electron nuclear double resonance ( endor ) spectroscopies have provided a unique opportunity to access mechanistic details if the intermediate radicals can be detected . the current model for this long - range oxidation it was originally proposed based on the x - ray structures of 2 and 2 , a docking model of these subunits based on their shape complementarity , and conserved residues . recent biophysical studies including pulsed electron electron double resonance ( peldor ) studies and small - angle x - ray scattering studies ( saxs ) and cryoem have established that the docking model is a reasonable representation of the active rnr structure in solution and that the oxidation occurs over > 35 . unfortunately there is little information about the molecular details of the interface between and , more specifically , the communication between y356 in 2 and y731 in 2 . the last 2530 amino acids of all 2 structures which include y356 or its equivalent are structurally disordered and the last 15 amino acids of escherichia coli 2 are largely responsible for the weak interaction between the subunits ( kd of 0.1 to 0.2 m ) . working model for pcet in e. coli rnr . ( a ) scheme for concerted pcet between pathway tyrosines within . ( b ) amino acids as well as protons ( blue ) proposed to be involved in radical transfer between the and subunits . w48 and d237 are shown in gray because there is currently no evidence for their participation in pcet . in , the protons are proposed to move orthogonal to the electrons ( blue vs black arrows ) and within they are proposed to move colinear with the electrons ( purple arrows ) . ( c ) overlay of residues in from the resting state x - ray structure ( pdb 2x0x gold / green ) with the density functional theory ( dft ) optimized structure for radical state y730 ( residues y731 , y730 and c439 in gray shade ) . position of the conserved water molecule wat1 as well as on - pathway ( yellow dots ) and other ( red dots ) hydrogen bonds are from the dft structure . the h bond distance of y731 to y730 is ro730o731 2.6 whereas the h bond distances of c439 and wat1 to y730 are rs439o730 3.3 and rowat1o730 2.8 respectively . inset : chemical structure of nh2y and ys in the pathway replaced by nh2y as well as the double mutants employed in this study . studies of this unprecedented oxidation catalyzed by wt rnr have not been possible as protein conformational change(s ) constitute the rate - limiting step(s ) and kinetically mask all of the detailed chemistry of the radical transfer ( rt ) and the nucleotide reduction step . however , use of technology in which the pathway tyrosines are replaced site - specifically with tyrosine analogues with altered pkas and reduction potentials , and the development of high frequency ( hf ) epr and endor methods to characterize y analogues generated with the mutant rnrs , are now unmasking the chemistry of this long - range oxidation . these studies taken together suggest that conformational gating is occurring within and that radical intermediates formed at y356 , y731 , y730 , c439 and substrate radical are likely in equilibrium along the pathway . their relative redox potentials are progressively uphill with corresponding lower concentrations . however , there is sufficient concentration of the most uphill radical ( substrate radical ) to drive the reaction to the right by a rapid , irreversible step proposed to be the loss of water during nucleotide reduction . once the chemical reaction is complete , the reformation of the y122 in is energetically downhill . the results from the incorporation of nh2y site - specifically at 356- , 731- and 730- , the focus of this paper , have played a critical role in our current understanding of specific steps of the rt pathway and the conformational gating process . in all three cases , nh2y-( ) is generated in 3040% yield upon incubation of the second subunit ( ) , substrate cdp and allosteric effector atp . the formation of the nh2y is a biphasic process with both phases kinetically competent in nucleotide reduction at 5 to 10% the rate of the wt - rnr . three recent experiments using this probe address the importance of pcet and the 22 conformational changes triggered by binding substrate and effector . insight into the initial rt event within has been studied by rapid freeze quench ( rfq ) mssbauer analysis using wt-2/nh2y730-2/cdp / atp . the results established that the proton of the water bound to fe1 ( figure 1b ) is transferred to y122 coupled with an electron , likely from y356 . using the nh2y730-2 only the forward radical step is observed and interestingly the fe1-oh now remains . these results support the model of orthogonal pcet in 2 and the exquisite control dependent on the proton s location ( figure 1b ) . using the same experimental design , analysis of the nh2y by hf epr and deuterium ( h ) endor provided the first spectroscopic insight suggesting the importance of colinear pcet within 2 and the structural relationship of y731 and y730 in the active 22 rnr complex . the link between epr data in the active intermediate state and the x - ray structural data in the inactive state was supported by quantum chemical calculations of the energy optimized structural models of y730- and nh2y730- including 211atoms ( figure 1c ) . the endor analysis in conjunction with density functional theory ( dft ) calculations revealed one strong ( ro o 2.7 ) and one weaker ( rs o 3.4 ) h bond proposed to be associated with residues adjacent to nh2y730 in the pathway , as well as another weak ( ro o 3.0 ) interaction with water . the water molecule was proposed to tune the rt rates in wt enzyme around y730 by about 1 order of magnitude . finally , our recent saxs and pull - down experiments of rnr from this reaction mixture established that a single h atom transfer from nh2y730 to y122 is sufficient to increase the weak / subunit affinity by a factor of 10 . this result again highlights the importance and subtlety of conformational changes in the rt process . in the current paper , experiments in which nh2y has been site specifically incorporated at nh2y356- , and at nh2y731 and nh2y730- ( figure 1c ) in conjunction with hf epr and hf h endor spectroscopies , density functional theory ( dft ) calculations using nh2y731(730)- structures , have been employed in an effort to understand the electrostatic and h bonding environment at these residues in the rt pathway . hf epr data were recorded at a microwave frequency of 263 ghz , which delivers unprecedented resolution in the spectra of the nh2ys . in addition , double mutants nh2y730/c439a- and nh2y731/y730f- were prepared and the h endor results compared to the single mutant results in an effort to assign the source of the h bonds observed to specific residues . our results support the colinear concerted pcet model within and provide spectroscopic evidence for the unusual stacking of y730/y731 in the active 22 complex . to probe the electrostatic environment of the radical intermediates at the three different positions in the rt pathway we examined the hf epr spectra formed after incubation of -nh2y356 , -nh2y731 , -nh2y730 with their respective complementary wt subunits , either or , cdp , and atp ( methods ) . figure 2 presents 263-ghz pulse epr spectra from the individually trapped nh2ys recorded with a prototype quasi - optical epr spectrometer ( methods ) . the buffer was exchanged with d2o to enhance epr resolution . at this frequency , all gx values of nh2ys ( nd2ys ) are clearly resolved and shifted by about 1 ppt ( or 0.001 ) from the value calculated for a free nh2y ( gx , free = 2.0061 ) . this shift is significant , on the order of the effect predicted from several hydrogen bonds.g values were calibrated with the spectrum of the stable radical y122 in 2 , which is present in all samples and visible only at low temperatures . additionally , gx slightly decreases starting from the most buried intermediate nh2y730 ( gx = 2.0054 ) to nh2y356 ( gx = 2.0049 ) located at the subunit interface . as a control , spectra were also recorded in protonated buffer and the g values were best reproduced ( supporting information , figure s1 ) . simulations of the 263 ghz spectra combined with 94 ghz spectra ( the latter frequency was used to constrain the simulation , figure s2 ) led to a consistent set of g values and c- proton hyperfine ( hf ) couplings ( see chemical structure in figure 1 inset ) that are summarized in table 1 . thus , the epr spectra of the single mutants in the active enzyme are contributed by a radical species ( per mutant ) with a well - defined microenvironment ( g value ) and molecular orientation . this finding underlines the importance of a specific structural arrangement in the active state to permit radical propagation . the observed shift of gx values is a hallmark for a substantial effect of either positive charges and/or hydrogen bond interactions . since hydrogen bonds are expected to have a predominant effect on g values , a possible correlation of the observed gx shifts with the number and strength of hydrogen bonds was examined . . electron spin echo ( ese ) detected spectra of intermediates without ( top spectrum for nh2y731/y122 , 10 k ) and with relaxation filtering ( bottom spectra for nh2ys , 70 k ) . echo sequence used to suppress the signal of y122 ( box ) is based on differences in the transverse relaxation times ( t2 ) of the radicals : nh2ys and y122. at t = 70 k , the signal associated with the y122 decays during the acquisition ( box , purple dotted line ) and does not contribute to the spin echo signal . ese detected epr spectra of the nh2ys radicals in h / d exchanged buffer : nd2y730 ( black ) , nd2y731 ( red ) and nd2y356 ( blue ) . experimental conditions : ese ( /2echo ) spectra : /2 = 60110 ns , = 290 ns , 250500 averages / point , acquisition time / spectrum = 1.53 days , t = 10 and 70 k. the values were obtained from combined simulations of the 263 and 94 ghz spectra and compared with those obtained from dft calculations . the n hyperfine tensor of the nh2y was not varied in the simulations and kept ax = 2.4 mhz , ay = 1.6 - 5 mhz , az = 30.7 mhz . uncertainty in g values is about 0.05 ppt for the experiments and 0.5 ppt for dft calculations . we probed exchangeable hydrogen bonds around the trapped nh2y intermediates by endor spectroscopy at 94 ghz . endor reveals the spectrum of magnetic nuclei that are in the coordination sphere ( usually 5 ) of the observed radical . after buffer exchange , exchangeable protons are substituted by deuterons ( abbreviated d , with the nucleus denoted h ) , which become visible by endor in the h resonance region . figure 3a illustrates the h mims endor spectra of the three nh2y intermediates ( nd2y ) trapped under comparable conditions as in the epr experiments . the spectrum of nd2y730 was initially reported in ref ( 25 ) , and it is included here for further analysis . all three spectra contain a broad , almost featureless background extending over 2 mhz , which arises from the strongly coupled amino deuterons . additionally , two samples , nd2y730 and nd2y731 , show pairs of sharp peaks in the region 1 mhz that is usually dominated by deuterons in the hydrogen bond range ( figure 3a , peaks marked in red and blue ) . the spectra of nd2y730 and nd2y731 look quite similar , with the peaks from nd2y731 being slightly shifted to larger couplings . the sharp peaks are split by an additional small coupling , i.e. , the quadrupol coupling . the endor spectrum can also detect weak couplings ( ro h 2.1 ) if they can be resolved from matrix deuterons ( matrix line ) . indeed , additional small differences are observed here also in the central resonance region ( 0.3 mhz ) . in nd2y730 , this region was proposed to be associated with a water molecule ( wat1 , figure 1 ) conserved in the structure of wt 2 and nd2y730-2 . interestingly the spectrum of nd2y356 lacks any sharp peaks and is associated with resonances of the amino deuteron and a matrix line . ( a ) comparison between spectra of nd2y730 ( black ) , nd2y731 ( red ) , and nd2y356 ( blue ) . simulation of the spectrum for nd2y731 ( gray ) is representatively decomposed into the contributions of the amino deuterons ( blue dashed and dotted lines ) and of one hydrogen bond ( red dashed line ) . spectrum of nd2y730 is contributed by one strong ( red peaks ) and one moderate ( blue peaks ) h bond . ( b ) spectra of double mutants nd2y730/c439a and nd2y731/y730f ( gray and red , respectively ) . simulation of the spectrum of the retained h bond ( red peaks ) with parameters reported in table s2 . the contributions from hydrogen bonds , as assigned here , are illustrated on the right . parameters : mims endor with /2 = 20 ns , = 200 ns , shot repetition time = 10 ms , random rf acquisition at 1 shot / point , acquisition time = 2455 h , t = 70 k. the same relaxation filtering mechanism was used for the endor experiments at 70 k as for the hf epr spectra ( cf . endor spectrum is centered at the larmor frequency 0 of h , i.e. , 21.9 mhz at a field of 3.3 t. further experimental evidence for the hydrogen bond at nh2y731 was derived from orientation selective hf h endor spectra recorded at different field positions in the epr line . figure 4a shows mims endor spectra recorded at the canonical orientations b0 gx , gy and gz within the region of 1.5 mhz . powder patterns are still observed at orientations b0 gx and gz as orientation selection is moderate for the large excitation bandwidth of the pulses ( 1.8 mt ) as compared to the total epr line width ( 8 mt ) ; however , clear differences in the line shapes are visible . the smallest hf coupling , taken as the center of the sharp peak , is observed at b0 gz and the hf tensor displays a form |az| |ay| > |ax| ( using the definition |ax| < |ay| < |az| ) . previous dft calculations have indicated that such a tensor form is typical for a deuteron directed almost perpendicular to the tyrosine ring plane , as previously reported also for nd2y730 , and having the smallest component along the h bond . such a tensor reflects couplings that still contain some contribution from a scalar interaction arising from orbital overlap . in the following , we define hydrogen bonds with these tensor properties as strong to moderate ( ro h 1.72.0 ) . the definition used here is consistent with ref ( 10 ) but expanded by the definition of moderate bonds . considering that the gx tensor component lies along the c o bond and gy points to the side of the amino group , the euler angles , , between hyperfine a and g tensor obtained from the simulation ( table 2 ) are consistent with a hydrogen bond directed almost perpendicular to the plane of the nh2y731 . simulations of the entire h endor spectrum with the parameters obtained here additionally indicate that the spectrum of a nd2y731 can be well reproduced by the contribution of a single hydrogen bond ( figure 3a ) according to the observed intensity ratio of the sharp peaks with respect to the amino deuteron resonances . h 94-ghz mims endor spectra ( black lines ) were recorded at field positions in the epr line parallel to the canonical orientations of the g tensor , i.e. , for b0 gx , gy , gz . simulations of the spectra ( red dashed - dotted curves ) were performed as described in the methods section . = 320 ns , rf pulse length = 40 s , shot repetition time = 150 ms , acquisition time = 50 h / spectrum , random rf acquisition , t = 10 k. the low s / n required operation at very low t and contribution of y122 could not be separated here . however , y122 does not display any hydrogen bonds but only a matrix line , as also discussed in ref ( 25 ) . the inset shows the orientation of the h hf tensor from the simulation . in an effort to assign the h bonding interactions observed with nh2y731- and nh2y730-2 by h endor , double mutants nh2y731/y730f and nh2y730/c439a were generated , in which one of the proposed h bonds was removed . the proteins were expressed , purified to homogeneity and characterized by stopped flow - vis spectroscopy monitoring the rate of loss of y122 ( 410 nm ) and rate of formation of the nh2y ( 320 or 325 nm ) in the presence of wt-2/cdp / atp , figure s3 . the results are summarized in table s1 and compared with results from similar experiments on the single mutants . with nh2y731/y730f-2 , nh2y was formed with biphasic kinetics resulting in 34 3% conversion , similar to the amount formed with nh2y731-2 ( 32 3% ) . the kinetics of both phases , however , were slower : 1.5 0.1 and 0.3 0.03 s compared to 9.6 0.6 and 0.8 0.1 s. similar studies with nh2y730/c439a-2 resulted in formation of nh2y in only 14 1% conversion , compared with 39% in the single mutant . furthermore , the rate constant for its formation was decreased 10 fold for the double mutant : from 12 1 to 0.13 0.01 s and only a single kinetic phase was measured . both double mutants were also characterized by 9 ghz epr spectroscopy in samples frozen at 30 s ( figure s4 ) . in the case of both double mutants , the altered kinetics of pathway radical formation and its altered amplitude in the case of nh2y730/c439a-2 are likely reflective of the importance of these h bonding interactions in tuning of pcet within 2 . to test for possible conformational differences between the nh2y intermediates in the double vs the single mutants , we measured their inter spin distances to the diagonalized y122 by peldor spectroscopy ( figure s5 and s6 ) . the experiments revealed distances of 3.8 and 3.9 nm , respectively , consistent within error ( 1 ) with the distances observed in the single mutants and from the 22 docking model in wt ; however , the assignment of the 3.9 nm distance in the mutant nh2y730/c439a is more uncertain due to the low radical yield and s / n ratio of the peldor traces hf epr spectra ( 263 ghz ) of nh2y trapped in the double mutants also revealed gx values ( figure s7 ) different from those in the single mutants , i.e. , shifted by + 0.30.4 ppt in the direction of a free nh2y as expected after removal of a h bond ( table 1 ) . h - endor spectra of both double mutants ( figure 3b ) display that the sharp peaks at 0.60.7 mhz have dramatically decreased . in the spectrum of nh2y731/y730f the peaks are now entirely absent , whereas residual peaks ( 30% of initial intensity ) are observed in nh2y730/c439a mutant . these results are consistent with the assignment of one h bond to nh2y731 associated with y730 and one hydrogen bond to nh2y730 associated with c439 . importantly , the spectrum of nh2y731/y730f gives no evidence for any additional strong h bonds . the nh2y730/c439a mutant , on the other hand , still reveals a contribution of a strong hydrogen bond presumably from y731 as previously proposed by dft calculations . we note also that the removal of the hydrogen bond is manifested in the hf coupling of the amino deuterons , which slightly decreases as compared to the single mutants due to a change in the spin density distribution on nd2y. to obtain a model structure of the radical localized at nh2y731 , dft calculations on representative model systems for nh2y731 were performed and the obtained magnetic parameters for gx and for h bonds were compared with the experimental values . as a starting point , we used the dft energy optimized structure of y731 that was previously reported ( methods ) . three large models ( up to 216 atoms , figure s8 ) were considered , which differed by the inclusion of zero , one or two water molecules . the models have taken into account all residues in an interaction sphere of about 5 around the oxygen of y731 . dft optimized structures of nh2y731. left : model 1 includes the water molecule wat1 . model 3 contains a second water molecule wat2 , which is observed in some x - ray structures ( see text ) . we found that the presence or absence of the water molecules has an impact on some residues arrangement around nh2y731 but not on the formation of a strong h bond with y730 . in optimized models 1 and 2 , the distance from the oxygen of nh2y731 to the hydrogen of the phenol group of y730 is 1.7 . in absence of wat1 ( model 2 ) , r411 approaches nh2y731 with hydrogens from its guanidinium group at distances of 2.1 and 2.6 from the oxygen of nh2y731. the calculated gx values from the two models differ by about 0.5 ppt , with the gx values of model 2 being closer to the experimental ones ( table 1 ) . in model 3 a second water molecule ( wat2 ) was included as observed in some x - ray structures of wt- and nh2y731/730- ( figure s9 ) in the vicinity ( ro o 2.63.6 ) of residue 731 . the optimized structure of model 3 finds wat2 within hydrogen bond distance ( ro h 1.9 ) to nh2y731 ; however , a stronger h bond to the phenoxyl hydrogen of y730 remains ( ro h 1.6 ) . the gx value for the model 3 structure is consistent with the experimental value ( table 1 ) . the computed epr parameters for the h bond distances and orientations relative to y730 are listed in table 2 . particularly , all models display an orientation of the strong h bond to nh2y731 provided by y730 , which is compatible with the endor data within an uncertainty of about 20% ( table 2 , figure s10 ) . parameters were obtained from simulations of the orientation selective 94-ghz endor spectra and comparison with the dft models . the signs of the couplings from the simulation are only relative to each other within one tensor . the euler angles ( , , ) are defined from the a or q to the g tensor based on the y convention ( positive sign for a rotation is counterclockwise , second rotation is around the y axis ) . the a and q ( quadrupole ) tensor are chosen such that |ax| < |ay| < |az| . within this definition , for both the amino deuterons and the h bond deuteron the ax direction results along the bond direction . euler angles from dft ( in the orca output positive rotations are defined clockwise ) were transformed into the magnetic resonance convention , for comparison . uncertainty in the parameters from the dft and endor simulations is estimated up to about 20% . considering the accuracy of the calculations , on the order of 0.5 ppt , all three models give gx values in principle compatible with the experiment . however , considering that the error in the trend of the calculation is smaller than 0.5 ppt , model 2 and 3 better reproduce the experimental g values . the results indicate that the strong gx shift at nh2y731 can be contributed by the combined effect of a strong h bond to y730 and the interaction either to a water molecule ( model 3 ) or alternatively to the positively charged arginine r411 ( model 2 ) . the findings are further supported by calculations on small model systems ( figure s11 ) , in which the effect of the individual residues r411 or y730 was systematically tested . both scenarios represented in models 2 and 3 find some precedents in the literature . studies on cation interactions revealed that these are common between amino acids like arginine and aromatic amino acids like tyrosine . other studies on small peptides showed that tyrosine / arginine interactions can alter the reduction potential of tyrosines . on the other hand , in ps ii two water molecules have been proposed to interact with the redox active yz during pcet and affect their g values . we note that protons of either wat2 or r411 possibly located at distances 1.9 from the phenoxy oxygen might not be resolved in an endor spectrum . finally we note that the g values for these nh2y can also be affected by the subunit , which can not be modeled at present . the electronic and structural features of the nh2ys trapped in the rt pathway of e. coli rnr report on how the local protein environment has reorganized after a pcet step to stabilize these intermediates . at each radical state , the protein has rearranged to accommodate a released electron and a proton , a scenario that can be probed by two epr parameters : the shift in the gx value caused by positive charges and the hyperfine interaction of protons forming hydrogen bonds of varying strengths . the first parameter is directly accessible from high - frequency epr spectra but contains only indirect information on hydrogen bond interactions . the second parameter , i.e. , the hyperfine coupling to protons exchanged by deuterons , requires endor spectroscopy and much more extensive analysis , which in turn , when attainable , uniquely delivers a high resolution structure of the h bond partners . in this study we report both parameters for the nh2ys trapped within the rt pathway of rnr subunit . all gx values of the nh2ys in the three pathway positions are similar and show a considerable shift from the calculated value for a free nh2y ( table 1 ) , consistent with a strongly perturbed electrostatic environment . however , our data reveal that the number , orientation and strength of exchangeable hydrogen bonds at these residues are intrinsically different , reflecting the subtle difference in protein architecture and giving direct insight into the hydrogen bond network involved in pcet . the endor data display one strong hydrogen bond and one moderate h bond with nh2y730 and one strong hydrogen bond with nh2y731. in both cases , the hyperfine couplings and tensor orientations of the two strong bonds are very similar ( tables 2 and s2 ) and indicate that the hydrogen must reside between the two tyrosyl rings ( figures 3 and 4 ) . the direction of the hydrogen bond to nh2y731 extracted from the present hyperfine and quadrupole tensors in the endor data ( figure 4 ) are consistent with the directions predicted by the dft calculations for a bond with y730 ( figure 5 ) no matter the number of water molecules considered in the calculation . moreover , the endor peaks ( figure 3b ) of this strong bond disappear when y730 is mutated to f730 . these results taken together provide strong evidence for a direct hydrogen bond from y730 to nh2y731 and strongly suggest that the proton is directly transferred via colinear pcet , as was proposed by previous quantum chemical calculations . thus , the results support the current model for pcet ( figure 1 ) , that is , that y731 functions as a proton acceptor for the subsequent rt step to y730 . they reveal that in each case , the h bond must be directed almost perpendicular to the tyrosyl system to reproduce the size of the hyperfine tensors and the gx shift of each nh2y. the results together provide strong support for the stacking of y731/y730 in the active it should be pointed out that this stacking is observed in some , but not all of the resting2 structures ( yeast and nh2y structures show residue 731 in multiple conformations ) and that currently no atomic resolution structural data are available for the complex . the unusual y / y stacking and the implications of this design relative to other configurations for optimum efficiency of transfer has been studied by theorists . the work of kaila and hummer indicates that this configuration results in strong electronic coupling and adiabatic , colinear pcet , consistent with our data . finally , the x - ray structure of the resting states of the wt , nh2y730- and nh2y731-s and our previous dft optimized structure of nh2y730 and y730-2 , show that residue c439 is in hydrogen bond distance to y730 ( figure 1c ) . the present experiments with the double mutant nh2y730/c439a corroborate the assignment of c439 as a second moderate hydrogen bond partner of y730. thus , the most direct interpretation of our data combined with the calculated reduction potentials in ref ( 25 ) . is that residue y730 acts as the direct hydrogen bond acceptor for c439 . in principle it still remains possible that water does provide the h - bond , and that the c439a mutant disrupts the h - bonding pattern and eliminates it ; however , our experimental data provide no evidence for the participation of a water in this proton transfer step , in contrast to a recent proposal of bu et al . this group using dft and qm / mm calculations on several model systems , in which cys could transfer a proton to y including one with the real protein environment for c439 , y730 , suggested that the conserved water molecule ( h2o138 in their notation is wat1 here ) moves and inserts itself between these residues promoting a double proton - coupled electron transfer step . however , their calculated barrier for direct pcet between c439 and y730 of 60 kcal / mol is inconsistent with ours and siegbahn and co - workers calculations , which delivered a barrier of 89 kcal / mol . intriguingly , in one of their model systems with a cys and tyr located on different peptide chains mimicking the rnr configuration , the barrier for pcet is almost identical ( 9 kcal / mol ) to our data in the real system . the studies with nh2y731/y730f ( figure 3b ) failed to reveal a h bonding interaction with a second proton as observed with nh2y730-hsc439 . however , in the center of the h - endor spectrum , the broad matrix line could be indeed contributed by one or several weak h bonds , which are not resolved . nevertheless , the dft calculation revealed that r411 is capable of rearranging to form weak hydrogen bonds ( 2.1 ) if it is not sterically hindered by water molecules , and that this configuration , along with the strong h bond to y730 , can reproduce the observed gx value . however , a model with two waters ( figure 5 ) , also is capable of recreating the experimentally measured gx value . the absence of structural insight about the / subunit interface and the ability to use the protein environment ( r411 or two waters ) to subtly alter the electrostatic environment , suggests multiple factors contribute to gx in addition to the strong h bond and our methods can not currently distinguish between them . perhaps the most unexpected observation from the current studies are the results with nh2y356-2 . in contrast with the pathway residues in 2 , no exchangeable moderate h bonds to nh2y356 are observed outside the matrix line , yet the gx value is perturbed to a similar extent as those for the nh2ys in 2 . our inability to observe crystallographically y356 within 2 alone or in 22 prevents any specific conclusions about the origin of the gx shift . however , the data still contribute to our understanding of pcet at this position . for example , one possible mode of communication between y356 and y731 could involve stacking similar to that observed between y731 and y730 . this type of interaction is unlikely , however , given the absence of any moderate h bond . a second possible model of communication for pcet between y356 and y731 , could involve a water molecule or network of water molecules at the subunit interface . the amino protons of nh2y356 would likely obscure these weak h bonds as they are adjacent to the matrix line , precluding their detection . the greatly perturbed gx value of nh2y356 in the absence of strong or moderate h bonds requires an altered electrostatic environment provided by the protein . whether this environment could be provided by water clusters at the interface or perhaps by binding of mg , long been known to play an important , but still poorly defined role in / , / and structure / chemistry , potentially in the interface region of the active complex , requires further analysis . in conclusion , the present data establish a hydrogen bond network between residues 731730439 in the 2 subunit . the observed hydrogen bonds and directions provide very strong support for a colinear pcet mechanism , consistent with the recent finding that the turnover rate constants within are very fast , > 14000 s , when conformational gating from is removed by photoinitation . these results differ dramatically from the pcet process at the / interface and within . our data indicate that colinear pcet and stacking between y356 and y731 are unlikely , and also reveal the importance of the electrostatic protein environment . additional spectroscopic experiments could be informative , but structural insight is also essential . within 2 the combination of protein engineering , spectroscopic data , and quantum chemical calculations has provided much insight into the pcet process within this subunit . nature appears to have utilized multiple pcet strategies to achieve this long - range oxidation over 35 . adenosine-5-triphosphate ( atp ) , cytidine-5-diphosphate ( cdp ) , reduced -nicotinamide adenine dinucleotide phosphate ( nadph ) , hydroxyurea ( hu ) , kanamycin ( km ) , chloramphenicol ( cm ) , 2xyt media , m9 minimal salts , l - arabinose ( ara ) , -mercaptoethanol ( -me ) , streptomycin sulfate and nh2y were purchased from sigma - aldrich . isopropyl--d - thiogalactopyranoside ( iptg ) and 1,4-dithiothreotol ( dtt ) were purchased from promega . nucleotide primers were purchased from invitrogen and pfu ultra ii polymerase was purchased from stratagene . ( his)6-wt-2 ( 2200 nmol / min / mg ) and wt-2 ( 7000 nmol / min / mg and 1.2 y /2 were expressed and purified by standard protocols . all 2 mutants were prereduced with 30 mm dtt and 15 mm hu before use.e . coli thioredoxin ( tr , 40 u / mg ) and thioredoxin reductase ( trr , 1800 u / mg ) used in assays were isolated as previously described . ( his)6-nh2y730-2 and ( his)6-nh2y731-2 were purified as previously described . the quikchange kit ( stratagene ) the template pet - nrda with the appropriate stop codon was amplified with primers 1 , 2 , and 3 and their reverse complements were used to insert a ttt ( phe ) at position 730 ; gcc ( ala ) at position 439 and agc ( ser ) at position 439 ( table 3 ) . all constructs contain an n - terminal ( his)6-tag with a 10 amino acid linker , as described previously . expression and purification of nh2y730-2 followed previous protocols except that the purification buffer ( 50 mm tris , 5% glycerol , 1 mm pmsf , ph 7.6 ) contained 1 mm tcep . -nh2y730 , -nh2y731 , -nh2y356 and their double mutants were combined with the corresponding wt( / ) 1:1 at final complex concentrations of 100200 m in d2o and h2o assay buffer as previously described . the reaction was initiated at 25 c by adding cdp and atp with final concentrations of 2 and 6 mm , respectively , and manually freeze quenched after 1020 s inside the epr tube with liquid n2 . echo - detected epr spectra at 263 ghz were recorded on a bruker elexsys e780 quasi optical spectrometer using a single mode ( te011 ) cylindrical resonator ( bruker biospin ) with a typical quality factor of 5001000 . maximum microwave power coupled to the resonator was about 15 mw . the electron spin echo ( /2echo ) the epr spectrum of y122 in wt- was used as a reference ( figure s1 ) at 250 m concentration in assay buffer to calibrate magnetic field . samples for 263 ghz epr were inserted in capillaries ( 0.33 od , vitrocom cv2033s / q ) in typical volumes of 50 nl . samples for 94 ghz spectroscopy contained typical volumes of 2 l in 0.9 mm od capillaries . samples frozen in liquid nitrogen were loaded into the resonator immersed in liquid nitrogen and then transferred into the precooled epr cryostat . 94 ghz pulse epr and endor spectra were recorded on a bruker e680 w - band spectrometer with 400 mw microwave output power ( bruker power upgrade 2 ) . 94 ghz h mims endor ( /2/2rf/2 echo ) was carried out using random radio frequency irradiation and 40 s rf pulses produced with a 250 w rf - amplifier ( 250a250a , amplifier research ) . all derivatives were obtained by fitting every four points with a second order polynomial and differentiating the function in matlab ( version 7.10 ) . the line width was set to 3 g with a line broadening contribution of a 1:1 gaussian to lorenzian . 94-ghz h endor spectra were simulated by using a matlab routine developed in house that is based on a first order hamiltonian ( high field condition ) for the hyperfine and quadrupolar interaction . the blind spots produced by the mims endor sequence were included by multiplying the calculated endor powder pattern with an envelope function given by iendor = 1 cos(2a ) . this is valid for the i = 1 nuclei considered here , as all quadrupole couplings are much smaller than the hyperfine values . the initial model structures were based on the large models 7 and 8 used in ref ( 25 ) augmented by the amino group at y731 . geometry optimizations have been performed using the bp86 gradient corrected density functional in combination with ahlrichs tzvp basis set of triple- quality . the resolution of the identity approximation with the corresponding auxiliary basis sets has been employed throughout . cartesian constraints were imposed on the position c of y730 , y731 and c439 as well as c and c of all surrounding residues . additionally the cartesian coordinates of the hydrogen atoms in the truncated gpd model replacing the bonds between c4 and c5 of the ribose as well as the bond between c1 of the ribose and the base were kept fixed . the epr calculations and geometry optimization of the small models were carried out with the b3lyp hybrid density functional in combination with the rijcosx approximation . here only the dihedral angle of the peptide bond of y730 and y731 was fixed and the cartesian restrains for all surrounding c s were kept . in the small models a solvation model ( cosmo ) with polarity = 24 was used to compensate protein influences . for all dft / epr calculations , barone s epr - ii ( iglo - ii for sulfur ) basis set of double- quality has been used in combination with the def2-tzvpp / jk auxiliary basis set for all atoms .

ribonucleotide reductases ( rnrs ) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all organisms . in all class ia rnrs , initiation of nucleotide diphosphate ( ndp ) reduction requires a reversible oxidation over 35 by a tyrosyl radical ( y122 , escherichia coli ) in subunit of a cysteine ( c439 ) in the active site of subunit . this radical transfer ( rt ) occurs by a specific pathway involving redox active tyrosines ( y122 y356 in to y731 y730 c439 in ) ; each oxidation necessitates loss of a proton coupled to loss of an electron ( pcet ) . to study these steps , 3-aminotyrosine was site - specifically incorporated in place of y356- , y731- and y730- , and each protein was incubated with the appropriate second subunit ( ) , cdp and effector atp to trap an amino tyrosyl radical ( nh2y ) in the active 22 complex . high - frequency ( 263 ghz ) pulse electron paramagnetic resonance ( epr ) of the nh2ys reported the gx values with unprecedented resolution and revealed strong electrostatic effects caused by the protein environment . 2h electron nuclear double resonance ( endor ) spectroscopy accompanied by quantum chemical calculations provided spectroscopic evidence for hydrogen bond interactions at the radical sites , i.e. , two exchangeable h bonds to nh2y730 , one to nh2y731 and none to nh2y356. similar experiments with double mutants -nh2y730/c439a and -nh2y731/y730f allowed assignment of the h bonding partner(s ) to a pathway residue(s ) providing direct evidence for colinear pcet within . the implications of these observations for the pcet process within and at the interface are discussed .

Introduction Results Discussion Conclusion Methods

ribonucleotide reductases ( rnrs ) catalyze the conversion of four nucleotides ( cdp , udp , adp , gdp ; ndps ) into the corresponding deoxynucleotides ( dndps ) . class ia rnrs are found in nearly all eukaryotic and some prokaryotic organisms and are composed of two homodimeric subunits , 2 and 2 , which form an active , transient 22 complex . subunit 2 houses the catalytic substrate binding site and the binding sites for the allosteric effectors that govern specificity and activity of nucleotide diphosphate ( ndp ) reduction . during each turnover , the fe2-y-2 oxidizes c439 in the active site of 2 where dndp is produced , and then it is subsequently reoxidized . the oxidation occurs via a radical hopping mechanism over 35 between the two subunits along a specific pathway comprised of redox active amino acids ( y122 [ w48 ? ] y356 in 2 y731 y730 c439 in 2 ) . reversible oxidation and reduction of y during turnover requires release of the phenolic proton to an acceptor concomitant with the oxidation ( figure 1a ) , a mechanistic strategy to avoid formation of high - energy intermediates . while the long - range oxidation through aromatic amino acid residues in ribonucleotide reductase ( rnr ) is unprecedented in biology , proton - coupled electron transfer ( pcet ) mechanisms are involved in many fundamental processes in biology including photosynthesis , respiration and nitrogen fixation . investigation of the electron and proton pathways has required rnrs with site - specifically incorporated unnatural amino acids and electron paramagnetic resonance ( epr ) combined with electron nuclear double resonance ( endor ) spectroscopies have provided a unique opportunity to access mechanistic details if the intermediate radicals can be detected . recent biophysical studies including pulsed electron electron double resonance ( peldor ) studies and small - angle x - ray scattering studies ( saxs ) and cryoem have established that the docking model is a reasonable representation of the active rnr structure in solution and that the oxidation occurs over > 35 . unfortunately there is little information about the molecular details of the interface between and , more specifically , the communication between y356 in 2 and y731 in 2 . studies of this unprecedented oxidation catalyzed by wt rnr have not been possible as protein conformational change(s ) constitute the rate - limiting step(s ) and kinetically mask all of the detailed chemistry of the radical transfer ( rt ) and the nucleotide reduction step . however , use of technology in which the pathway tyrosines are replaced site - specifically with tyrosine analogues with altered pkas and reduction potentials , and the development of high frequency ( hf ) epr and endor methods to characterize y analogues generated with the mutant rnrs , are now unmasking the chemistry of this long - range oxidation . however , there is sufficient concentration of the most uphill radical ( substrate radical ) to drive the reaction to the right by a rapid , irreversible step proposed to be the loss of water during nucleotide reduction . the results from the incorporation of nh2y site - specifically at 356- , 731- and 730- , the focus of this paper , have played a critical role in our current understanding of specific steps of the rt pathway and the conformational gating process . in all three cases , nh2y-( ) is generated in 3040% yield upon incubation of the second subunit ( ) , substrate cdp and allosteric effector atp . using the same experimental design , analysis of the nh2y by hf epr and deuterium ( h ) endor provided the first spectroscopic insight suggesting the importance of colinear pcet within 2 and the structural relationship of y731 and y730 in the active 22 rnr complex . the link between epr data in the active intermediate state and the x - ray structural data in the inactive state was supported by quantum chemical calculations of the energy optimized structural models of y730- and nh2y730- including 211atoms ( figure 1c ) . in the current paper , experiments in which nh2y has been site specifically incorporated at nh2y356- , and at nh2y731 and nh2y730- ( figure 1c ) in conjunction with hf epr and hf h endor spectroscopies , density functional theory ( dft ) calculations using nh2y731(730)- structures , have been employed in an effort to understand the electrostatic and h bonding environment at these residues in the rt pathway . hf epr data were recorded at a microwave frequency of 263 ghz , which delivers unprecedented resolution in the spectra of the nh2ys . in addition , double mutants nh2y730/c439a- and nh2y731/y730f- were prepared and the h endor results compared to the single mutant results in an effort to assign the source of the h bonds observed to specific residues . our results support the colinear concerted pcet model within and provide spectroscopic evidence for the unusual stacking of y730/y731 in the active 22 complex . to probe the electrostatic environment of the radical intermediates at the three different positions in the rt pathway we examined the hf epr spectra formed after incubation of -nh2y356 , -nh2y731 , -nh2y730 with their respective complementary wt subunits , either or , cdp , and atp ( methods ) . this shift is significant , on the order of the effect predicted from several hydrogen bonds.g values were calibrated with the spectrum of the stable radical y122 in 2 , which is present in all samples and visible only at low temperatures . additionally , gx slightly decreases starting from the most buried intermediate nh2y730 ( gx = 2.0054 ) to nh2y356 ( gx = 2.0049 ) located at the subunit interface . simulations of the 263 ghz spectra combined with 94 ghz spectra ( the latter frequency was used to constrain the simulation , figure s2 ) led to a consistent set of g values and c- proton hyperfine ( hf ) couplings ( see chemical structure in figure 1 inset ) that are summarized in table 1 . thus , the epr spectra of the single mutants in the active enzyme are contributed by a radical species ( per mutant ) with a well - defined microenvironment ( g value ) and molecular orientation . this finding underlines the importance of a specific structural arrangement in the active state to permit radical propagation . the observed shift of gx values is a hallmark for a substantial effect of either positive charges and/or hydrogen bond interactions . echo sequence used to suppress the signal of y122 ( box ) is based on differences in the transverse relaxation times ( t2 ) of the radicals : nh2ys and y122. ese detected epr spectra of the nh2ys radicals in h / d exchanged buffer : nd2y730 ( black ) , nd2y731 ( red ) and nd2y356 ( blue ) . endor reveals the spectrum of magnetic nuclei that are in the coordination sphere ( usually 5 ) of the observed radical . after buffer exchange , exchangeable protons are substituted by deuterons ( abbreviated d , with the nucleus denoted h ) , which become visible by endor in the h resonance region . additionally , two samples , nd2y730 and nd2y731 , show pairs of sharp peaks in the region 1 mhz that is usually dominated by deuterons in the hydrogen bond range ( figure 3a , peaks marked in red and blue ) . endor spectrum is centered at the larmor frequency 0 of h , i.e. , 21.9 mhz at a field of 3.3 t. further experimental evidence for the hydrogen bond at nh2y731 was derived from orientation selective hf h endor spectra recorded at different field positions in the epr line . powder patterns are still observed at orientations b0 gx and gz as orientation selection is moderate for the large excitation bandwidth of the pulses ( 1.8 mt ) as compared to the total epr line width ( 8 mt ) ; however , clear differences in the line shapes are visible . considering that the gx tensor component lies along the c o bond and gy points to the side of the amino group , the euler angles , , between hyperfine a and g tensor obtained from the simulation ( table 2 ) are consistent with a hydrogen bond directed almost perpendicular to the plane of the nh2y731 . simulations of the entire h endor spectrum with the parameters obtained here additionally indicate that the spectrum of a nd2y731 can be well reproduced by the contribution of a single hydrogen bond ( figure 3a ) according to the observed intensity ratio of the sharp peaks with respect to the amino deuteron resonances . h 94-ghz mims endor spectra ( black lines ) were recorded at field positions in the epr line parallel to the canonical orientations of the g tensor , i.e. in an effort to assign the h bonding interactions observed with nh2y731- and nh2y730-2 by h endor , double mutants nh2y731/y730f and nh2y730/c439a were generated , in which one of the proposed h bonds was removed . the proteins were expressed , purified to homogeneity and characterized by stopped flow - vis spectroscopy monitoring the rate of loss of y122 ( 410 nm ) and rate of formation of the nh2y ( 320 or 325 nm ) in the presence of wt-2/cdp / atp , figure s3 . in the case of both double mutants , the altered kinetics of pathway radical formation and its altered amplitude in the case of nh2y730/c439a-2 are likely reflective of the importance of these h bonding interactions in tuning of pcet within 2 . the experiments revealed distances of 3.8 and 3.9 nm , respectively , consistent within error ( 1 ) with the distances observed in the single mutants and from the 22 docking model in wt ; however , the assignment of the 3.9 nm distance in the mutant nh2y730/c439a is more uncertain due to the low radical yield and s / n ratio of the peldor traces hf epr spectra ( 263 ghz ) of nh2y trapped in the double mutants also revealed gx values ( figure s7 ) different from those in the single mutants , i.e. these results are consistent with the assignment of one h bond to nh2y731 associated with y730 and one hydrogen bond to nh2y730 associated with c439 . we note also that the removal of the hydrogen bond is manifested in the hf coupling of the amino deuterons , which slightly decreases as compared to the single mutants due to a change in the spin density distribution on nd2y. to obtain a model structure of the radical localized at nh2y731 , dft calculations on representative model systems for nh2y731 were performed and the obtained magnetic parameters for gx and for h bonds were compared with the experimental values . in model 3 a second water molecule ( wat2 ) was included as observed in some x - ray structures of wt- and nh2y731/730- ( figure s9 ) in the vicinity ( ro o 2.63.6 ) of residue 731 . the optimized structure of model 3 finds wat2 within hydrogen bond distance ( ro h 1.9 ) to nh2y731 ; however , a stronger h bond to the phenoxyl hydrogen of y730 remains ( ro h 1.6 ) . the gx value for the model 3 structure is consistent with the experimental value ( table 1 ) . particularly , all models display an orientation of the strong h bond to nh2y731 provided by y730 , which is compatible with the endor data within an uncertainty of about 20% ( table 2 , figure s10 ) . considering the accuracy of the calculations , on the order of 0.5 ppt , all three models give gx values in principle compatible with the experiment . the results indicate that the strong gx shift at nh2y731 can be contributed by the combined effect of a strong h bond to y730 and the interaction either to a water molecule ( model 3 ) or alternatively to the positively charged arginine r411 ( model 2 ) . the electronic and structural features of the nh2ys trapped in the rt pathway of e. coli rnr report on how the local protein environment has reorganized after a pcet step to stabilize these intermediates . at each radical state , the protein has rearranged to accommodate a released electron and a proton , a scenario that can be probed by two epr parameters : the shift in the gx value caused by positive charges and the hyperfine interaction of protons forming hydrogen bonds of varying strengths . the first parameter is directly accessible from high - frequency epr spectra but contains only indirect information on hydrogen bond interactions . all gx values of the nh2ys in the three pathway positions are similar and show a considerable shift from the calculated value for a free nh2y ( table 1 ) , consistent with a strongly perturbed electrostatic environment . the direction of the hydrogen bond to nh2y731 extracted from the present hyperfine and quadrupole tensors in the endor data ( figure 4 ) are consistent with the directions predicted by the dft calculations for a bond with y730 ( figure 5 ) no matter the number of water molecules considered in the calculation . these results taken together provide strong evidence for a direct hydrogen bond from y730 to nh2y731 and strongly suggest that the proton is directly transferred via colinear pcet , as was proposed by previous quantum chemical calculations . thus , the results support the current model for pcet ( figure 1 ) , that is , that y731 functions as a proton acceptor for the subsequent rt step to y730 . they reveal that in each case , the h bond must be directed almost perpendicular to the tyrosyl system to reproduce the size of the hyperfine tensors and the gx shift of each nh2y. the results together provide strong support for the stacking of y731/y730 in the active it should be pointed out that this stacking is observed in some , but not all of the resting2 structures ( yeast and nh2y structures show residue 731 in multiple conformations ) and that currently no atomic resolution structural data are available for the complex . the present experiments with the double mutant nh2y730/c439a corroborate the assignment of c439 as a second moderate hydrogen bond partner of y730. in principle it still remains possible that water does provide the h - bond , and that the c439a mutant disrupts the h - bonding pattern and eliminates it ; however , our experimental data provide no evidence for the participation of a water in this proton transfer step , in contrast to a recent proposal of bu et al . this group using dft and qm / mm calculations on several model systems , in which cys could transfer a proton to y including one with the real protein environment for c439 , y730 , suggested that the conserved water molecule ( h2o138 in their notation is wat1 here ) moves and inserts itself between these residues promoting a double proton - coupled electron transfer step . however , in the center of the h - endor spectrum , the broad matrix line could be indeed contributed by one or several weak h bonds , which are not resolved . the absence of structural insight about the / subunit interface and the ability to use the protein environment ( r411 or two waters ) to subtly alter the electrostatic environment , suggests multiple factors contribute to gx in addition to the strong h bond and our methods can not currently distinguish between them . in contrast with the pathway residues in 2 , no exchangeable moderate h bonds to nh2y356 are observed outside the matrix line , yet the gx value is perturbed to a similar extent as those for the nh2ys in 2 . the greatly perturbed gx value of nh2y356 in the absence of strong or moderate h bonds requires an altered electrostatic environment provided by the protein . whether this environment could be provided by water clusters at the interface or perhaps by binding of mg , long been known to play an important , but still poorly defined role in / , / and structure / chemistry , potentially in the interface region of the active complex , requires further analysis . our data indicate that colinear pcet and stacking between y356 and y731 are unlikely , and also reveal the importance of the electrostatic protein environment . within 2 the combination of protein engineering , spectroscopic data , and quantum chemical calculations has provided much insight into the pcet process within this subunit . the quikchange kit ( stratagene ) the template pet - nrda with the appropriate stop codon was amplified with primers 1 , 2 , and 3 and their reverse complements were used to insert a ttt ( phe ) at position 730 ; gcc ( ala ) at position 439 and agc ( ser ) at position 439 ( table 3 ) .

mammals express three aurora kinases , aurora a , aurora b , and aurora c , encoded by the genes aurka , aurkb , and aurkc . aurora c expression is mainly restricted to testis , where it functions during meiosis , whereas aurora a and b play key functions regulating mitosis . aurora a is localized to the centrosome and spindle poles , where it drives centrosome maturation , separation , and bipolar spindle assembly . aurora b is the central component of the chromosomal passenger complex ( cpc ) that also contains the inner centromere protein ( incenp ) , borealin , and survivin . these nonenzymatic components of the cpc play a key role in regulating kinase activity and localization . the aurora kinases are related to the agc ( protein kinase a , protein kinase g , and protein kinase c ) branch of protein kinases . part of the activation mechanism for most agc kinases is mediated by binding of the phosphorylated c - terminal hydrophobic motif , fxxf(t / s)f , to the n - terminal lobe of the kinase catalytic domain . however , aurora kinases lack canonical c - terminal hydrophobic motifs and are activated in trans through binding of regulatory proteins to their n - terminal lobe . c , the c - terminal section of the incenp protein ( the in - box ) performs a similar regulatory function . the structure of human aurora a has been determined both without tpx2 and in complex with tpx2 . the structure of aurora b has previously been determined from xenopus laevis , in complex with incenp and the aurora b inhibitor hesperadin as well as other inhibitors . human aurora b is 80% identical to x. laevis aurora b over the kinase domain ( 55344 ) and 72% identical to human aurora a over the kinase domain . human incenp is 50% identical to x. laevis incenp over the region 835903 that is involved in binding to aurora b. aurora a phosphorylated on the activation loop in the absence of tpx2 has been observed in both active and inactive conformations . aurora a has been cocrystallized together with 1 ( vx-680 , figure 1 ) in the presence of tpx2 , which caused binding to an active kinase conformation , and also in the absence of tpx2 , which caused 1 to bind to an inactive conformation of the kinase domain , although this latter structure is not yet available in the protein data bank . aurora b is activated by phosphorylation of both aurora b and incenp . first , phosphorylation of incenp at the tss motif of the c - terminal in - box region , which can be performed by aurora b itself , is important for full activity . second , autophosphorylation on the activation loop of aurora b ( for human aurora b at thr232 ) produces the active kinase . a recent study showed that phosphorylation on ser331 of aurora b by chk1 was essential for phosphorylation of the tss motif of incenp and also for full activation of aurora b. although most genetic alterations identified in cancer involve the aurora a locus , high expression levels of aurora b have been detected and associated with poor prognosis in glioblastoma , ovarian carcinoma , and hepatocellular carcinoma . inhibition of aurora b leads to failure to biorientate chromosomes and massive polyploidization . as a result of the induced chromosomal defects , both p53-proficient and p53-deficient cells aurora b has therefore become an extensively pursued anticancer target , and many aurora inhibitors have been developed , including recently some isoform - specific inhibitors . the aurora kinase inhibitors ( second generation type 2 inhibitors ) such as 1(23 ) have considerable antitumor activity . compound 1 also inhibits some other kinases , notably the tyrosine kinase abl , and crystal structures have been solved for 1 bound to abl1 and abl2 . to provide a model for structure - based design and to gain insight into the molecular mechanism of human aurora b activation , we determined the structure of human aurora b kinase domain , in complex with the c - terminal in - box section of human incenp ( residues 835903 ) and the type ii inhibitor 1 ( figure 1 ) . we began structural studies on human aurora b by preparing a set of constructs spanning different ranges of the gene , all of which included the kinase domain , for overexpression in escherichia coli . from this set , three constructs ( 55344 , 65344 , and 70344 ) expressed large amounts of soluble protein . therefore , a bacterial expression construct for the c - terminal part of human incenp was prepared , spanning residues 835903 , which is the aurora - binding region . this expression construct contained a glutathione - s - transferase purification tag to enable sequential affinity purification of the heterodimeric aurora b : incenp complex by sequential ni - affinity followed by glutathione sepharose affinity chromatography . the two proteins were expressed separately , and the cells were mixed before lysis and subsequent protein purification . the aurora b protein was monophosphorylated according to electrospray ionization mass spectrometry ( data not shown ) . using this purified protein , we were able to cocrystallize the aurora b incenp heterodimer in complex with the type ii inhibitor 1 and determine the structure to a resolution of 2.75 ( table 1 ) . values in parentheses are for the highest resolution shell . as expected from the primary sequence similarities , the overall structure of human aurora b resembles the structures of x. laevis aurora b and human aurora a , with the exception of the conformation of the activation loop ( figure 2 ) . unexpectedly , a domain swap was observed ; part of the kinase activation loop was exchanged between two aurora b molecules in the crystal lattice , resulting in similar binding of the activation loop against the c - terminal lobe of the kinase but different conformations around the dfg ( asp - phe - gly ) motif at the start of the activation loop ( figure 3a ) . although this is a feature that has been observed before in kinase crystal structures , to our knowledge , this is the first time it has been observed in an aurora kinase structure . activation loop exchange has been suggested as an activation mechanism for kinases that autoactivate on nonconsensus binding sites in their activation segment . in the aurora b : incenp complex , the activation segment is disordered after the dfg motif , including the phosphorylation site thr232 . the c - terminal part of the activation segment is dislodged from the lower lobe and interacts with a deep groove generated by the displaced activation segment in the interacting protomer . overall , there is a buried surface area of interaction of 3460 , and the segment is anchored mainly by aromatic and hydrophobic interactions of y239 , l237 , pro242 , and i245 ( figure 3c , d ) . overview of human aurora b kinase as compared to x. laevis aurora b and human aurora a. ( a ) human aurora b with incenp colored green and the activation loop colored red . ( b ) x. laevis aurora b complex with hesperadin ( pdb i d : 2bfy ) , with incenp colored green and the activation loop colored red . ( c ) human aurora a complex with 1 ( pdb i d : 3e5a ) , with tpx2 colored green and the activation loop colored red . ( a ) the two aurora b molecules in the dimer are colored in yellow and blue , incenp is green , and the activation loops are colored red with dashed lines to indicate where the disordered residues would form a connection . ( b ) the electron density at the cross - over point for the domain swap . the dashed lines in red indicate the connections that would be made between met249 and his250 in the absence of a domain swap . ( c ) as in panel a but with one aurora b : incenp molecule displayed as a surface , showing the binding of the activation loop in the dimeric arrangement . the electron density unambiguously favored the loop - exchanged arrangement over the alternative model of a conventional arrangement ( figure 3b ) . the residues at the point of the domain swap form more favorable interactions than they would have in the case of a nonexchanged arrangement ( figure 3b ) . in particular , there is a favorable hydrophobic packing of the side chain of met249 with met244 and a favorable interaction of his250 with his198 . met249 , which is important for the formation of the domain - swapped form , is not conserved in x. laevis aurora b , which has thr265 instead . apart from one conservative substitution of an arginine for a lysine , this is the only difference in the activation loop between the human and x. laevis enzymes , which is one of the most highly conserved regions in the whole protein . given the very favorable nature of the hydrophobic packing of the met249 side chain , it may be that this single amino acid difference is responsible for the observation of a domain swapped dimeric form of aurora b only for crystals of the human orthologue . in this domain - swapped arrangement , part of the activation loop packs against the outside of helix c , which is important for the activity and regulation of protein kinases . whether this arrangement has any effect on the activity or regulation of human aurora b kinase is an open question , but it may be partly responsible for the outward movement of c relative to the structures of x. laevis aurora b with 1 and abl1/2 with 1 ( discussed below ) . after observing the activation loop in the domain swapped conformation , to see if the dimeric state was also observed in solution , we analyzed the oligomeric state of the complex by analytical ultracentrifugation ( auc ) . a sedimentation velocity experiment showed the presence of both monomer and dimer , with the majority of the sample being dimeric ( figure 4 ) . as the monomer and dimer had different frictional ratios ( f / f0 ) , the data were modeled as a dual distribution of s and f / f0 . the sedimentation velocity analysis gave molecular weights of 43307 and 81882 for the two species present , which compared well with the expected values of 41849 and 83680 for monomer and dimer based on the protein sequences . the frictional ratio for the dimeric species was higher at 2.09 , as compared to 1.94 for the monomeric species , corresponding to a less spherical dimeric arrangement . integration of the distribution resulted in values of 54% dimer and 31% monomer , plus 11% of a species of approximately 49 kda with a significantly lower frictional ratio than the peaks assigned as monomer and dimer . sedimentation velocity analysis of aurorab : incenp , showing a 2d plot of f / f0 on the y - axis and s on the x - axis . the smaller and larger peaks correspond to monomeric and dimeric aurorab : incenp , respectively . peak height is indicated by color on a scale from blue to red ( strongest ) . the c - terminal part of incenp , the in - box , wraps around the n - terminal lobe of the aurora b kinase domain in a manner similar to that seen previously with the x. laevis orthologue . the two proteins are 50% identical over the region used in the construct for crystallization ( 835903 ) ( figure 5a ) . however , although the binding of the n - terminal part of the n - box ( residues 842863 , site 1 ) closely resembles that of the x. laevis aurora b incenp structure ( figure 5b ) , the c - terminal part ( residues 868881 , site 2 ) is mostly different , with a substantial variation in the backbone conformation of the incenp residues at site 2 ( figure 5c ) . at site 1 , the incenp backbone adopts the same -helical arrangement , with the only significant differences in binding being for the nonconserved residues ile856 of incenp and leu210 of aurora b , which are arg812 and tyr228 in the x. laevis orthologue . the human complex therefore has a hydrophobic interaction at this point in place of the hydrogen bond between the tyr and the arg side chains in x. laevis . ( a ) sequence alignment of human and x. laevis incenp over the c - terminal in - box region . ( b ) comparison of human and x. laevis aurora b : incenp binding at site 1 ( residues 842863 of human incenp ) showing the mostly conserved interactions , with two exceptions highlighted . the x. laevis aurora b and incenp are colored blue and purple , respectively ( x. laevis coordinates were taken from pdb i d 2bfx(8 ) ) . ( c ) comparison of human and x. laevis aurora b : incenp binding at site 2 ( residues 868881 of human incenp ) showing the mostly nonconserved binding conformation , with the exception of the terminal phe881 . ( d ) binding detail for site 2 of human aurora b : incenp . at site 2 , the incenp backbone adopts a different conformation with only the binding of the very c - terminal phe881 conserved ( figure 5c ) . furthermore , the aurora c helix is displaced relative to the rest of the aurora structure . the binding is largely composed of hydrophobic interactions , particularly those around incenp leu875 and leu877 ( figure 5d ) . there is also a hydrogen bond between the aurora b tyr145 ( a histidine in x. laevis aurora b ) and the incenp backbone ( figure 5d ) . compound 1 binds to human aurora b in a similar conformation to that seen in complexes with other kinases . a structure of x. laevis aurora b bound to 1 is not available , so the structural comparisons of 1 binding considered here are restricted to the published structures of aurora a:1 ( pdb i d : 3e5a ) and abl2:1 ( pdb i d : 2xyn ) . a structure of an imatinib - resistant mutant abl1 in complex with 1 is also available . the glycine - rich loop of aurora b is in a different conformation to that of aurora a in the region around phe88 ( figure 6a , b ) . it adopts a conformation similar to the glycine - rich loop of abl2 when bound to 1 ( figure 6a , c ) , although abl2 has tyr299 in place of phe88 of aurora b. however , the most significant difference between aurora a and aurora b when bound to 1 is in the arrangement of the dfg motif . in aurora a , the dfg motif is arranged with the phe underneath the c helix , while in aurora b , the c helix is moved further out from the atp site and the dfg phe is to the side of the helix with the asp not well - ordered in the structure ( figure 6d ) . as abl1/2 also have a similar arrangement of the dfg phe and c as aurora a , aurora b differs from all of these when bound to 1 . the movement out of c allows more positional options for the aurora b dfg motif and also a potentially bigger back pocket for inhibitor binding than in aurora a , although due to the placement of the dfg phe the pocket in this particular structure is slightly smaller . comparison of binding to 1 by aurora b , aurora a , and abl2 . ( d ) overlay of the dfg motifs of the complexes with 1 of aurora b ( in yellow ) and aurora a ( in pink ) . ( e ) overlay of the dfg motifs of the complexes with 1 of aurora b ( in yellow ) and abl2 ( in blue ) . comparison of the ic50 values for inhibition x. laevis aurora b by three common aurora kinase inhibitors with the previously published ic50 values for inhibition of human aurora b by the same three inhibitors shows that aurora b from both species behaves almost identically ( table 2 ) . aurora kinases have been pursued as drug targets for some time , particularly in oncology . x - ray crystal structures of aurora a and aurora b have been used extensively to advise medicinal chemistry efforts , either through ligand - bound structures or through their use in molecular modeling . until now , there has not been a structure of human aurora b kinase in the public domain , and the x. laevis orthologue has been used instead . it has been predicted previously that the binding site of aurora a : tpx2 has a smaller hydrophobic back pocket than that of aurora b : incenp . efforts to develop aurora isoform - specific inhibitors have naturally focused on this region as well as on the differences at the entrance to the atp binding site . the aurora b structure presented here allows direct comparison with an aurora a structure with the same inhibitor and confirms that the hydrophobic back pocket of aurora b is indeed larger but with the caveat that the more outward position of c allows other conformations of the dfg motif , which may favor or disfavor particular inhibitors . the more outward position of c is so far unique to human aurora b ; all of the x. laevis aurora b cocrystal structures so far deposited in the pdb have a similar c position , even with the inhibitor zm447439 , which binds much further into the hydrophobic pocket than 1 ( pdb i d : 2vrx(9 ) ) , although it should be noted that the structure with zm447439 was obtained by soaking aurora b : incenp crystals with the compound , and a different structure could possibly have arisen by cocrystallization . for aurora a in the absence of tpx2 , even further outward positions of c have been observed , both with type ii compounds that extend significantly into the hydrophobic pocket [ e.g. , pdb i d : 3dj6 ( mouse aurora a ) ] and with compounds that do not ( e.g. , pdb i d : 2wtv or 3unz ) . in both of the structures of aurora a in complex with tpx2 ( which binds on the outside of c ) , c adopts an inward position . the dimerization of aurora b : incenp that we have observed both in the crystal structure and in solution is probably the most significant factor in the different dfg motif arrangement of this complex with 1 as compared to those of aurora a or abl . the domain swap involves a very different conformation of the activation loop , which is most likely the cause of the different dfg position . it is unclear to what extent different inhibitors might promote or hinder dimerization . the auc experiments showed dimerization in the absence of an inhibitor , so compound 1 was not the cause of the observed dimerization in the crystal structure . however , because the dimerization interface involves c and is dependent on the activation loop , an inhibitor that promotes a substantially different c position or dfg position could conceivably promote or hinder dimerization . the variation of incenp binding between the human and the x. laevis orthologues at the second site next to c is probably a feature of the irregular backbone conformation . by contrast , at the first binding site , incenp has an invariant -helical backbone conformation , which inevitably leads to a more conserved binding arrangement . whether incenp can adopt different conformations at the second site to accommodate movements of c during activation ( or inhibition ) of aurora b is a question that has to be explored in the future . dna for residues 55344 of the kinase domain of human aurkb ( gi|83776600 ) was pcr amplified from dna in the mammalian gene collection ( image i d 2819846 ) and inserted into vector pnic28-bsa4 ( which carries kanamycin resistance ) in - frame with an n - terminal hexahistidine purification tag and tev protease cleavage site , by ligation - independent cloning . dna for residues 835903 of human incenp ( gi|102467235 , inner centromere protein isoform 1 ) was pcr amplified from incenp cdna and inserted into vector pgtvl1-sgc ( which carries ampicillin resistance ) in - frame with an n - terminal glutathione s - transferase purification tag and tev protease cleavage site , also by ligation - independent cloning . these plasmids were each transformed separately into chemically competent e. coli bl21 ( de3 ) containing a plasmid for expression of rare trnas . colonies from the transformation were used to inoculate lb media containing 34 g / ml chloramphenicol and either 100 g / ml ampicillin ( for incenp ) or 50 g / ml kanamycin ( for aurora b ) . the cultures were grown overnight in baffled shaker flasks at 37 c with shaking . these cultures were used to inoculate tb media by adding 15 ml of culture to 1 l of tb ( containing either 80 g / ml ampicillin or 40 g / ml kanamycin ) in baffled shaker flasks . when the cultures had an od600 of approximately 1.2 , the temperature was reduced to 20 c . after a further 25 min , the cultures were left shaking at 20 c overnight before the cell pellets were harvested by centrifugation . the cells were resuspended in binding buffer ( 20 mm imidazole , 200 mm nacl , and 50 mm tris , ph 7.8 ) with the addition of 0.5 mm tris(2-carboxyethyl)phosphine ( tcep ) and protease inhibitor cocktail ( sigma ) . the resuspended cells were stored at 20 c . for protein purification , the resuspended cells for the aurora b and incenp expressions were combined and thawed together ( 4 l of culture of aurora b and 6 l of culture of incenp ) . the combined cells were lysed with a high - pressure homogenizer ( avestin ) . polyethyleneimine was added to a concentration of 0.15% , and the lysate was centrifuged at 38000 g for 20 min at 4 c . the supernatant was filtered and loaded onto 10 ml of nickel - chelating resin . the resin was washed with binding buffer and with binding buffer containing 40 mm imidazole . the protein was eluted with binding buffer containing 250 mm imidazole , and the eluate was loaded onto a column of 10 ml of glutathione sepharose 4b resin ( ge healthcare ) . this resin was washed with binding buffer before the protein was eluted with binding buffer containing 10 mm reduced glutathione . the hexahistidine and gst tags were removed by overnight treatment with tev protease at 4 c . the digested sample was concentrated to 4 ml volume and loaded onto a superdex75 gel filtration column ( hiload 16/60 , ge healthcare ) pre - equilibrated in gf buffer ( 20 mm hepes , ph 7.5 , 200 mm nacl , and 0.5 mm tcep ) . fractions from the gel filtration containing the desired aurorab : incenp complex were pooled and passed through a column of 1 ml of glutathione sepharose 4b and then loaded onto a column of 0.7 ml of nickel - chelating resin . this resin was eluted with 5 ml each of gf buffer containing 20 , 40 , 60 , 80 , 100 , and finally 120 mm imidazole . these fractions were pooled , compound 1 was added , and the sample was twice concentrated to 250 l and then diluted to 4 ml with gf buffer . finally , the sample was concentrated to 200 l volume at which the protein concentration was 6 mg / ml . the protein identities were verified by electrospray ionization time - of - flight mass spectrometry ( agilent lc / msd ) . the aurorab : incenp:1 complex ( pdb i d : 4af3 ) was crystallized at 20 c in 300 nl drops from a 2:1 ratio of aurorab : incenp:1 ( 6 mg / ml protein ) and reservoir solution ( 10% w / v peg3350 , 0.2 m kscn , 10% ethylene glycol , and 0.1 m bistrispropane , ph 6.15 ) . the crystals were cryoprotected in reservoir solution with 25% ( v / v ) ethylene glycol and flash - frozen in liquid nitrogen . x - ray diffraction data were collected at 100 k on beamline i04 at diamond . the diffraction images were processed using xds and the ccp4 suite of programs . the structure was solved by molecular replacement using phaser with the structure of x. laevis aurora b ( pdb i d : 2bfx ) as a search model . the structure was refined against maximum likelihood targets using restrained refinement and tls parameters , as implemented in the program refmac . positive difference electron density for the incenp protein component and the inhibitor 1 was visible in the initial maps . iterative rounds of refinement were interspersed with manual rebuilding of the model using coot . progress of the refinement was judged throughout by following a reduction in rfree ( calculated from a random 5% of the data that was excluded from the refinement ) . sedimentation velocity measurements were made at 40000 rpm with a beckman an50 ti rotor in a beckman xl-1 ultracentrifuge at 4 c . the protein sample was dialyzed overnight in 20 mm hepes , ph 7.5 , 200 mm nacl , and 0.5 mm tcep , and the protein concentration was 1.4 mg / ml for measurements . kinase assays were performed as previously described . briefly , reaction mixes contained 50 m atp , 1 mm dtt , 1 mm na3vo4 , 5 ci -[p]atp , 1 g of histone h3 as substrate , 1 l of dmso or drugs dissolved in dmso , and the indicated kinases . reaction mixes were incubated for 1 h at 30 c , quenched with sds loading buffer , and resolved on 14% sds - page . ic50 values were calculated from log dose response curves using prism 4 software ( graphpad software , inc . ) .

we present the structure of the human aurora b kinase domain in complex with the c - terminal aurora - binding region of human incenp and the aurora kinase inhibitor vx-680 . the structure unexpectedly reveals a dimeric arrangement of the aurora b : incenp complex , which was confirmed to exist in solution by analytical ultracentrifugation . the dimerization involves a domain swap of the activation loop , resulting in a different conformation of the dfg motif as compared to that seen in other kinase complexes with vx-680 . the binding of incenp differs significantly from that seen in the xenopus laevis aurora b : incenp complex currently used as a model for structure - based design for this important oncology target .

Introduction Results Discussion Materials and Methods

aurora b is the central component of the chromosomal passenger complex ( cpc ) that also contains the inner centromere protein ( incenp ) , borealin , and survivin . part of the activation mechanism for most agc kinases is mediated by binding of the phosphorylated c - terminal hydrophobic motif , fxxf(t / s)f , to the n - terminal lobe of the kinase catalytic domain . however , aurora kinases lack canonical c - terminal hydrophobic motifs and are activated in trans through binding of regulatory proteins to their n - terminal lobe . c , the c - terminal section of the incenp protein ( the in - box ) performs a similar regulatory function . the structure of human aurora a has been determined both without tpx2 and in complex with tpx2 . the structure of aurora b has previously been determined from xenopus laevis , in complex with incenp and the aurora b inhibitor hesperadin as well as other inhibitors . human aurora b is 80% identical to x. laevis aurora b over the kinase domain ( 55344 ) and 72% identical to human aurora a over the kinase domain . human incenp is 50% identical to x. laevis incenp over the region 835903 that is involved in binding to aurora b. aurora a phosphorylated on the activation loop in the absence of tpx2 has been observed in both active and inactive conformations . aurora a has been cocrystallized together with 1 ( vx-680 , figure 1 ) in the presence of tpx2 , which caused binding to an active kinase conformation , and also in the absence of tpx2 , which caused 1 to bind to an inactive conformation of the kinase domain , although this latter structure is not yet available in the protein data bank . first , phosphorylation of incenp at the tss motif of the c - terminal in - box region , which can be performed by aurora b itself , is important for full activity . second , autophosphorylation on the activation loop of aurora b ( for human aurora b at thr232 ) produces the active kinase . a recent study showed that phosphorylation on ser331 of aurora b by chk1 was essential for phosphorylation of the tss motif of incenp and also for full activation of aurora b. although most genetic alterations identified in cancer involve the aurora a locus , high expression levels of aurora b have been detected and associated with poor prognosis in glioblastoma , ovarian carcinoma , and hepatocellular carcinoma . as a result of the induced chromosomal defects , both p53-proficient and p53-deficient cells aurora b has therefore become an extensively pursued anticancer target , and many aurora inhibitors have been developed , including recently some isoform - specific inhibitors . the aurora kinase inhibitors ( second generation type 2 inhibitors ) such as 1(23 ) have considerable antitumor activity . to provide a model for structure - based design and to gain insight into the molecular mechanism of human aurora b activation , we determined the structure of human aurora b kinase domain , in complex with the c - terminal in - box section of human incenp ( residues 835903 ) and the type ii inhibitor 1 ( figure 1 ) . we began structural studies on human aurora b by preparing a set of constructs spanning different ranges of the gene , all of which included the kinase domain , for overexpression in escherichia coli . therefore , a bacterial expression construct for the c - terminal part of human incenp was prepared , spanning residues 835903 , which is the aurora - binding region . this expression construct contained a glutathione - s - transferase purification tag to enable sequential affinity purification of the heterodimeric aurora b : incenp complex by sequential ni - affinity followed by glutathione sepharose affinity chromatography . the aurora b protein was monophosphorylated according to electrospray ionization mass spectrometry ( data not shown ) . using this purified protein , we were able to cocrystallize the aurora b incenp heterodimer in complex with the type ii inhibitor 1 and determine the structure to a resolution of 2.75 ( table 1 ) . as expected from the primary sequence similarities , the overall structure of human aurora b resembles the structures of x. laevis aurora b and human aurora a , with the exception of the conformation of the activation loop ( figure 2 ) . unexpectedly , a domain swap was observed ; part of the kinase activation loop was exchanged between two aurora b molecules in the crystal lattice , resulting in similar binding of the activation loop against the c - terminal lobe of the kinase but different conformations around the dfg ( asp - phe - gly ) motif at the start of the activation loop ( figure 3a ) . in the aurora b : incenp complex , the activation segment is disordered after the dfg motif , including the phosphorylation site thr232 . the c - terminal part of the activation segment is dislodged from the lower lobe and interacts with a deep groove generated by the displaced activation segment in the interacting protomer . overview of human aurora b kinase as compared to x. laevis aurora b and human aurora a. ( a ) human aurora b with incenp colored green and the activation loop colored red . ( b ) x. laevis aurora b complex with hesperadin ( pdb i d : 2bfy ) , with incenp colored green and the activation loop colored red . ( c ) human aurora a complex with 1 ( pdb i d : 3e5a ) , with tpx2 colored green and the activation loop colored red . ( a ) the two aurora b molecules in the dimer are colored in yellow and blue , incenp is green , and the activation loops are colored red with dashed lines to indicate where the disordered residues would form a connection . the dashed lines in red indicate the connections that would be made between met249 and his250 in the absence of a domain swap . ( c ) as in panel a but with one aurora b : incenp molecule displayed as a surface , showing the binding of the activation loop in the dimeric arrangement . the residues at the point of the domain swap form more favorable interactions than they would have in the case of a nonexchanged arrangement ( figure 3b ) . met249 , which is important for the formation of the domain - swapped form , is not conserved in x. laevis aurora b , which has thr265 instead . apart from one conservative substitution of an arginine for a lysine , this is the only difference in the activation loop between the human and x. laevis enzymes , which is one of the most highly conserved regions in the whole protein . given the very favorable nature of the hydrophobic packing of the met249 side chain , it may be that this single amino acid difference is responsible for the observation of a domain swapped dimeric form of aurora b only for crystals of the human orthologue . in this domain - swapped arrangement , part of the activation loop packs against the outside of helix c , which is important for the activity and regulation of protein kinases . whether this arrangement has any effect on the activity or regulation of human aurora b kinase is an open question , but it may be partly responsible for the outward movement of c relative to the structures of x. laevis aurora b with 1 and abl1/2 with 1 ( discussed below ) . after observing the activation loop in the domain swapped conformation , to see if the dimeric state was also observed in solution , we analyzed the oligomeric state of the complex by analytical ultracentrifugation ( auc ) . a sedimentation velocity experiment showed the presence of both monomer and dimer , with the majority of the sample being dimeric ( figure 4 ) . the sedimentation velocity analysis gave molecular weights of 43307 and 81882 for the two species present , which compared well with the expected values of 41849 and 83680 for monomer and dimer based on the protein sequences . the frictional ratio for the dimeric species was higher at 2.09 , as compared to 1.94 for the monomeric species , corresponding to a less spherical dimeric arrangement . the c - terminal part of incenp , the in - box , wraps around the n - terminal lobe of the aurora b kinase domain in a manner similar to that seen previously with the x. laevis orthologue . however , although the binding of the n - terminal part of the n - box ( residues 842863 , site 1 ) closely resembles that of the x. laevis aurora b incenp structure ( figure 5b ) , the c - terminal part ( residues 868881 , site 2 ) is mostly different , with a substantial variation in the backbone conformation of the incenp residues at site 2 ( figure 5c ) . at site 1 , the incenp backbone adopts the same -helical arrangement , with the only significant differences in binding being for the nonconserved residues ile856 of incenp and leu210 of aurora b , which are arg812 and tyr228 in the x. laevis orthologue . the human complex therefore has a hydrophobic interaction at this point in place of the hydrogen bond between the tyr and the arg side chains in x. laevis . ( a ) sequence alignment of human and x. laevis incenp over the c - terminal in - box region . ( b ) comparison of human and x. laevis aurora b : incenp binding at site 1 ( residues 842863 of human incenp ) showing the mostly conserved interactions , with two exceptions highlighted . the x. laevis aurora b and incenp are colored blue and purple , respectively ( x. laevis coordinates were taken from pdb i d 2bfx(8 ) ) . ( c ) comparison of human and x. laevis aurora b : incenp binding at site 2 ( residues 868881 of human incenp ) showing the mostly nonconserved binding conformation , with the exception of the terminal phe881 . ( d ) binding detail for site 2 of human aurora b : incenp . at site 2 , the incenp backbone adopts a different conformation with only the binding of the very c - terminal phe881 conserved ( figure 5c ) . furthermore , the aurora c helix is displaced relative to the rest of the aurora structure . there is also a hydrogen bond between the aurora b tyr145 ( a histidine in x. laevis aurora b ) and the incenp backbone ( figure 5d ) . compound 1 binds to human aurora b in a similar conformation to that seen in complexes with other kinases . a structure of x. laevis aurora b bound to 1 is not available , so the structural comparisons of 1 binding considered here are restricted to the published structures of aurora a:1 ( pdb i d : 3e5a ) and abl2:1 ( pdb i d : 2xyn ) . a structure of an imatinib - resistant mutant abl1 in complex with 1 is also available . the glycine - rich loop of aurora b is in a different conformation to that of aurora a in the region around phe88 ( figure 6a , b ) . it adopts a conformation similar to the glycine - rich loop of abl2 when bound to 1 ( figure 6a , c ) , although abl2 has tyr299 in place of phe88 of aurora b. however , the most significant difference between aurora a and aurora b when bound to 1 is in the arrangement of the dfg motif . in aurora a , the dfg motif is arranged with the phe underneath the c helix , while in aurora b , the c helix is moved further out from the atp site and the dfg phe is to the side of the helix with the asp not well - ordered in the structure ( figure 6d ) . as abl1/2 also have a similar arrangement of the dfg phe and c as aurora a , aurora b differs from all of these when bound to 1 . the movement out of c allows more positional options for the aurora b dfg motif and also a potentially bigger back pocket for inhibitor binding than in aurora a , although due to the placement of the dfg phe the pocket in this particular structure is slightly smaller . ( d ) overlay of the dfg motifs of the complexes with 1 of aurora b ( in yellow ) and aurora a ( in pink ) . ( e ) overlay of the dfg motifs of the complexes with 1 of aurora b ( in yellow ) and abl2 ( in blue ) . comparison of the ic50 values for inhibition x. laevis aurora b by three common aurora kinase inhibitors with the previously published ic50 values for inhibition of human aurora b by the same three inhibitors shows that aurora b from both species behaves almost identically ( table 2 ) . until now , there has not been a structure of human aurora b kinase in the public domain , and the x. laevis orthologue has been used instead . it has been predicted previously that the binding site of aurora a : tpx2 has a smaller hydrophobic back pocket than that of aurora b : incenp . the aurora b structure presented here allows direct comparison with an aurora a structure with the same inhibitor and confirms that the hydrophobic back pocket of aurora b is indeed larger but with the caveat that the more outward position of c allows other conformations of the dfg motif , which may favor or disfavor particular inhibitors . the more outward position of c is so far unique to human aurora b ; all of the x. laevis aurora b cocrystal structures so far deposited in the pdb have a similar c position , even with the inhibitor zm447439 , which binds much further into the hydrophobic pocket than 1 ( pdb i d : 2vrx(9 ) ) , although it should be noted that the structure with zm447439 was obtained by soaking aurora b : incenp crystals with the compound , and a different structure could possibly have arisen by cocrystallization . in both of the structures of aurora a in complex with tpx2 ( which binds on the outside of c ) , c adopts an inward position . the dimerization of aurora b : incenp that we have observed both in the crystal structure and in solution is probably the most significant factor in the different dfg motif arrangement of this complex with 1 as compared to those of aurora a or abl . the domain swap involves a very different conformation of the activation loop , which is most likely the cause of the different dfg position . the auc experiments showed dimerization in the absence of an inhibitor , so compound 1 was not the cause of the observed dimerization in the crystal structure . however , because the dimerization interface involves c and is dependent on the activation loop , an inhibitor that promotes a substantially different c position or dfg position could conceivably promote or hinder dimerization . the variation of incenp binding between the human and the x. laevis orthologues at the second site next to c is probably a feature of the irregular backbone conformation . whether incenp can adopt different conformations at the second site to accommodate movements of c during activation ( or inhibition ) of aurora b is a question that has to be explored in the future . dna for residues 55344 of the kinase domain of human aurkb ( gi|83776600 ) was pcr amplified from dna in the mammalian gene collection ( image i d 2819846 ) and inserted into vector pnic28-bsa4 ( which carries kanamycin resistance ) in - frame with an n - terminal hexahistidine purification tag and tev protease cleavage site , by ligation - independent cloning . dna for residues 835903 of human incenp ( gi|102467235 , inner centromere protein isoform 1 ) was pcr amplified from incenp cdna and inserted into vector pgtvl1-sgc ( which carries ampicillin resistance ) in - frame with an n - terminal glutathione s - transferase purification tag and tev protease cleavage site , also by ligation - independent cloning . for protein purification , the resuspended cells for the aurora b and incenp expressions were combined and thawed together ( 4 l of culture of aurora b and 6 l of culture of incenp ) . fractions from the gel filtration containing the desired aurorab : incenp complex were pooled and passed through a column of 1 ml of glutathione sepharose 4b and then loaded onto a column of 0.7 ml of nickel - chelating resin . the structure was solved by molecular replacement using phaser with the structure of x. laevis aurora b ( pdb i d : 2bfx ) as a search model . positive difference electron density for the incenp protein component and the inhibitor 1 was visible in the initial maps .

mitochondria are key organelles in intermediate cellular metabolism , energy conversion , and calcium homeostasis ( dimmer and scorrano , 2006 ) . they also integrate and amplify apoptosis induced by intrinsic stimuli , releasing cytochrome c and other proapoptotic factors required for the activation of caspases ( green and kroemer , 2004 ) . cytochrome c release is regulated by proteins of the bcl-2 family that control the permeabilization of the outer membrane ( omm ) ( danial and korsmeyer , 2004 ) . energy conversion occurs at the inner mitochondrial membrane ( imm ) that can be further divided into two subcompartments : the so - called boundary membrane and the cristae , separated from the former by narrow tubular junctions ( frey and mannella , 2000 ) . mitochondria become condensed , with an expanded cristae space ( hackenbrock , 1966 ) . during apoptosis , the curvature of the cristae membrane is inverted in a remodeling process required for the complete release of cytochrome c , normally confined in the cristae ( scorrano et al . , 2002 ; frezza et al . , 2006 ; yamaguchi et al . , cristae remodeling occurs in response to proapoptotic bh3-only bcl-2 family members , such as bid , bim - s , and bnip3 , and independently of the outer membrane multidomain bcl-2 family members bax and bak ( scorrano et al . , 2002 ; cipolat et al . , 2006 ; whether changes in morphology of the cristae , where respiratory chain complexes ( rccs ) mainly localize ( vogel et al . , 2006 ) , affect oxidative phosphorylation efficiency , as originally predicted ( hackenbrock , 1966 ) , is unclear . this issue is further complicated by the assembly of rcc in supercomplexes ( rcs ) ( schgger , 1995 ; acn - prez et al . , 2008 ) , quaternary supramolecular structures that , by channeling electrons among individual rccs , allow the selective use of rcc subsets for nicotine adenine dinucleotide ( nadh)- or flavin adenine dinucleotide - derived electrons ( lapuente - brun et al . , 2013 ) . such a supramolecular organization is common in cristae : also , the mitochondrial atp synthase is assembled into dimers with greater adenosine triphosphatase ( atpase ) activity ( campanella et al . interestingly , cristae shape and atpase dimers are linked : in yeast mutants where the atpase can not dimerize , cristae are disorganized ( paumard et al . , 2002 ; , 2005 ; strauss et al . , 2008 ) , whereas in mammalian cells , increased cristae density favors atpase dimerization during autophagy ( gomes et al . , 2011 ) . on the contrary , despite their importance in mitochondrial bioenergetics , the relationship between rcs and cristae shape remains unclear . mitochondrial morphology and ultrastructure depends on mitochondria - shaping proteins that regulate organellar fusion and fission ( griparic and van der bliek , 2001 ) . mitofusins ( mfn ) 1 and 2 , highly homologous dynamin - related proteins of the omm , orchestrate fusion ( santel and fuller , 2001 ; legros et al . , 2002 ; chen et al . , 2003 ; santel et al . , mfn1 primarily participates in fusion , cooperating with the imm dynamin - related protein optic atrophy 1 ( opa1 ) ( cipolat et al . , 2004 ) , whereas mfn2 also tethers mitochondria to the endoplasmic reticulum ( de brito and scorrano , 2008 ) . mitochondrial fission is regulated by the cytoplasmic dynamin - related protein 1 that , upon calcineurin - dependent dephosphorylation , translocates to mitochondria ( yoon et al . , 2001 ; genetic depletion of opa1 leads to disorganization of the cristae ( frezza et al . , 2006 ) , and oligomers that contain a soluble and a membrane - bound form of opa1 keep the cristae junctions tight , independently from opa1 role in fusion ( frezza et al . , 2006 ; during apoptosis , these oligomers are early targets of bid , bim - s , and bnip3 , as well as of intrinsic death stimuli ( frezza et al . , 2006 ; whereas our knowledge of the molecular determinants of cristae shape and their role in apoptosis is increasing , the relationship between cristae morphology and mitochondrial function remains unexplored . we therefore set out to genetically dissect whether and how cristae shape regulates mitochondrial respiration . we show that cristae morphology determines assembly and stability of rcs and hence optimal mitochondrial respiratory function during life and death of the cell . whether apoptotic cristae remodeling that maximizes cytochrome c release from mitochondria affects mitochondrial function is unclear , mainly because it occurs around the same time as outer membrane permeabilization ( scorrano et al . , 2002 ) . in order to genetically dissociate the two processes , we inspected the primary structure of the prototypical cristae remodeling inducer bcl-2 family member bid for homology with peptides known to perturb the mitochondrial inner membrane , like mastoparan , a 14 amino acid wasp venom component ( pfeiffer et al . , 1995 ) . interestingly , bid membrane inserting 6 helix as well as the transmembrane domains of bnip3 and bims that also remodel cristae ( yamaguchi et al . 2010 ) displayed homology to mastoparan ( figures s1a and s1b available online ) . to exploit the role of this homologous sequence in cristae remodeling , we mutagenized the two highly conserved 157 and 158 lys h. sapiens bid residues to ala ( bid ) ( figure s1c ) . because this mutation did not impair caspase-8 cleaved recombinant bid ( cbid ) integration in purified mouse liver mitochondria ( mlm ) ( wei et al . , 2000 ; figure s1d ) , we could measure its biological activity using an established quantitative , specific cytochrome c release elisa ( scorrano et al . , 2002 ) . cbid efficiently released cytochrome c from purified mitochondria , whereas a bh3 domain g94e mutant was , as expected , inactive ( wei et al . , 2000 ) and the cbid mutant released 25%30% more cytochrome c than the baseline ( figure 1a ) , a figure close to the amount of free intermembrane space cytochrome c ( scorrano et al . , 2002 ) . bak oligomerization was superimposable in cbid or cbid - treated mitochondria ( figure 1b ) ; conversely , two established assays of intramitochondrial cytochrome c redistribution , the cytochrome b5-dependent extramitochondrial nadh oxidation and the ratio of ascorbate - driven over tetramethyl - p - phenylenediamine ( tmpd)-driven respiration ( scorrano et al . , 2002 ) , indicated that cbid mobilized the cristae cytochrome c pool less efficiently than cbid ( figures 1c and 1d ) . indeed , cbid was unable to remodel mitochondrial cristae , as indicated by morphometric analysis of electron micrographs of mitochondria treated with the bid mutants ( figures 1e and 1f ) ( scorrano et al . , 2002 ) . cristae remodeling is associated with the disruption of high molecular weight ( hmw ) opa1 oligomers ( frezza et al . , 2006 ) . western blots of blue native gel electrophoresis ( bnge ) of mitochondrial proteins revealed four major opa1-containing complexes . upon treatment with cbid , opa1 rapidly disappeared from 720 kda hmw complexes ( figures 1 g , s1e , and s1f ) . these hmw forms of opa1 were similarly targeted by cbid but significantly less by cbid , as determined by bnge ( figure 1h , quantification in [ i ] ) . chemical crosslinking experiments ( frezza et al . , 2006 ) further confirmed that the opa1-containing oligomer is disrupted by the mutants of cbid able to induce cristae remodeling ( figures s1 g and s1h ) . finally , we measured the killing efficiency of these truncated bid ( tbid ) mutants expressed in mouse embryonic fibroblasts ( mefs ) . only tbid efficiently killed mefs : tbid and tbid elicited comparable low levels of cell death , whereas the double tbid mutant appeared completely ineffective ( figure 1j ) , suggesting that both outer membrane permeabilization and mitochondrial cristae remodeling are required for bid - induced apoptosis . in conclusion , bid is deficient in cristae remodeling , cytochrome c release , and induction of apoptosis . the bid mutant dissociates outer membrane permeabilization from cristae remodeling and can be used to investigate the relationship between the latter and mitochondrial function . we therefore measured the effect of the bid mutants on the respiratory control ratio ( rcr ) , an index of respiratory efficiency , of mitochondria incubated with excess exogenous cytochrome c and nadh ( to compensate for the potential effects of inner membrane or outer membrane [ om ] permeabilization ) . cbid reduced rcr only when mitochondria were energized with substrates for complex i ( glutamate / malate ) but not when they were fed with substrates entering the electron transport chain at complex ii ( succinate ) or complex iv ( ascorbate + tmpd ) ( figure 2a ; data not shown ) . interestingly , these changes were recapitulated by the bh3 domain mutant cbid that does not permeabilize the om but not by the cristae remodeling - deficient mutants cbid and cbid ( figure 2b ) . maximal ( uncoupled ) respiration was similarly affected by the cbid mutants tested , ruling out that bid alters rcr , because it affects atpase or activity or atp / adp exchange ( figure s2a ) . these experiments suggest that cristae remodeling causes complex i - dependent changes in mitochondrial bioenergetics . complex i is further assembled in quaternary functional rcs with complexes iii and iv ( i + iii and i + iii + iv ) , whereas most complex ii is not found in rcs ( acn - prez et al . , 2008 ) . thus , the reduction in complex i - supported respiration could be a consequence of specific inhibition of complex i or of issues in rcs function . even after 30 min of acute bid treatment , the specific complex i nadh - ubiquinone reductase activity of purified mitochondria was unaltered ( data not shown ) , prompting us to investigate rcs assembly and stability in situ . we therefore took advantage of bax , bak ( dko ) mefs , resistant to mitochondrial permeabilization , cytochrome c release , and apoptosis triggered by expression of tbid ( wei et al . , 2001 ) . upon transduction of metabolically labeled dko mefs with tbid but not with tbid that does not cause cristae remodeling ( figure s2b ) , the rcs radioactivity signal as well as the rcs / complex v radioactivity ratio were reduced ( figures 2c and 2d ) , and we observed a reduction in the autoradiographic signal of cytochrome b retrieved in rcs compared to that in free complex iii ( figures 2e and 2f ) . whereas this result could suggest that complex iii was incorporated less efficiently into rcs , immunoblotting for the complex i subunit ndufa9 revealed that rcss were also destabilized in dko cells ( figure 2 g ) . functionally , only cbid and cbid that cause cristae remodeling but not cbid reduced glutamate - supported rcr in dko mitochondria ( figure 2h ) . whether rcs disorganization was a general consequence of altered cristae shape , we turned to cells lacking opa1 , a key regulator of cristae morphology ( frezza et al . , however , chronic opa1 depletion impaired mitochondrial dna ( mtdna ) levels and translation ( figures s3a and s3b ) , complicating the analysis of the relationship between opa1 and rcs and calling for a model of conditional opa1 ablation . we produced , by homologous recombination , c57bl6/j embryonic stem cells with loxp sites inserted in the opa1 gene ( opa1 ) , which were then microinjected in c57bl6/j blastocysts to generate opa1 mice . following cre - mediated recombination , the deletion of exons 2 and 3 resulted in an aberrant exon1exon4 transcript with a stop codon immediately after exon 1 , producing a predicted 10 amino acid ( aa ) residual protein ( figures 3a and 3b ) . chimerism and germ - line transmission of the offspring was tested by pcr , and germ - line transmittants were bred to homozygosity ( figure 3c ) . fibroblasts isolated from the diaphragm of homozygous opa1 7-week - old male mice ( mafs ) were immortalized and used for subsequent analysis . opa1 was completely ablated 24 hr after adenoviral delivery of cre recombinase ( figure 4a ) and , as expected , mitochondria were fragmented ( figures 4b and 4c ) with defects in cristae shape ( figures 4d and 4e ) . four days after cre - mediated opa1 ablation , mtdna copy number ( figure 4f ) and translation ( figures 4 g and 4h ) were unaffected , allowing us to specifically address the role of opa1 and cristae shape in rcs assembly using an assay based on the incorporation of radiolabeled mtdna - encoded proteins into rcc and rcs ( acn - prez et al . , 2008 ) . upon acute opa1 ablation , the assembly of mtdna - encoded subunits into rcc was not affected ( data not shown ) . we therefore followed the rcs assembly rate ( measured as the ratio between rcs and complex v radioactivity throughout the chase period ) that resulted 8-fold slower when opa1 was ablated from opa1 mafs ( figures 4i and 4j ) . a similar reduction in the rcs assembly rate was observed in opa1 mefs ( figure s3c ) , suggesting that , in absence of opa1 , rcc are less superassembled , irrespective of their initial levels . to test if acute opa1 ablation altered rcs in vivo , we tail vein - injected cre - expressing adenoviruses in opa1 animals . after 72 hr , s4a ) , cristae morphology was abnormal ( figure s4b ) , rcs were reduced ( figure s4c ) , and glutamate / malate rcr was impaired ( figure 4k ) . these experiments of conditional ablation of opa1 identify a role for cristae shape in rcs assembly in vitro and in vivo . the model linking cristae shape to rcs predicts that higher opa1 levels should favor rcs assembly . to verify this hypothesis , we wanted to generate a mouse model of opa1 overexpression . very high opa1 levels are , however , toxic , causing paradoxical mitochondrial fragmentation ( cipolat et al . , 2004 ) : we therefore targeted , by homologous recombination in the murine x chromosome hprt region , a transgene - carrying mouse variant 1 opa1 under the human -actin promoter ( opa1 ) ( figure 3d ) . the integration into microinjected bpes embryonic stem cells was verified by pcr ( figure 3e ) , and the cells were microinjected into c57bl6/j blastocysts . six generated agouti chimeras with a chimerism exceeding 90% were bred with c57bl6/j mates and tested for germline transmission by fur color and transgene pcr analysis ( figure 3f ) . mice were viable , fertile , grew normally , and resisted to different forms of tissue damage ( t.v . r. menab , m. sandri , f. di lisa , and l.s . , unpublished data ) . immortalized mafs prepared from the diaphragm of hemizygous opa1 7-week - old c57bl6/j male mice displayed an 1.5 increase in opa1 levels compared to age- and sex - matched littermate controls mafs ( wild - type [ wt ] ; figure 5a ) . mitochondria were slightly elongated ( figures 5b and 5c ) and cristae tighter ( figures 5d and 5e ) , without any difference in mtdna levels ( figure 5f ) and translation ( figures 5 g and 5h ) . importantly , in opa1 mafs , rcs assembly ( figures 5i and 5j ) and glutamate - supported rcr ( figure an 50% increase in liver mitochondria opa1 levels ( figure s5a ) was similarly associated to tighter cristae ( figure s5b ) and increased rcs levels ( figure s5c ) . taken together , these results indicate that rcs assembly is facilitated by increased opa1 levels and tighter cristae . we therefore measured the growth of dko cells ( resistant to bid - induced outer membrane permeabilization , caspase - dependent mitochondrial damage , and apoptosis ) in galactose media , where most of cellular atp comes from the respiratory chain ( acn - prez et al . , 2004 ) . wt and g94e tbid impaired growth in galactose , whereas cells transduced with tbid that does not cause cristae remodeling did not display any defect ( figure 6a ) . growth in galactose - containing media was impaired upon acute ablation of opa1 in mafs , whereas it was normal for fusion - deficient mfn1 , mfn2 mefs ( figures 6b and 6c ) , where mitochondrial fusion is also inhibited . in mfn1 , mfn2 mefs , mtdna copy number was reduced ( figure s6a ) , but cristae shape ( figures s6b and s6c ) , mtdna translation ( figure s6d ) , rcs stability ( figure s6e ) , and assembly ( figure s6f ) were not affected . thus , the galactose growth defect is not the consequence of impaired fusion but correlates with altered cristae shape and rcs . finally , opa1 mafs grew faster than their wt counterparts in galactose media ( figure 6d ) , further confirming the link between cristae shape , rcs levels , and mitochondria - dependent cellular growth . in conclusion , respiratory chain supercomplexes have been considered bnge artifacts until direct respirometric experiments on purified rcs identified them as the functional mitochondrial respiratory units ( acn - prez et al . , 2008 ) . since then , rcs have been directly visualized in intact cristae by electron tomography ( davies et al . , 2011 ) , complex iv assembly factors that favor rcs formation have been identified ( chen et al . , 2012 ; 2013 ) , and the role of rcs in mitochondrial utilization of reducing equivalents has been demonstrated ( lapuente - brun et al . , 2013 ) . however , the relationship between cristae shape and rcs , as well as between rcs and mitochondrial function , remained obscure . our results demonstrate that cristae shape regulates respiratory chain supercomplexes stability and assembly , impacting on respiratory efficiency and respiratory cell growth . to dissect the role of cristae shape in rcs structure and function , we genetically ablated the master cristae shape regulator opa1 . individual respiratory chain units associate with opa1 ( zanna et al . , 2008 ) , and mitochondrial metabolism is deranged in dominant optic atrophy caused by opa1 mutations ( lodi et al . , 2004 ) . however , the defect in atp production in opa1 haploinsufficient cells was unexplained : opa1 is not essential for assembly of respiratory chain complexes and mtdna levels as well as activities of individual respiratory chain complexes are normal in dominant optic atrophy ( zanna et al . , 2008 ) . conversely , the reduction in mtdna copy number has been invoked to explain the mitochondrial dysfunction of fusion - deficient cells from mfn1 , mfn2-deficient mice ( chen et al . , 2010 ) . our results challenge this hypothesis : upon acute opa1 ablation , mtdna levels are normal , whereas cristae shape , rcs , complex - i - dependent respiration , and respiratory growth are impaired . conversely , in mfn1 , mfn2 cells , mtdna copy number is reduced , but cristae shape , rcs , and respiratory growth are normal . thus , rcs disorganization shall be regarded as a key mechanism of mitochondrial dysfunction accompanying altered organelle morphology . the role of opa1 and cristae shape in rcs organization is further supported by mouse models of opa1 conditional ablation and mild overexpression . the first tool allowed us to dissociate cristae biogenesis from mtdna maintenance : whereas chronic opa1 depletion reduces mtdna copy number and translation , upon acute opa1 ablation , mtdna levels are normal , but cristae are disorganized , impacting on rcs assembly and respiratory function and growth . thus , mtdna reduction appears to be a consequence of chronic fusion inhibition in opa1 ( and double mfn ) cells . we can therefore predict that the opa1 cells will be useful to elucidate how prolonged inhibition of mitochondrial fusion results in mtdna levels reduction . opa1 mild overexpression lends further support to the model linking rcs organization to cristae shape : rcs assembly and respiratory function and growth are increased in opa1 cells without any measurable change in mtdna levels and translation . the opa1 mouse will be instrumental to investigate the role of opa1 and cristae shape in vivo . the role and mechanisms of cristae remodeling in apoptosis are controversial ( scorrano et al . , 2002 ; germain et al . , 2005 ; frezza et al . , 2006 ; yamaguchi et al . , 2008 ; 2010 ) , cristae remodeling has been reckoned as a mere feedback mechanism in situ , occurring after caspase activation ( sun et al . , 2007 ) . our results suggest that , in addition to its role in cytochrome c release , cristae remodeling also impairs mitochondrial function to precipitate apoptosis . the bid 6 mutant generated here , which does not induce cristae changes and cytochrome c mobilization but permeabilizes the outer membrane , can be a useful tool to dissect in vivo the involvement of cristae remodeling in developmental and homeostatic apoptosis . we think that cristae remodeling influences rcs by targeting opa1 ( frezza et al . , 2006 ) , not by altering membrane potential that is normal during cristae remodeling ( scorrano et al . , 2002 ) or by inhibiting mtdna translation and insertion of mtdna - encoded subunits that similarly appear normal in dko cell - expressing bid ( data not shown ) . our work unravels a role for cristae shape in rcs assembly and stability , mitochondrial respiratory efficiency , and respiratory growth , suggesting that shape of biological membranes can influence membrane protein complexes . moreover , our data highlight the importance of rcs in respiration by complex i - feeding substrates . the pathogenesis of dominant optic atrophy where opa1 is mutated ( alexander et al . , 2000 ) or of other mitochondrial diseases where opa1 is degraded ( duvezin - caubet et al . , 2006 ) could also depend on this unexpected opa1 function . in these latter settings , stabilization of opa1 could correct rcs and therefore mitochondrial dysfunction , opening novel therapeutic perspectives for currently intractable diseases . to generate opa1 mice , a mouse bac clone containing the opa1 gene was isolated from the c57bl/6j es bac clone library . an 11 kb hpai dna restriction fragment containing the opa1 fragment was excised with ecorv and xmal to generate blunt ends and inserted into a pko4.4a - loxp cut with xhoi and sali . a loxp site was introduced between intron1 and exon2 of opa1 and a phosphoglycerate kinase ( pgk ) promoter - driven neomycin resistance gene , flanked by two frt sequences and with one loxp sequence downstream , was inserted in intron3 . the targeting vector was linearized and electroporated into c57bl6 embryonic stem cells ( escs ) . three mutated esc lines were microinjected into c57bl6 blastocytes and implanted in host mice to obtain chimeric mice , which were then bred with c57bl6 mates and their offspring tested by pcr for germline transmission . the human -actin promoter was extracted from pdrive - h-actin ( invivogen ) using spei and ncoi and cloned in pentry . the complementary dna of mouse isoform 1 opa1 and polya extracted from pcdna3.1-opa1 ( cipolat et al . , 2004 ) using nhe1 and ecii was ligated into pentry using quick ligase ( ozyme ) . the transgene was then inserted by homologous recombination in a pdest vector containing part of the human hypoxanthine phosphoribosyltransferase locus . the resulting vector was linearized using pvui and electroporated into c57bl6 bpes cells by nucleis ( france ) . three positive esc recombinant clones were microinjected into c57bl6 blastocysts and implanted into host pseudopregnant female c57bl6 to obtain chimeric mice . six chimeras ( identified by fur agouti color ) were bred with c57bl6 mates , and germline transmission was verified by fur color and pcr . mitochondria ( 10 mg / ml ) were suspended in buffer d ( 1 m 6-aminohexanoic acid , 1.25% v / v digitonin , 50 mm bis - tris - hcl , ph 7 ) and centrifuged . the supernatant was collected , and 5% serva blue g dye in 1 m 6-aminohexanoic acid was added to 1/3 of the final sample volume . equal amounts ( 100 g ) of mitochondrial proteins were separated by 3%13% gradient bnge ( schgger , 1995 ) . for rcs detection , the concentration of digitonin in buffer d was 4% ( v / v ) . for two - dimensional ( 2d ) blue native ( bn)/bnge , the lane cut from the first - dimension bnge was casted on top of a native 3%14% gradient gel in 1% ( v / v ) dodecyl maltoside . for 2d bn / sds page , the lane cut from the first - dimension bnge was incubated for 1 hr at 25c in 1% sds and 1% -mercaptoethanol and then casted on top of an 8% or a 16.5% denaturing gel . after electrophoresis , the complexes were electroblotted on a polyvinylidene fluoride ( pvdf ) membrane and probed with the indicated antibodies . to detect rcs from radiolabelled cells , samples were treated as described above , and after electrophoresis , the gels were dried and the signal was detected following exposure for 36 days . labeling of mtdna - encoded proteins was performed with [ s]-methionine and cysteine ( express protein labeling mix , perkin elmer life sciences ) . cells were preincubated for 12 hr in the presence of 40 g / ml chloramphenicol in uridine - supplemented medium and then exposed for 2 hr to the [ s ] protein labeling mix ( pulse ) in the presence of 50 g / ml cycloheximide . cells were washed for four times with pbs and cold dulbecco s modified eagle s medium ( dmem ) and then cultured for the indicated time ( chase ) prior to lysis and protein separation by bnge . to assay basal mtdna translation , equal protein amounts ( 40 g ) from cells metabolically labeled as described above treated for 30 min with 50 g / ml emetine were separated by sds - page . mitochondria from liver of mice and from the indicated cell lines were isolated as in frezza et al . cytochrome c release , ascorbate / tmpd - driven respiration and cytochrome b5-dependent nadh oxidation were determined as described ( scorrano et al . , crosslinking , mitochondria treated as indicated were incubated with 1 mm 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride ( edc ) or 1 mm bismaleimidohexane ( bmh ) , as previously described ( frezza et al . , 2006 ) . carbonate extraction was performed as previously described ( dimmer et al . , 2008 ) . details and procedures for sds - page and immunoblotting can be found in the extended experimental procedures . the retroviral vector pmig - tbid was described previously ( cheng et al . , 2001 ) . kkaa , g94e , and g94ekkaa mutants were generated by site - direct mutagenesis using kod polymerase ( biolabs ) . to measure mtdna copy number , total cellular dna was amplified using specific oligodeoxynucleotides for mt - co2 and sdha by real - time pcr . mefs and human embryonic kidney 293 cells ( hek293 ) cells were cultured as described ( gomes et al . , 2011 ) . when indicated , in dmem , glucose was substituted with 0.9 mg / ml galactose . ecotropic viruses were generated as described ( cheng et al . , 2001 ) . wt , opa1 , opa1 , and mafs sv40 transformed cell lines were generated from the diaphragm of the respective 7-week - old mouse killed by cervical dislocation . acute opa1 ablation in opa1 mafs was obtained by infection with adenoviruses expressing cytomegalovirus ( cmv)-cre - gfp ( ad - cre ; 300 pfu / cell ; vector biolabs ) . opa1 mafs were infected and dko mefs were transduced 24 or 16 hr before the growth was assessed . apoptosis was measured by flow cytometric detection ( facscalibur ) of the annexin - v - pe positive events in the gfp - positive population . details can be found in the extended experimental procedures . for mitochondrial imaging , cells stained with a rabbit polyclonal anti - tom20 ( santa cruz ; 1:200 ) , as previously described ( frezza et al . , 2006 ) electron microscopy ( em ) was performed as described ( scorrano et al . , 2002 ) . details can be found in the extended experimental procedures . extended experimental proceduresmouse handlingfor genotyping of the opa1 mice , the following primers were used : primer ck1 5-cag tgt tga tga cag ctc ag-3 ; primer ck2 5-cat cac aca cta gct tac att tgc-3. for genotyping of the opa1 mice , the following primers were used : primer tgf 5-gca atg acg tgg tcc tgt tttg-3 ; primer tgr 5-gat agg tca ggt aag caa gca ac-3 ; primer wtr 5-gag gga gaa aaa tgc gga gtg-3 ; primer wtf 5-ctc cgg aaa gca gtg agg taa g-3.all mice procedures were performed according to protocols approved by the local ethic committees ( protocol 32/2011 ceasa university of padova , venetian institute of molecular medicine ; license 1034/3703/02 cantonal veterinarian authority , university of geneva ) . opa1 mice were tail injected with adenoviruses ( 2x10 pfu ) ( vector biolabs ) . after 3 days , they were killed by cervical dislocation and the liver was extracted.mitochondrial assaysmitochondria from mouse liver and from the indicated cell lines were isolated as described ( frezza et al . , 2007 ) . mitochondria were isolated from opa1 mice 3 days after the tail - vein injection of the indicated adenoviruses . cytochrome c release , ascorbate / tmpd - driven respiration and cytochrome b5-dependent nadh oxidation were determined as described ( scorrano et al . , 2002 ) . to measure respiration , mitochondria ( 1 mg / ml ) were incubated in experimental buffer ( eb : 150 mm kcl , 10 mm tris mops , 10 m egta - tris , 10m atp ) . to determine bid effect of rcr , eb was supplemented with 5 m cytochrome c , 2 mm nadh and 3.2 pmol mg cbid . when indicated , mitochondria were transferred into a clark s type oxygen electrode chamber and 5 mm glutamate /2.5 mm malate or 2 m rotenone/10 mm succinate were added . basal o2 consumption was recorded ( state 2 ) and after 2 min 100 m adp was added ( state 3 ) , followed by 2.5 g / ml oligomycin ( state 4 ) and 200 m fccp ( state 3u ) . for complex iv dependent - o2 consumption , mitochondria ( 1 mg / ml ) were incubated for the indicated time in eb supplemented with 5 m cytochrome c. mitochondria were treated with 1 nmol / mg antimycin a and 2 nm rotenone and they were transferred into a clark s type oxygen electrode chamber . after 2 min 6 mm ascorbate and 300 m tmpd were added and the complex iv - dependent o2 consumption rate was measured.the enzymatic activity of complex i and citrate synthase was determined as described ( spinazzi et al . , 2012).transmission electron microscopyem of cells and isolated mitochondria were performed as described ( scorrano et al . , 2002 ) and thin sections were imaged on a tecnai-20 electron microscope ( philips - fei ) . for morphometric analysis of mitochondrial cristae , cristae width was measured using the image j multimeasure plug-in.measurement of mitochondrial dna copy numbertotal cellular dna was isolated using phenol / chlorophorm 24:25 ( v / v ) and was amplified using specific oligodeoxynucleotides for mt - co2 and sdha by real - time pcr using platinum sybr green qpcr supermix ( invitrogen ) following manufacturer s indications . the mtdna copy number per cell was calculated using sdha amplification as a reference for nuclear dna content.imaging4x10 cells seeded on 13 mm round glass coverslips were fixed , permeabilized and blocked as previously described ( frezza et al . , 2006 ) and then stained with a rabbit polyclonal anti - tom20 ( santa cruz , 1:200 ) overnight at 4c . after washing with pbs , cells were incubated with texasred conjugated goat anti - rabbit ( 1:500 , invitrogen ) for 1 hr at room temperature and coverslips were mounted on slides using prolong gold antifade reagent ( invitrogen ) and analyzed by confocal microscopy . adenovirus infected cells were identified by expression of gfp.confocal images were acquired using a nikon eclipse te300 inverted microscope equipped with a spinning - disk perkinelmer ultraview lci confocal system , a piezoelectric z axis motorized stage ( pifoc , physik instrumente ) , and an orca er 12-bit ccd camera ( hamamatsu photonics ) . for detection of gfp or texasred fluorescence , samples were excited using the 488 nm or the 543 nm lines of a he ne laser ( perkinelmer , waltham , ma , usa ) with exposure times of 100 - 200 ms using a 60x , 1.4 na plan apo objective ( nikon ) . measurement of mitochondrial major axis length was performed in at least five mitochondria per cell , in a minimum of 50 cells / experiment ( 250 mitochondria / experiment ) . mitochondrial length was then quantified using the roi manager plug - in of imagej software.biochemistryfor protein crosslinking , mitochondria treated as indicated were incubated with 1 mm edc or 1 mm bmh as previously described ( frezza et al . , 2006 ) . for sds - page , mitochondrial proteins ( 20 g ) were separated on 3%8% tris - acetate or 4%12% tris - glycine ( nupage , invitrogen ) polyacrilamide gels , transferred onto pvdf membranes ( biorad ) and probed using the indicated primary antibodies and isotype matched secondary antibodies conjugated to horseradish peroxidase whose signal was detected with ecl ( amersham ) . the following primary antibodies were employed : monoclonal anti - opa1 ( 1:1000 bd pharmingen ) , rabbit polyclonal anti - bak - nt ( 1:1000 upstate ) , mouse monoclonal anti - fp subunit ( 1:5000 ) , mouse monoclonal anti - ndufa9 subunit ( 1:1000 ) , mouse monoclonal anti - core2 subunit ( 1:1000 ) , mouse monoclonal anti - co1subunit ( 1:1000 ) ( mitoscience ) , mouse monoclonal anti - vdac1 ( 1:1000 abcam ) , rabbit polyclonal anti - tom20 ( 1:1000 santa cruz ) , mouse monoclonal anti - tim23 ( 1:1000 bd pharmingen ) , rabbit polyclonal anti - bid ( 1:1,000 , a kind gift of a. gross , weizmann institute , rehovot ) . densitometry was performed by analyzing optical density using gel pro analyzer.carbonate extraction was performed as previously described ( dimmer et al . , 2008).molecular biologythe retroviral vector pmig - tbid was described previously ( cheng et al . , 2001 ) . kkaa , g94e and g94ekkaa mutants were generated by site - direct mutagenesis using kod polymerase ( biolabs ) and the following primers : for bid forward 5-aca atg ctg ttg gcc gcc gcc gtg gcc agt cac-3 and reverse 5-gtg act ggc cac ggc ggc ggc caa cag cat tgt-3 ; for bid forward 5-ctc gcc caa ata gaa gat gag atg gac cac aac-3 and reverse 5-gtt gtg gtc cat ctc atc ttc tat ttg ggc gag-3.recombinant protein expressionp7/p15 recombinant bid was produced , purified and cleaved with caspase-8 as described ( frezza et al . , 2006).assays of cell growth and deaththe growth of wt , opa1 mafs was determined by counting viable cells ( as determined by trypan blue exclusion ) daily for 5 days . 1.5x10 opa1 mafs were infected with the indicated adenoviruses and after 24 hr the growth was determined by counting viable cells ( as determined by trypan blue exclusion ) daily for 4 days . 1.5x10 dko mefs were transduced with the indicated retroviruses and after 16 hr the growth was determined by counting viable gfp positive cells by flow cytometry.for measurements of apoptosis , 1.5x10 cells of the indicated genotype grown in 6 well - plates were transfected with the indicated vector . after 48 hr apoptosis was measured by flow cytometric detection ( facscalibur ) of the annexin - v - pe positive events in the gfp - positive population.cell culture , transfection , virus production , and transductiondko mefs and hek293 cells were cultured as previously described ( gomes et al . , 2011 ) . when indicated , glucose in dmem was substituted with 0.9 mg / ml galactose . cells were transfected using transfectin ( biorad ) following manufacturer s instruction.ecotropic viruses were generated by cotransfecting the hek293 packaging cell lines with the packaging vector pik and the appropriate pmig as previously described ( cheng et al . , 2001 ) . viral supernatant were retrieved and used to transduce dko mefs in the presence of 4 g / ml of hexadimethrine bromide ( sigma ) . following an over - night transduction , the rate of gfp expression was typically around 70% as determined by flow cytometry.wt , opa1 , opa1 , mafs sv40 transformed cell lines were generated respectively from wt , opa1 , opa1 mice . male 7 weeks old mice were killed by cervical dislocation and their diaphragm were carefully dissected , washed in dmem , minced and then enzymatically digested with a collagenase 0.5% ( sigma ) solution for 10 min at 37c . cell were spun at 200 g for 3 min , resuspended in dmem containing 4.5 mg / ml glucose , 10% fetal bovine serum ( fbs ) , 50 u / ml penicillin , 50 g / ml streptomycin , 100 m nonessential aminoacids ( gibco ) , 50 g / ml uridine , 25 mm hepes ( gibco ) , 2.5 g / ml fungizone ( gibco ) and seeded on a 0.1% gelatin ( sigma ) precoated plate . after 15 min the supernatant was removed and the adherent fibroblasts were left growing in complete dmem . mafs were transformed using an sv40 large t antigen expressing plasmid as previously described ( cipolat et al . , 2006 ) and for genotyping of the opa1 mice , the following primers were used : primer ck1 5-cag tgt tga tga cag ctc ag-3 ; primer ck2 5-cat cac aca cta gct tac att tgc-3. for genotyping of the opa1 mice , the following primers were used : primer tgf 5-gca atg acg tgg tcc tgt tttg-3 ; primer tgr 5-gat agg tca ggt aag caa gca ac-3 ; primer wtr 5-gag gga gaa aaa tgc gga gtg-3 ; primer wtf 5-ctc cgg aaa gca gtg agg taa g-3. all mice procedures were performed according to protocols approved by the local ethic committees ( protocol 32/2011 ceasa university of padova , venetian institute of molecular medicine ; license 1034/3703/02 cantonal veterinarian authority , university of geneva ) . opa1 mice were tail injected with adenoviruses ( 2x10 pfu ) ( vector biolabs ) . after 3 days , they were killed by cervical dislocation and the liver was extracted . mitochondria from mouse liver and from the indicated cell lines were isolated as described ( frezza et al . , 2007 ) . mitochondria were isolated from opa1 mice 3 days after the tail - vein injection of the indicated adenoviruses . cytochrome c release , ascorbate / tmpd - driven respiration and cytochrome b5-dependent nadh oxidation were determined as described ( scorrano et al . , 2002 ) . to measure respiration , mitochondria ( 1 mg / ml ) were incubated in experimental buffer ( eb : 150 mm kcl , 10 mm tris mops , 10 m egta - tris , 10m atp ) . to determine bid effect of rcr , eb was supplemented with 5 m cytochrome c , 2 mm nadh and 3.2 pmol mg cbid . when indicated , mitochondria were transferred into a clark s type oxygen electrode chamber and 5 mm glutamate /2.5 mm malate or 2 m rotenone/10 mm succinate were added . basal o2 consumption was recorded ( state 2 ) and after 2 min 100 m adp was added ( state 3 ) , followed by 2.5 g / ml oligomycin ( state 4 ) and 200 m fccp ( state 3u ) . for complex iv dependent - o2 consumption , mitochondria ( 1 mg / ml ) were incubated for the indicated time in eb supplemented with 5 m cytochrome c. mitochondria were treated with 1 nmol / mg antimycin a and 2 nm rotenone and they were transferred into a clark s type oxygen electrode chamber . after 2 min 6 mm ascorbate and 300 m tmpd were added and the complex iv - dependent o2 consumption rate was measured . the enzymatic activity of complex i and citrate synthase was determined as described ( spinazzi et al . em of cells and isolated mitochondria were performed as described ( scorrano et al . , 2002 ) and thin sections were imaged on a tecnai-20 electron microscope ( philips - fei ) . for morphometric analysis of mitochondrial cristae , cristae width was measured using the image j multimeasure plug - in . total cellular dna was isolated using phenol / chlorophorm 24:25 ( v / v ) and was amplified using specific oligodeoxynucleotides for mt - co2 and sdha by real - time pcr using platinum sybr green qpcr supermix ( invitrogen ) following manufacturer s indications . the mtdna copy number per cell was calculated using sdha amplification as a reference for nuclear dna content . 4x10 cells seeded on 13 mm round glass coverslips were fixed , permeabilized and blocked as previously described ( frezza et al . , 2006 ) and then stained with a rabbit polyclonal anti - tom20 ( santa cruz , 1:200 ) overnight at 4c . after washing with pbs , cells were incubated with texasred conjugated goat anti - rabbit ( 1:500 , invitrogen ) for 1 hr at room temperature and coverslips were mounted on slides using prolong gold antifade reagent ( invitrogen ) and analyzed by confocal microscopy . confocal images were acquired using a nikon eclipse te300 inverted microscope equipped with a spinning - disk perkinelmer ultraview lci confocal system , a piezoelectric z axis motorized stage ( pifoc , physik instrumente ) , and an orca er 12-bit ccd camera ( hamamatsu photonics ) . for detection of gfp or texasred fluorescence , samples were excited using the 488 nm or the 543 nm lines of a he ne laser ( perkinelmer , waltham , ma , usa ) with exposure times of 100 - 200 ms using a 60x , 1.4 na plan apo objective ( nikon ) . measurement of mitochondrial major axis length was performed in at least five mitochondria per cell , in a minimum of 50 cells / experiment ( 250 mitochondria / experiment ) . mitochondrial length was then quantified using the roi manager plug - in of imagej software . for protein crosslinking , mitochondria treated as indicated were incubated with 1 mm edc or 1 mm bmh as previously described ( frezza et al . , 2006 ) . for sds - page , mitochondrial proteins ( 20 g ) were separated on 3%8% tris - acetate or 4%12% tris - glycine ( nupage , invitrogen ) polyacrilamide gels , transferred onto pvdf membranes ( biorad ) and probed using the indicated primary antibodies and isotype matched secondary antibodies conjugated to horseradish peroxidase whose signal was detected with ecl ( amersham ) . the following primary antibodies were employed : monoclonal anti - opa1 ( 1:1000 bd pharmingen ) , rabbit polyclonal anti - bak - nt ( 1:1000 upstate ) , mouse monoclonal anti - fp subunit ( 1:5000 ) , mouse monoclonal anti - ndufa9 subunit ( 1:1000 ) , mouse monoclonal anti - core2 subunit ( 1:1000 ) , mouse monoclonal anti - co1subunit ( 1:1000 ) ( mitoscience ) , mouse monoclonal anti - vdac1 ( 1:1000 abcam ) , rabbit polyclonal anti - tom20 ( 1:1000 santa cruz ) , mouse monoclonal anti - tim23 ( 1:1000 bd pharmingen ) , rabbit polyclonal anti - bid ( 1:1,000 , a kind gift of a. gross , weizmann institute , rehovot ) . carbonate extraction was performed as previously described ( dimmer et al . , 2008 ) . the retroviral vector pmig - tbid was described previously ( cheng et al . , 2001 ) . kkaa , g94e and g94ekkaa mutants were generated by site - direct mutagenesis using kod polymerase ( biolabs ) and the following primers : for bid forward 5-aca atg ctg ttg gcc gcc gcc gtg gcc agt cac-3 and reverse 5-gtg act ggc cac ggc ggc ggc caa cag cat tgt-3 ; for bid forward 5-ctc gcc caa ata gaa gat gag atg gac cac aac-3 and reverse 5-gtt gtg gtc cat ctc atc ttc tat ttg ggc gag-3. p7/p15 recombinant bid was produced , purified and cleaved with caspase-8 as described ( frezza et al . , 2006 ) . the growth of wt , opa1 mafs was determined by counting viable cells ( as determined by trypan blue exclusion ) daily for 5 days . 1.5x10 opa1 mafs were infected with the indicated adenoviruses and after 24 hr the growth was determined by counting viable cells ( as determined by trypan blue exclusion ) daily for 4 days . 1.5x10 dko mefs were transduced with the indicated retroviruses and after 16 hr the growth was determined by counting viable gfp positive cells by flow cytometry . for measurements of apoptosis , 1.5x10 cells of the indicated genotype grown in 6 well - plates were transfected with the indicated vector . after 48 hr apoptosis was measured by flow cytometric detection ( facscalibur ) of the annexin - v - pe positive events in the gfp - positive population . dko mefs and hek293 cells were cultured as previously described ( gomes et al . , 2011 ) . when indicated , glucose in dmem was substituted with 0.9 mg / ml galactose . ecotropic viruses were generated by cotransfecting the hek293 packaging cell lines with the packaging vector pik and the appropriate pmig as previously described ( cheng et al . , 2001 ) . viral supernatant were retrieved and used to transduce dko mefs in the presence of 4 g / ml of hexadimethrine bromide ( sigma ) . following an over - night transduction , wt , opa1 , opa1 , mafs sv40 transformed cell lines were generated respectively from wt , opa1 , opa1 mice . male 7 weeks old mice were killed by cervical dislocation and their diaphragm were carefully dissected , washed in dmem , minced and then enzymatically digested with a collagenase 0.5% ( sigma ) solution for 10 min at 37c . cell were spun at 200 g for 3 min , resuspended in dmem containing 4.5 mg / ml glucose , 10% fetal bovine serum ( fbs ) , 50 u / ml penicillin , 50 g / ml streptomycin , 100 m nonessential aminoacids ( gibco ) , 50 g / ml uridine , 25 mm hepes ( gibco ) , 2.5 g / ml fungizone ( gibco ) and seeded on a 0.1% gelatin ( sigma ) precoated plate . after 15 min the supernatant was removed and the adherent fibroblasts were left growing in complete dmem . mafs were transformed using an sv40 large t antigen expressing plasmid as previously described ( cipolat et al . , 2006 ) and

summaryrespiratory chain complexes assemble into functional quaternary structures called supercomplexes ( rcs ) within the folds of the inner mitochondrial membrane , or cristae . here , we investigate the relationship between respiratory function and mitochondrial ultrastructure and provide evidence that cristae shape determines the assembly and stability of rcs and hence mitochondrial respiratory efficiency . genetic and apoptotic manipulations of cristae structure affect assembly and activity of rcs in vitro and in vivo , independently of changes to mitochondrial protein synthesis or apoptotic outer mitochondrial membrane permeabilization . we demonstrate that , accordingly , the efficiency of mitochondria - dependent cell growth depends on cristae shape . thus , rcs assembly emerges as a link between membrane morphology and function .

Introduction Results Discussion Experimental Procedures

cytochrome c release is regulated by proteins of the bcl-2 family that control the permeabilization of the outer membrane ( omm ) ( danial and korsmeyer , 2004 ) . energy conversion occurs at the inner mitochondrial membrane ( imm ) that can be further divided into two subcompartments : the so - called boundary membrane and the cristae , separated from the former by narrow tubular junctions ( frey and mannella , 2000 ) . during apoptosis , the curvature of the cristae membrane is inverted in a remodeling process required for the complete release of cytochrome c , normally confined in the cristae ( scorrano et al . , cristae remodeling occurs in response to proapoptotic bh3-only bcl-2 family members , such as bid , bim - s , and bnip3 , and independently of the outer membrane multidomain bcl-2 family members bax and bak ( scorrano et al . , 2006 ; whether changes in morphology of the cristae , where respiratory chain complexes ( rccs ) mainly localize ( vogel et al . this issue is further complicated by the assembly of rcc in supercomplexes ( rcs ) ( schgger , 1995 ; acn - prez et al . such a supramolecular organization is common in cristae : also , the mitochondrial atp synthase is assembled into dimers with greater adenosine triphosphatase ( atpase ) activity ( campanella et al . on the contrary , despite their importance in mitochondrial bioenergetics , the relationship between rcs and cristae shape remains unclear . mitochondrial morphology and ultrastructure depends on mitochondria - shaping proteins that regulate organellar fusion and fission ( griparic and van der bliek , 2001 ) . mitofusins ( mfn ) 1 and 2 , highly homologous dynamin - related proteins of the omm , orchestrate fusion ( santel and fuller , 2001 ; legros et al . mitochondrial fission is regulated by the cytoplasmic dynamin - related protein 1 that , upon calcineurin - dependent dephosphorylation , translocates to mitochondria ( yoon et al . , 2001 ; genetic depletion of opa1 leads to disorganization of the cristae ( frezza et al . , 2006 ) , and oligomers that contain a soluble and a membrane - bound form of opa1 keep the cristae junctions tight , independently from opa1 role in fusion ( frezza et al . , 2006 ; whereas our knowledge of the molecular determinants of cristae shape and their role in apoptosis is increasing , the relationship between cristae morphology and mitochondrial function remains unexplored . we show that cristae morphology determines assembly and stability of rcs and hence optimal mitochondrial respiratory function during life and death of the cell . whether apoptotic cristae remodeling that maximizes cytochrome c release from mitochondria affects mitochondrial function is unclear , mainly because it occurs around the same time as outer membrane permeabilization ( scorrano et al . in order to genetically dissociate the two processes , we inspected the primary structure of the prototypical cristae remodeling inducer bcl-2 family member bid for homology with peptides known to perturb the mitochondrial inner membrane , like mastoparan , a 14 amino acid wasp venom component ( pfeiffer et al . to exploit the role of this homologous sequence in cristae remodeling , we mutagenized the two highly conserved 157 and 158 lys h. sapiens bid residues to ala ( bid ) ( figure s1c ) . , 2000 ; figure s1d ) , we could measure its biological activity using an established quantitative , specific cytochrome c release elisa ( scorrano et al . bak oligomerization was superimposable in cbid or cbid - treated mitochondria ( figure 1b ) ; conversely , two established assays of intramitochondrial cytochrome c redistribution , the cytochrome b5-dependent extramitochondrial nadh oxidation and the ratio of ascorbate - driven over tetramethyl - p - phenylenediamine ( tmpd)-driven respiration ( scorrano et al . indeed , cbid was unable to remodel mitochondrial cristae , as indicated by morphometric analysis of electron micrographs of mitochondria treated with the bid mutants ( figures 1e and 1f ) ( scorrano et al . finally , we measured the killing efficiency of these truncated bid ( tbid ) mutants expressed in mouse embryonic fibroblasts ( mefs ) . only tbid efficiently killed mefs : tbid and tbid elicited comparable low levels of cell death , whereas the double tbid mutant appeared completely ineffective ( figure 1j ) , suggesting that both outer membrane permeabilization and mitochondrial cristae remodeling are required for bid - induced apoptosis . the bid mutant dissociates outer membrane permeabilization from cristae remodeling and can be used to investigate the relationship between the latter and mitochondrial function . we therefore measured the effect of the bid mutants on the respiratory control ratio ( rcr ) , an index of respiratory efficiency , of mitochondria incubated with excess exogenous cytochrome c and nadh ( to compensate for the potential effects of inner membrane or outer membrane [ om ] permeabilization ) . these experiments suggest that cristae remodeling causes complex i - dependent changes in mitochondrial bioenergetics . thus , the reduction in complex i - supported respiration could be a consequence of specific inhibition of complex i or of issues in rcs function . even after 30 min of acute bid treatment , the specific complex i nadh - ubiquinone reductase activity of purified mitochondria was unaltered ( data not shown ) , prompting us to investigate rcs assembly and stability in situ . we therefore took advantage of bax , bak ( dko ) mefs , resistant to mitochondrial permeabilization , cytochrome c release , and apoptosis triggered by expression of tbid ( wei et al . upon transduction of metabolically labeled dko mefs with tbid but not with tbid that does not cause cristae remodeling ( figure s2b ) , the rcs radioactivity signal as well as the rcs / complex v radioactivity ratio were reduced ( figures 2c and 2d ) , and we observed a reduction in the autoradiographic signal of cytochrome b retrieved in rcs compared to that in free complex iii ( figures 2e and 2f ) . whether rcs disorganization was a general consequence of altered cristae shape , we turned to cells lacking opa1 , a key regulator of cristae morphology ( frezza et al . , however , chronic opa1 depletion impaired mitochondrial dna ( mtdna ) levels and translation ( figures s3a and s3b ) , complicating the analysis of the relationship between opa1 and rcs and calling for a model of conditional opa1 ablation . following cre - mediated recombination , the deletion of exons 2 and 3 resulted in an aberrant exon1exon4 transcript with a stop codon immediately after exon 1 , producing a predicted 10 amino acid ( aa ) residual protein ( figures 3a and 3b ) . opa1 was completely ablated 24 hr after adenoviral delivery of cre recombinase ( figure 4a ) and , as expected , mitochondria were fragmented ( figures 4b and 4c ) with defects in cristae shape ( figures 4d and 4e ) . four days after cre - mediated opa1 ablation , mtdna copy number ( figure 4f ) and translation ( figures 4 g and 4h ) were unaffected , allowing us to specifically address the role of opa1 and cristae shape in rcs assembly using an assay based on the incorporation of radiolabeled mtdna - encoded proteins into rcc and rcs ( acn - prez et al . upon acute opa1 ablation , the assembly of mtdna - encoded subunits into rcc was not affected ( data not shown ) . we therefore followed the rcs assembly rate ( measured as the ratio between rcs and complex v radioactivity throughout the chase period ) that resulted 8-fold slower when opa1 was ablated from opa1 mafs ( figures 4i and 4j ) . a similar reduction in the rcs assembly rate was observed in opa1 mefs ( figure s3c ) , suggesting that , in absence of opa1 , rcc are less superassembled , irrespective of their initial levels . to test if acute opa1 ablation altered rcs in vivo , we tail vein - injected cre - expressing adenoviruses in opa1 animals . after 72 hr , s4a ) , cristae morphology was abnormal ( figure s4b ) , rcs were reduced ( figure s4c ) , and glutamate / malate rcr was impaired ( figure 4k ) . these experiments of conditional ablation of opa1 identify a role for cristae shape in rcs assembly in vitro and in vivo . the model linking cristae shape to rcs predicts that higher opa1 levels should favor rcs assembly . to verify this hypothesis , we wanted to generate a mouse model of opa1 overexpression . importantly , in opa1 mafs , rcs assembly ( figures 5i and 5j ) and glutamate - supported rcr ( figure an 50% increase in liver mitochondria opa1 levels ( figure s5a ) was similarly associated to tighter cristae ( figure s5b ) and increased rcs levels ( figure s5c ) . we therefore measured the growth of dko cells ( resistant to bid - induced outer membrane permeabilization , caspase - dependent mitochondrial damage , and apoptosis ) in galactose media , where most of cellular atp comes from the respiratory chain ( acn - prez et al . in mfn1 , mfn2 mefs , mtdna copy number was reduced ( figure s6a ) , but cristae shape ( figures s6b and s6c ) , mtdna translation ( figure s6d ) , rcs stability ( figure s6e ) , and assembly ( figure s6f ) were not affected . thus , the galactose growth defect is not the consequence of impaired fusion but correlates with altered cristae shape and rcs . finally , opa1 mafs grew faster than their wt counterparts in galactose media ( figure 6d ) , further confirming the link between cristae shape , rcs levels , and mitochondria - dependent cellular growth . , 2012 ; 2013 ) , and the role of rcs in mitochondrial utilization of reducing equivalents has been demonstrated ( lapuente - brun et al . however , the relationship between cristae shape and rcs , as well as between rcs and mitochondrial function , remained obscure . our results demonstrate that cristae shape regulates respiratory chain supercomplexes stability and assembly , impacting on respiratory efficiency and respiratory cell growth . to dissect the role of cristae shape in rcs structure and function , we genetically ablated the master cristae shape regulator opa1 . however , the defect in atp production in opa1 haploinsufficient cells was unexplained : opa1 is not essential for assembly of respiratory chain complexes and mtdna levels as well as activities of individual respiratory chain complexes are normal in dominant optic atrophy ( zanna et al . conversely , the reduction in mtdna copy number has been invoked to explain the mitochondrial dysfunction of fusion - deficient cells from mfn1 , mfn2-deficient mice ( chen et al . our results challenge this hypothesis : upon acute opa1 ablation , mtdna levels are normal , whereas cristae shape , rcs , complex - i - dependent respiration , and respiratory growth are impaired . conversely , in mfn1 , mfn2 cells , mtdna copy number is reduced , but cristae shape , rcs , and respiratory growth are normal . thus , rcs disorganization shall be regarded as a key mechanism of mitochondrial dysfunction accompanying altered organelle morphology . the role of opa1 and cristae shape in rcs organization is further supported by mouse models of opa1 conditional ablation and mild overexpression . the first tool allowed us to dissociate cristae biogenesis from mtdna maintenance : whereas chronic opa1 depletion reduces mtdna copy number and translation , upon acute opa1 ablation , mtdna levels are normal , but cristae are disorganized , impacting on rcs assembly and respiratory function and growth . thus , mtdna reduction appears to be a consequence of chronic fusion inhibition in opa1 ( and double mfn ) cells . opa1 mild overexpression lends further support to the model linking rcs organization to cristae shape : rcs assembly and respiratory function and growth are increased in opa1 cells without any measurable change in mtdna levels and translation . the opa1 mouse will be instrumental to investigate the role of opa1 and cristae shape in vivo . the role and mechanisms of cristae remodeling in apoptosis are controversial ( scorrano et al . , 2008 ; 2010 ) , cristae remodeling has been reckoned as a mere feedback mechanism in situ , occurring after caspase activation ( sun et al . the bid 6 mutant generated here , which does not induce cristae changes and cytochrome c mobilization but permeabilizes the outer membrane , can be a useful tool to dissect in vivo the involvement of cristae remodeling in developmental and homeostatic apoptosis . we think that cristae remodeling influences rcs by targeting opa1 ( frezza et al . our work unravels a role for cristae shape in rcs assembly and stability , mitochondrial respiratory efficiency , and respiratory growth , suggesting that shape of biological membranes can influence membrane protein complexes . moreover , our data highlight the importance of rcs in respiration by complex i - feeding substrates . in these latter settings , stabilization of opa1 could correct rcs and therefore mitochondrial dysfunction , opening novel therapeutic perspectives for currently intractable diseases . the transgene was then inserted by homologous recombination in a pdest vector containing part of the human hypoxanthine phosphoribosyltransferase locus . for rcs detection , the concentration of digitonin in buffer d was 4% ( v / v ) . for 2d bn / sds page , the lane cut from the first - dimension bnge was incubated for 1 hr at 25c in 1% sds and 1% -mercaptoethanol and then casted on top of an 8% or a 16.5% denaturing gel . after electrophoresis , the complexes were electroblotted on a polyvinylidene fluoride ( pvdf ) membrane and probed with the indicated antibodies . to detect rcs from radiolabelled cells , samples were treated as described above , and after electrophoresis , the gels were dried and the signal was detected following exposure for 36 days . apoptosis was measured by flow cytometric detection ( facscalibur ) of the annexin - v - pe positive events in the gfp - positive population . extended experimental proceduresmouse handlingfor genotyping of the opa1 mice , the following primers were used : primer ck1 5-cag tgt tga tga cag ctc ag-3 ; primer ck2 5-cat cac aca cta gct tac att tgc-3. for genotyping of the opa1 mice , the following primers were used : primer tgf 5-gca atg acg tgg tcc tgt tttg-3 ; primer tgr 5-gat agg tca ggt aag caa gca ac-3 ; primer wtr 5-gag gga gaa aaa tgc gga gtg-3 ; primer wtf 5-ctc cgg aaa gca gtg agg taa g-3.all mice procedures were performed according to protocols approved by the local ethic committees ( protocol 32/2011 ceasa university of padova , venetian institute of molecular medicine ; license 1034/3703/02 cantonal veterinarian authority , university of geneva ) . mitochondria were isolated from opa1 mice 3 days after the tail - vein injection of the indicated adenoviruses . after 2 min 6 mm ascorbate and 300 m tmpd were added and the complex iv - dependent o2 consumption rate was measured.the enzymatic activity of complex i and citrate synthase was determined as described ( spinazzi et al . the mtdna copy number per cell was calculated using sdha amplification as a reference for nuclear dna content.imaging4x10 cells seeded on 13 mm round glass coverslips were fixed , permeabilized and blocked as previously described ( frezza et al . , 2006).assays of cell growth and deaththe growth of wt , opa1 mafs was determined by counting viable cells ( as determined by trypan blue exclusion ) daily for 5 days . after 48 hr apoptosis was measured by flow cytometric detection ( facscalibur ) of the annexin - v - pe positive events in the gfp - positive population.cell culture , transfection , virus production , and transductiondko mefs and hek293 cells were cultured as previously described ( gomes et al . following an over - night transduction , the rate of gfp expression was typically around 70% as determined by flow cytometry.wt , opa1 , opa1 , mafs sv40 transformed cell lines were generated respectively from wt , opa1 , opa1 mice . , 2006 ) and for genotyping of the opa1 mice , the following primers were used : primer ck1 5-cag tgt tga tga cag ctc ag-3 ; primer ck2 5-cat cac aca cta gct tac att tgc-3. for genotyping of the opa1 mice , the following primers were used : primer tgf 5-gca atg acg tgg tcc tgt tttg-3 ; primer tgr 5-gat agg tca ggt aag caa gca ac-3 ; primer wtr 5-gag gga gaa aaa tgc gga gtg-3 ; primer wtf 5-ctc cgg aaa gca gtg agg taa g-3. mitochondria were isolated from opa1 mice 3 days after the tail - vein injection of the indicated adenoviruses . after 2 min 6 mm ascorbate and 300 m tmpd were added and the complex iv - dependent o2 consumption rate was measured . the enzymatic activity of complex i and citrate synthase was determined as described ( spinazzi et al . for measurements of apoptosis , 1.5x10 cells of the indicated genotype grown in 6 well - plates were transfected with the indicated vector .

diabetes is thought to be a stronger cardiovascular disease ( cvd ) risk factor in women than in men because women with type 2 diabetes ( dm2 ) are more likely to develop heart failure ( 1 ) and die of a cvd event than those without diabetes or men of the same age with or without diabetes ( 2 ) . however , the reasons for the sex difference in diabetes - associated cvd are not well understood . diabetes usually occurs in the presence of other cardiometabolic factors , such as hypertension , adiposity , dyslipidemia , and insulin resistance , which all appear to be accentuated in dm2 and may predispose to a greater cvd risk . meta - analyses suggest , however , that the vulnerability to cvd among individuals with dm2 may exist after accounting for these factors ( 35 ) and may reflect interacting effects of estrogens and insulin on cardiomyocytes ( 5 ) or a disparity in the recognition and care for heart disease in dm2 ( 6 ) . androgens are thought to contribute to the earlier development of heart disease in men ( 7 ) . testosterone levels are higher in postmenopausal women with diabetes compared with those without diabetes independent of age or adiposity ( 710 ) , and several reports have suggested that androgen production may play a role in their elevated cvd risk ( 8,9 ) . lower levels of sex - hormone binding globulin ( shbg ) in dm2 further increase the non - shbg ( bioavailable ) testosterone and may amplify its actions . androgens have been associated with subclinical cvd ( 11,12 ) , but evidence for an association between androgens and incident cardiovascular end points among women with diabetes in longitudinal studies is lacking . the source of excess androgens in dm2 has not been studied in detail ; however , results in women with polycystic ovary syndrome ( pcos ) , who are also obese and insulin resistant , suggest a predominant ovarian source ( 13 ) , although adrenal steroid production might also be increased ( 14 ) . bilateral salpingo oophorectomy ( bso ) some studies found bso is associated with cvd risk ( 1618 ) , but others did not ( 1921 ) , and no studies have focused on dm2 . if elevated androgen levels increase the risk for cvd in dm2 , we tested the hypothesis that bso would be associated with a reduced risk of cvd mortality among women with dm2 using data from the study of osteoporotic fractures ( sof ) . sof is a multicenter prospective observational study of women aged 65 or older , with clinical sites at baltimore , md ; minneapolis mn ; portland , or ; and monongahela valley near pittsburgh , pa . originally , 9,704 women , who were predominantly caucasian , were recruited from september 1986 to october 1988 through population - based listings ( 22 ) . women who had a history of bilateral hip replacement or were unable to walk without assistance were excluded ( 23 ) . follow - up contacts for ascertaining vital status were performed every 4 months . to date all participants provided written informed consent , and human subject research approval was obtained by each clinical center 's institutional review board . for the present analyses , we excluded women who had missing or unknown age at hysterectomy , oophorectomy , or last menstrual period ( n = 87 ) ; unilateral or unknown oophorectomy status ( n = 795 ) ; or intact uterus but underwent bso ( n = 43 ) and those with prevalent cvd ( n = 802 ) , as defined by a physician - diagnosed heart attack , coronary , or myocardial infarction , or stroke , yielding an analytic sample of 7,977 women . sex hormone measurements were assayed in a random subsample of the cohort and were restricted to women who reported they were not taking postmenopausal hormone therapy ( n = 954 ) . for this present analysis of sex hormones , only women who had no missing baseline values for androgens were included ( n = 564 ) . participants in this analytic subsample were not different from those excluded in age , education , smoking status , bmi , waist girth , bso status , parity , physical activity , age at menarche , and age at menopause . information on age , race , education , and smoking status was self - reported . height , weight , bmi , waist circumference , hip girth , and waist - to - hip ratio were measured according to standard protocols ( 22 ) . recreational and sports activities from the previous 12 months were recorded using a modified version of the harvard alumni questionnaire and were converted into weekly caloric expenditure by intensity level ( 23 ) . participants were asked at baseline and follow - up about their medical and reproductive health histories , including gynecological surgeries . age at menarche and natural menopause , hysterectomy status , parity , and hormone therapy use were all self - reported . women in whom both ovaries were surgically removed were defined to have bso , with the age at which this occurred recorded . participants were asked had a doctor had ever told you that you have diabetes or women who answered yes were also asked about current insulin use . a systolic blood pressure above 140 mmhg and/or diastolic blood pressure above 90 mmhg defined a diagnosis of hypertension . at baseline , nonfasting blood samples were drawn from all participants between the hours of 8:00 a.m. and 2:00 p.m. participants were instructed to avoid fatty foods on the morning of the examination to minimize lipemia , which may interfere with assays . blood was drawn after the participant had been seated for at least 10 min and was immediately frozen to 20c for up to 2 weeks and stored in liquid nitrogen at 190c until assays were done ( 2426 ) . total testosterone was measured by radioimmunoassay after extraction and aluminum oxide column chromatography with interassay coefficient of variation of 6.113.4% ( 2426 ) . free testosterone was measured using an ammonium sulfate precipitation procedure with an interassay coefficient of variation of 10.715.5% ( 2426 ) , with adjustment for the albumin concentration ( coefficient of variation for intra - assay and total assay 5% and 5.4% , respectively ; sensitivity 34.7 pmol / l ) ( 27 ) . dehydroepiandrosterone sulfate ( dheas ) was measured using radioimmunoassay with sample dilution ( coefficient of variation for intra - assay and interassay 611% and 912% , respectively ( 27 ) . all biochemical analyses were performed at nichols institute ( san juan capistrano , ca ) or endocrine sciences esoterix ( calabasas hills , ca ) . in both laboratories , the intra - assay and interassay coefficients of variation , respectively , for total testosterone ranged from 4 to 12% and from 9 to 11% for the assays done by nichols and from 3 to 13% and from 9 to 14% for assays done by endocrine sciences esoterix ( 2426 ) . blood samples from 51 women were selected to determine the stability of these hormones in stored serum . accordingly , samples obtained at baseline and after 3.5 years of follow - up stored at 190c were analyzed . the correlations between testosterone measured at these two time points was r = 0.99 ( p < 0.001 ) ( 26 ) . we obtained copies of original death certificates of participants who died during follow - up . cvd - related mortality was defined using icd-9 codes 402 , 404 , 410414 , and 426445 , which represented deaths from atherosclerosis , stroke , or coronary heart disease , respectively . comparisons for categorical variables were assessed using , and continuous variables were tested using the t test and anova . analyses of covariance with adjustment for age and bmi were used for comparison of sex hormones . quantitative measures that were skewed were normalized by natural log transformation . the tukey adjustment method was used for multiple comparisons of sex hormones among the groups with diabetes - bso . in time - to - event analysis , the kaplan - meier method was used to estimate the risk of cvd mortality . survival curves were produced to graphically depict incident events with differences among groups assessed by means of the log - rank test . estimation of adjusted hazard ratios ( hr ) and 95% ci for cvd mortality were calculated using cox proportional hazards regression models . the proportionality assumption was tested using cumulative sums of martingale residuals with a kolmogorov - type supremum test and also by visually inspecting plots of schoenfeld residuals versus time . because testosterone levels decrease during the menopausal transition ( 28 ) and our previous study of women in the national health and nutrition examination survey ( 29 ) suggested that bso before age 45 years increased the cvd risk for dm2 , we examined all - cause and cvd mortality in women stratified by age at menopause ( 45 vs. > 45 years ) , a cut point that others have also used ( 30 ) . a two - tailed p value of < 0.05 was considered statistically significant . in sensitivity analyses , we calculated hrs using fine and gray methodology ( 31 ) for competing risks , taking into account the competing risk of death from other non - cvd causes . sof is a multicenter prospective observational study of women aged 65 or older , with clinical sites at baltimore , md ; minneapolis mn ; portland , or ; and monongahela valley near pittsburgh , pa . originally , 9,704 women , who were predominantly caucasian , were recruited from september 1986 to october 1988 through population - based listings ( 22 ) . women who had a history of bilateral hip replacement or were unable to walk without assistance were excluded ( 23 ) . follow - up contacts for ascertaining vital status were performed every 4 months . to date all participants provided written informed consent , and human subject research approval was obtained by each clinical center 's institutional review board . for the present analyses , we excluded women who had missing or unknown age at hysterectomy , oophorectomy , or last menstrual period ( n = 87 ) ; unilateral or unknown oophorectomy status ( n = 795 ) ; or intact uterus but underwent bso ( n = 43 ) and those with prevalent cvd ( n = 802 ) , as defined by a physician - diagnosed heart attack , coronary , or myocardial infarction , or stroke , yielding an analytic sample of 7,977 women . sex hormone measurements were assayed in a random subsample of the cohort and were restricted to women who reported they were not taking postmenopausal hormone therapy ( n = 954 ) . for this present analysis of sex hormones , only women who had no missing baseline values for androgens were included ( n = 564 ) . participants in this analytic subsample were not different from those excluded in age , education , smoking status , bmi , waist girth , bso status , parity , physical activity , age at menarche , and age at menopause . information on age , race , education , and smoking status was self - reported . height , weight , bmi , waist circumference , hip girth , and waist - to - hip ratio were measured according to standard protocols ( 22 ) . recreational and sports activities from the previous 12 months were recorded using a modified version of the harvard alumni questionnaire and were converted into weekly caloric expenditure by intensity level ( 23 ) . participants were asked at baseline and follow - up about their medical and reproductive health histories , including gynecological surgeries . age at menarche and natural menopause , hysterectomy status , parity , and hormone therapy use were all self - reported . women in whom both ovaries were surgically removed were defined to have bso , with the age at which this occurred recorded . participants were asked had a doctor had ever told you that you have diabetes or sugar diabetes ? women who answered yes were also asked about current insulin use . a systolic blood pressure above 140 mmhg and/or diastolic blood pressure above 90 mmhg defined a diagnosis of hypertension . at baseline , nonfasting blood samples were drawn from all participants between the hours of 8:00 a.m. and 2:00 p.m. participants were instructed to avoid fatty foods on the morning of the examination to minimize lipemia , which may interfere with assays . blood was drawn after the participant had been seated for at least 10 min and was immediately frozen to 20c for up to 2 weeks and stored in liquid nitrogen at 190c until assays were done ( 2426 ) . total testosterone was measured by radioimmunoassay after extraction and aluminum oxide column chromatography with interassay coefficient of variation of 6.113.4% ( 2426 ) . free testosterone was measured using an ammonium sulfate precipitation procedure with an interassay coefficient of variation of 10.715.5% ( 2426 ) , with adjustment for the albumin concentration ( coefficient of variation for intra - assay and total assay 5% and 5.4% , respectively ; sensitivity 34.7 pmol / l ) ( 27 ) . dehydroepiandrosterone sulfate ( dheas ) was measured using radioimmunoassay with sample dilution ( coefficient of variation for intra - assay and interassay 611% and 912% , respectively ( 27 ) . all biochemical analyses were performed at nichols institute ( san juan capistrano , ca ) or endocrine sciences esoterix ( calabasas hills , ca ) . in both laboratories , the intra - assay and interassay coefficients of variation , respectively , for total testosterone ranged from 4 to 12% and from 9 to 11% for the assays done by nichols and from 3 to 13% and from 9 to 14% for assays done by endocrine sciences esoterix ( 2426 ) . blood samples from 51 women were selected to determine the stability of these hormones in stored serum . accordingly , samples obtained at baseline and after 3.5 years of follow - up stored at 190c were analyzed . the correlations between testosterone measured at these two time points was r = 0.99 ( p < 0.001 ) ( 26 ) . we obtained copies of original death certificates of participants who died during follow - up . cvd - related mortality was defined using icd-9 codes 402 , 404 , 410414 , and 426445 , which represented deaths from atherosclerosis , stroke , or coronary heart disease , respectively . comparisons for categorical variables were assessed using , and continuous variables were tested using the t test and anova . analyses of covariance with adjustment for age and bmi were used for comparison of sex hormones . quantitative measures that were skewed were normalized by natural log transformation . the tukey adjustment method was used for multiple comparisons of sex hormones among the groups with diabetes - bso . in time - to - event analysis , survival curves were produced to graphically depict incident events with differences among groups assessed by means of the log - rank test . estimation of adjusted hazard ratios ( hr ) and 95% ci for cvd mortality were calculated using cox proportional hazards regression models . the proportionality assumption was tested using cumulative sums of martingale residuals with a kolmogorov - type supremum test and also by visually inspecting plots of schoenfeld residuals versus time . because testosterone levels decrease during the menopausal transition ( 28 ) and our previous study of women in the national health and nutrition examination survey ( 29 ) suggested that bso before age 45 years increased the cvd risk for dm2 , we examined all - cause and cvd mortality in women stratified by age at menopause ( 45 vs. > 45 years ) , a cut point that others have also used ( 30 ) . a two - tailed p value of < 0.05 was considered statistically significant . in sensitivity analyses , we calculated hrs using fine and gray methodology ( 31 ) for competing risks , taking into account the competing risk of death from other non - cvd causes . baseline characteristics of the participants are presented in table 1 according to diabetes and bso status . the mean sd age at baseline was 71.5 5.3 years , with a 6.3% prevalence of diabetes . approximately 37% of participants had undergone hysterectomy . of these , 49% had concomitant bso at a mean age of 45.6 years ( 95% ci 45.346.0 ) . a greater proportion of women with bso , compared with women with intact ovaries , were current users of hormone therapy ( 25.0 vs. 9.7% ) , nulliparous ( 22.1 vs. 18.3% ) , or hypertensive ( 41.0 vs. 37.4% ) . women with diabetes , compared with those without diabetes , were more likely to never use hormone therapy ( 67.1 vs. 52.8% ) or report a hysterectomy ( 42.0 vs. 36.9% ) and were hypertensive ( 54.3 vs. 37.0% ) . with regard to differences by ovarian status among dm2 , women with bso reached menopause at an earlier age ( 44.7 vs. 48.8 years ) , were more likely to be current hormone therapy users ( 15.8 vs. 4.0% ) , and were more often nulliparous ( 21.8 vs. 17.2% ) compared with those with intact ovaries , with all differences meeting statistical significance . baseline characteristics of 7,977 participants according to diabetes and bso status from the sof values are displayed as percentages or mean ( sd ) . p values are based on the test for categorical variables and anova for normally distributed continuous variables . p value comparing women with diabetes and no bso to women with diabetes and bso . sex - steroid hormones levels according to dm2 and oophorectomy status are reported in table 2 . on the one hand , women with diabetes had lower shbg ( p < 0.001 ) and higher free testosterone levels ( p = 0.026 ) than women without diabetes that was independent of age and bmi . on the other hand , women with dm2 with intact ovaries had higher estrone levels than women without dm2 ( p < 0.001 ) . as expected , total testosterone ( p < 0.001 ) and free testosterone ( p < 0.001 ) levels were lower in women with bso than in women with intact ovaries . bso was associated with slightly lower shbg levels in women with dm2 but with higher levels ( p = 0.001 ) in women without dm2 . no statistically significant differences were found for estrone or dheas levels by diabetes diagnosis or ovarian status . restricted to dm2 , those with bso had significantly lower levels of total testosterone and a higher percentage of free testosterone than women with dm2 and intact ovaries . however , after adjustment for age and bmi , only the variation in percentage of free testosterone by ovarian status persisted among women with dm2 . hormone levels according to diabetes and bso status among a subsample of participants ( n = 564 ) in the sof data are shown as the median ( interquartile range ) . * p value comparing all four categories based on anova for normally distributed continuous variables after log transformation . . p value for subgroup analyses restricted to only women with diabetes by bso status adjusted for age and bmi , based on ancova . p value for comparing women with and without diabetes regardless of bso status adjusted for age and bmi . after a mean follow - up of 15.1 years , 4,797 deaths occurred , with 1,638 ( 34.1% ) attributed to cvd . cvd - related mortality rates per 1,000 , according to diabetes and bso status , were no diabetes or bso , 12.9 ; bso without diabetes , 12.7 ; diabetes without bso , 25.9 ; and diabetes with bso , 31.4 . the incidence of cvd mortality was elevated in women with diabetes , with or without bso ( p < 0.001 ) ( fig . the risk of cvd mortality was elevated for those with intact ovaries ( hr 1.95 , 95% ci 1.622.35 ) and for those with bso ( hr 2.56 , 95% ci 1.793.65 ) ( table 3 ) . although cvd mortality in women with diabetes and bso was slightly higher compared with women with diabetes and intact ovaries , this difference did not meet statistical significance ( hr 1.49 , 95% ci 0.902.46 , p = 0.120 ) . overall , bso was not associated with cvd mortality ( hr 1.05 , 95% ci 0.891.23 ) . in addition , no significant interaction was found between diabetes - bso status and hormone therapy ( p = 0.340 ) . kaplan - meier cumulative incidence estimates of cvd mortality according to diabetes and oophorectomy status . all - cause and cvd mortality according to diabetes and sbo status and age at menopause from the sof * adjusted for age , education , age at menopause , hysterectomy , parity , postmenopausal hormone use , bmi , waist - to - hip ratio , hypertension , smoking status , and physical activity . we next performed analyses stratified by age at menopause ( 45 or > 45 years ) ( table 3 ) and found that cvd mortality risk was elevated for women with diabetes regardless of age at menopause . however , in analyses restricted to dm2 , with those who had intact ovaries as referent , the adjusted risk of cvd mortality was elevated for women with dm2 who had bso before or at 45 years of age ( hr 2.75 , 95% ci 1.246.11 , p = 0.012 ) , whereas the risk among women with dm2 who had surgery at or after age 45 years was not statistically different from that for women with dm2 with intact ovaries ( hr 0.94 , 95% ci 0.481.85 , p = 0.864 and p = 0.097 for interaction ) . similar results were obtained when the analyses were stratified by the average age ( 50 years ) at natural menopause ( data not shown ) . in a sensitivity analyses , we observed that these associations were similar when deaths from other non - cvd causes were censored or treated as competing events ( supplementary table 1 ) . sex - steroid hormones levels according to dm2 and oophorectomy status are reported in table 2 . on the one hand , women with diabetes had lower shbg ( p < 0.001 ) and higher free testosterone levels ( p = 0.026 ) than women without diabetes that was independent of age and bmi . on the other hand , women with dm2 with intact ovaries had higher estrone levels than women without dm2 ( p < 0.001 ) . as expected , total testosterone ( p < 0.001 ) and free testosterone ( p < 0.001 ) levels were lower in women with bso than in women with intact ovaries . bso was associated with slightly lower shbg levels in women with dm2 but with higher levels ( p = 0.001 ) in women without dm2 . no statistically significant differences were found for estrone or dheas levels by diabetes diagnosis or ovarian status . these observations remained even after adjusting for age and bmi . in analysis restricted to dm2 , those with bso had significantly lower levels of total testosterone and a higher percentage of free testosterone than women with dm2 and intact ovaries . however , after adjustment for age and bmi , only the variation in percentage of free testosterone by ovarian status persisted among women with dm2 . hormone levels according to diabetes and bso status among a subsample of participants ( n = 564 ) in the sof data are shown as the median ( interquartile range ) . * p value comparing all four categories based on anova for normally distributed continuous variables after log transformation . p value for subgroup analyses restricted to only women with diabetes by bso status adjusted for age and bmi , based on ancova . p value for comparing women with and without diabetes regardless of bso status adjusted for age and bmi . after a mean follow - up of 15.1 years , 4,797 deaths occurred , with 1,638 ( 34.1% ) attributed to cvd . cvd - related mortality rates per 1,000 , according to diabetes and bso status , were no diabetes or bso , 12.9 ; bso without diabetes , 12.7 ; diabetes without bso , 25.9 ; and diabetes with bso , 31.4 . the incidence of cvd mortality was elevated in women with diabetes , with or without bso ( p < 0.001 ) ( fig . the risk of cvd mortality was elevated for those with intact ovaries ( hr 1.95 , 95% ci 1.622.35 ) and for those with bso ( hr 2.56 , 95% ci 1.793.65 ) ( table 3 ) . although cvd mortality in women with diabetes and bso was slightly higher compared with women with diabetes and intact ovaries , this difference did not meet statistical significance ( hr 1.49 , 95% ci 0.902.46 , p = 0.120 ) . overall , bso was not associated with cvd mortality ( hr 1.05 , 95% ci 0.891.23 ) . in addition , no significant interaction was found between diabetes - bso status and hormone therapy ( p = 0.340 ) . kaplan - meier cumulative incidence estimates of cvd mortality according to diabetes and oophorectomy status . all - cause and cvd mortality according to diabetes and sbo status and age at menopause from the sof * adjusted for age , education , age at menopause , hysterectomy , parity , postmenopausal hormone use , bmi , waist - to - hip ratio , hypertension , smoking status , and physical activity . we next performed analyses stratified by age at menopause ( 45 or > 45 years ) ( table 3 ) and found that cvd mortality risk was elevated for women with diabetes regardless of age at menopause . however , in analyses restricted to dm2 , with those who had intact ovaries as referent , the adjusted risk of cvd mortality was elevated for women with dm2 who had bso before or at 45 years of age ( hr 2.75 , 95% ci 1.246.11 , p = 0.012 ) , whereas the risk among women with dm2 who had surgery at or after age 45 years was not statistically different from that for women with dm2 with intact ovaries ( hr 0.94 , 95% ci 0.481.85 , p = 0.864 and p = 0.097 for interaction ) . similar results were obtained when the analyses were stratified by the average age ( 50 years ) at natural menopause ( data not shown ) . in a sensitivity analyses , we observed that these associations were similar when deaths from other non - cvd causes were censored or treated as competing events ( supplementary table 1 ) . to our knowledge , this is the first study to assess the association between bso , serum androgens , and cvd mortality in a large population of women with or without diabetes . we found that diabetes in postmenopausal caucasian women was associated with a higher cvd mortality risk that appeared to be influenced by the age at surgical menopause . as in previous studies , androgen levels were higher in women with diabetes ( 710 ) . because androgens have been linked to cvd risk , possibly due to insulin resistance and glycemia , and to adverse effects on the coronary vasculature ( 32 ) , cardiac myocytes ( 33 ) , and carotid artery intima - media thickness ( 11,12 ) , we hypothesized that bso would actually reduce the cvd risk in women with dm2 . however , our analysis of this prospective cohort of 7,977 women aged 65 years and older does not support this hypothesis . the cvd risk among women with dm2 appeared to be greater in women who had bso before or at age 45 years . although this risk may be explained by estrogen deficiency ( 30,34 ) , bso before age 45 years did not increase cvd mortality risk in women without diabetes . thus it is intriguing to propose that a unique underlying disease process links bso and cvd in dm2 . on the one hand , no difference was found in bmi , waist circumference , age at menarche , or prevalence of hypertension between women with dm2 with bso and those without bso . on the other hand , parity was less among women with dm2 with bso , and shbg levels tended to be lower rather than increased as in women without diabetes . few studies ( 30,34 ) have included the reason for pelvic surgery in their analysis , but in women from olmstead county , mn ( 34 ) , elevated mortality was limited to those who were younger than age 45 years at bso and those who underwent bso for benign tumors or inflammation but not the larger group of women with endometriosis . we propose that further investigation that includes indications for bso in younger women with dm2 may provide insight into why they are at higher risk for cvd . further , we hypothesize that hysterectomies concomitant with bso may be performed in young women because of ovarian cysts in conjunction with irregular menstrual bleeding in whom the diagnosis is pcos , a high - risk group for insulin resistance , diabetes , and cvd . bso , particularly at a young age , has been associated with increased overall and cvd mortality in the general population in some studies but not in others ( 30,34 ) . the nurses health study ( 16 ) found a 17% higher risk for cvd in women with bso and a 44% higher risk among those women with bso before age 45 years . the women s health initiative ( 19 ) found no statistically significant differences in the rates of fatal and nonfatal chd , stroke , or total cvd in women with hysterectomy concomitant with bso compared with women with hysterectomy alone . the california teachers study found no elevated cvd risk or mortality with bso irrespective of hormone therapy use or age at bso ( 35 ) . the prevalence of diabetes in each of these studies was low compared with estimates from the current study . in this cohort of postmenopausal women , shbg levels were lower and free testosterone levels were higher among women with diabetes independent of age and adiposity . several previous studies have found similar results ( 8,9 ) ; however , the source and pathogenesis of the androgen excess are not well understood . by extrapolating the findings in insulin - resistant premenopausal women with pcos , korytkowski et al . ( 8) proposed that elevated androgens in postmenopausal women with diabetes are of ovarian origin . however , results after dexamethasone suppression and acth stimulation in postmenopausal women with a history of pcos suggest that the ovary and adrenal cortex both contribute to the elevated androgen levels in these subjects ( 14 ) . in the current study , free testosterone levels among women with bso were slightly but not statistically higher in women with diabetes than in those without diabetes after accounting for the effect of age and adiposity . the level of dheas ( an androgenic steroid produced by the adrenal cortex ) was similar in the two groups as in other studies ( 8) . these results favor the ovary as the predominant but not the sole source of androgens in postmenopausal women with diabetes . the use of a large sample of postmenopausal women with sufficiently long follow - up , coupled with minimal loss to follow - up of participants , enhances the assessment of mortality outcomes , which were adjudicated . in addition , total testosterone was measured by radioimmunoassay after extraction and aluminum oxide column chromatography , a method with good precision and sensitivity and a high accuracy comparable to liquid chromatography - tandem mass spectrometry for the low values in women ( 36 ) . the analytic sample consisted of caucasian women , limiting any generalization to women of other races . further , because sex hormones were measured in a small subset of only five women with diabetes and bso , we could not test whether sex steroids modify the relationship between bso and diabetes on mortality . a single measure of sex hormones at baseline was used to characterize each woman s hormonal status . however , a single measurement for plasma sex hormones in postmenopausal women can reliably categorize average levels over a period of at least 3 years ( 37 ) . adjustment for lipids in multivariable models was not feasible because only a subsample of women had measures of cholesterol and triglycerides . a failure to recall the actual age at menopause and oophorectomy occurring many years before study enrollment could potentially lead to misclassification . a woman s accuracy in recalling age at oophorectomy seems to surpass recall of age at natural menopause ( 38 ) . the use of death certificates to identify cardiovascular deaths may have resulted in some misclassification of cause of death ( 39 ) especially among elderly women , who often have multiple medical problems . our cvd findings were consistent , however , with all - cause mortality , suggesting that the influence of such misclassification on our results , if present , may be minimal . lastly , the diagnosis of diabetes was based on self - report of physician diagnosis and no doubt is an underestimate because many women could have had undiagnosed diabetes . regardless , this random misclassification would have been expected to bias the results toward the null given the observational design of our study . in summary , women with dm2 have a higher cvd mortality risk that is not attenuated by a history of bso . these findings imply that ovarian hyperandrogenemia may not be the primary mechanism to explain cvd mortality among postmenopausal women with dm2 . the apparent influence of age at menopause on the relation between bso and diabetes with cvd mortality raises the possibility that joint factors may influence the risk for diabetes , cvd , and the decision for bso in younger women . these results warrant further investigations among younger women with diabetes and bso to determine if this risk is due to bso or is a marker for risk factors that could be associated with pcos . as such , it is essential and prudent that we understand and identify the underlying factors that predispose women with diabetes to cardiovascular events to aid in the early detection and management of high - risk women in a timely fashion .

objectivediabetes elevates cardiovascular disease ( cvd ) risk more markedly in women than in men . because the high risk of cvd among women with type 2 diabetes ( dm2 ) may be partly due to increased ovarian androgen production , we investigated whether a history of bilateral salpingo oophorectomy ( bso ) is inversely associated with cvd mortality among women with dm2.research design and methodsdata were obtained from 7,977 women ( a random subset of 564 had measurements of sex - steroid hormones ) enrolled in the study of osteoporotic fractures ( sof ) , a community - based , multicenter study that monitored women aged 65 years for a mean of 15.1 years . adjusted hazard ratios ( hrs ) and 95% cis were calculated using cox proportional hazards regression.resultsthe average age at baseline was 71.5 years , with 6.3% and 18% of participants reporting a history of diabetes or bso , respectively . in the subset of the sof cohort with sex - steroid hormone measurements , those with dm2 had 43.6% significantly higher levels of free testosterone that were partly explained by age and adiposity , whereas total and free testosterone levels were lower in women with bso than in those with intact ovaries . cvd mortality was elevated in women with dm2 without bso ( hr 1.95 , 95% ci 1.622.35 ) as well as in women with dm2 and bso ( hr 2.56 , 95% ci 1.793.65 ; p = 0.190 for interaction ) . overall , bso was not associated with cvd mortality ( hr 1.05 , 95% ci 0.891.23).conclusionsthe association of diabetes with cvd was not reduced by bso , suggesting that ovarian hyperandrogenemia may not be a primary mechanism to explain the high risk for cvd among women with dm2 .

Introduction Research Design and Methods Study Population Determinants and Covariates Biochemical Analyses Outcome Statistical Analyses Results Sex-Steroid Hormone Levels Incident CVD Conclusions Supplementary Material

diabetes is thought to be a stronger cardiovascular disease ( cvd ) risk factor in women than in men because women with type 2 diabetes ( dm2 ) are more likely to develop heart failure ( 1 ) and die of a cvd event than those without diabetes or men of the same age with or without diabetes ( 2 ) . meta - analyses suggest , however , that the vulnerability to cvd among individuals with dm2 may exist after accounting for these factors ( 35 ) and may reflect interacting effects of estrogens and insulin on cardiomyocytes ( 5 ) or a disparity in the recognition and care for heart disease in dm2 ( 6 ) . testosterone levels are higher in postmenopausal women with diabetes compared with those without diabetes independent of age or adiposity ( 710 ) , and several reports have suggested that androgen production may play a role in their elevated cvd risk ( 8,9 ) . lower levels of sex - hormone binding globulin ( shbg ) in dm2 further increase the non - shbg ( bioavailable ) testosterone and may amplify its actions . androgens have been associated with subclinical cvd ( 11,12 ) , but evidence for an association between androgens and incident cardiovascular end points among women with diabetes in longitudinal studies is lacking . the source of excess androgens in dm2 has not been studied in detail ; however , results in women with polycystic ovary syndrome ( pcos ) , who are also obese and insulin resistant , suggest a predominant ovarian source ( 13 ) , although adrenal steroid production might also be increased ( 14 ) . bilateral salpingo oophorectomy ( bso ) some studies found bso is associated with cvd risk ( 1618 ) , but others did not ( 1921 ) , and no studies have focused on dm2 . if elevated androgen levels increase the risk for cvd in dm2 , we tested the hypothesis that bso would be associated with a reduced risk of cvd mortality among women with dm2 using data from the study of osteoporotic fractures ( sof ) . sof is a multicenter prospective observational study of women aged 65 or older , with clinical sites at baltimore , md ; minneapolis mn ; portland , or ; and monongahela valley near pittsburgh , pa . women who had a history of bilateral hip replacement or were unable to walk without assistance were excluded ( 23 ) . for the present analyses , we excluded women who had missing or unknown age at hysterectomy , oophorectomy , or last menstrual period ( n = 87 ) ; unilateral or unknown oophorectomy status ( n = 795 ) ; or intact uterus but underwent bso ( n = 43 ) and those with prevalent cvd ( n = 802 ) , as defined by a physician - diagnosed heart attack , coronary , or myocardial infarction , or stroke , yielding an analytic sample of 7,977 women . sex hormone measurements were assayed in a random subsample of the cohort and were restricted to women who reported they were not taking postmenopausal hormone therapy ( n = 954 ) . free testosterone was measured using an ammonium sulfate precipitation procedure with an interassay coefficient of variation of 10.715.5% ( 2426 ) , with adjustment for the albumin concentration ( coefficient of variation for intra - assay and total assay 5% and 5.4% , respectively ; sensitivity 34.7 pmol / l ) ( 27 ) . in time - to - event analysis , the kaplan - meier method was used to estimate the risk of cvd mortality . estimation of adjusted hazard ratios ( hr ) and 95% ci for cvd mortality were calculated using cox proportional hazards regression models . because testosterone levels decrease during the menopausal transition ( 28 ) and our previous study of women in the national health and nutrition examination survey ( 29 ) suggested that bso before age 45 years increased the cvd risk for dm2 , we examined all - cause and cvd mortality in women stratified by age at menopause ( 45 vs. > 45 years ) , a cut point that others have also used ( 30 ) . sof is a multicenter prospective observational study of women aged 65 or older , with clinical sites at baltimore , md ; minneapolis mn ; portland , or ; and monongahela valley near pittsburgh , pa . women who had a history of bilateral hip replacement or were unable to walk without assistance were excluded ( 23 ) . for the present analyses , we excluded women who had missing or unknown age at hysterectomy , oophorectomy , or last menstrual period ( n = 87 ) ; unilateral or unknown oophorectomy status ( n = 795 ) ; or intact uterus but underwent bso ( n = 43 ) and those with prevalent cvd ( n = 802 ) , as defined by a physician - diagnosed heart attack , coronary , or myocardial infarction , or stroke , yielding an analytic sample of 7,977 women . sex hormone measurements were assayed in a random subsample of the cohort and were restricted to women who reported they were not taking postmenopausal hormone therapy ( n = 954 ) . women in whom both ovaries were surgically removed were defined to have bso , with the age at which this occurred recorded . free testosterone was measured using an ammonium sulfate precipitation procedure with an interassay coefficient of variation of 10.715.5% ( 2426 ) , with adjustment for the albumin concentration ( coefficient of variation for intra - assay and total assay 5% and 5.4% , respectively ; sensitivity 34.7 pmol / l ) ( 27 ) . estimation of adjusted hazard ratios ( hr ) and 95% ci for cvd mortality were calculated using cox proportional hazards regression models . because testosterone levels decrease during the menopausal transition ( 28 ) and our previous study of women in the national health and nutrition examination survey ( 29 ) suggested that bso before age 45 years increased the cvd risk for dm2 , we examined all - cause and cvd mortality in women stratified by age at menopause ( 45 vs. > 45 years ) , a cut point that others have also used ( 30 ) . the mean sd age at baseline was 71.5 5.3 years , with a 6.3% prevalence of diabetes . a greater proportion of women with bso , compared with women with intact ovaries , were current users of hormone therapy ( 25.0 vs. 9.7% ) , nulliparous ( 22.1 vs. 18.3% ) , or hypertensive ( 41.0 vs. 37.4% ) . with regard to differences by ovarian status among dm2 , women with bso reached menopause at an earlier age ( 44.7 vs. 48.8 years ) , were more likely to be current hormone therapy users ( 15.8 vs. 4.0% ) , and were more often nulliparous ( 21.8 vs. 17.2% ) compared with those with intact ovaries , with all differences meeting statistical significance . sex - steroid hormones levels according to dm2 and oophorectomy status are reported in table 2 . on the one hand , women with diabetes had lower shbg ( p < 0.001 ) and higher free testosterone levels ( p = 0.026 ) than women without diabetes that was independent of age and bmi . on the other hand , women with dm2 with intact ovaries had higher estrone levels than women without dm2 ( p < 0.001 ) . as expected , total testosterone ( p < 0.001 ) and free testosterone ( p < 0.001 ) levels were lower in women with bso than in women with intact ovaries . bso was associated with slightly lower shbg levels in women with dm2 but with higher levels ( p = 0.001 ) in women without dm2 . restricted to dm2 , those with bso had significantly lower levels of total testosterone and a higher percentage of free testosterone than women with dm2 and intact ovaries . however , after adjustment for age and bmi , only the variation in percentage of free testosterone by ovarian status persisted among women with dm2 . hormone levels according to diabetes and bso status among a subsample of participants ( n = 564 ) in the sof data are shown as the median ( interquartile range ) . after a mean follow - up of 15.1 years , 4,797 deaths occurred , with 1,638 ( 34.1% ) attributed to cvd . cvd - related mortality rates per 1,000 , according to diabetes and bso status , were no diabetes or bso , 12.9 ; bso without diabetes , 12.7 ; diabetes without bso , 25.9 ; and diabetes with bso , 31.4 . the incidence of cvd mortality was elevated in women with diabetes , with or without bso ( p < 0.001 ) ( fig . the risk of cvd mortality was elevated for those with intact ovaries ( hr 1.95 , 95% ci 1.622.35 ) and for those with bso ( hr 2.56 , 95% ci 1.793.65 ) ( table 3 ) . although cvd mortality in women with diabetes and bso was slightly higher compared with women with diabetes and intact ovaries , this difference did not meet statistical significance ( hr 1.49 , 95% ci 0.902.46 , p = 0.120 ) . overall , bso was not associated with cvd mortality ( hr 1.05 , 95% ci 0.891.23 ) . all - cause and cvd mortality according to diabetes and sbo status and age at menopause from the sof * adjusted for age , education , age at menopause , hysterectomy , parity , postmenopausal hormone use , bmi , waist - to - hip ratio , hypertension , smoking status , and physical activity . we next performed analyses stratified by age at menopause ( 45 or > 45 years ) ( table 3 ) and found that cvd mortality risk was elevated for women with diabetes regardless of age at menopause . however , in analyses restricted to dm2 , with those who had intact ovaries as referent , the adjusted risk of cvd mortality was elevated for women with dm2 who had bso before or at 45 years of age ( hr 2.75 , 95% ci 1.246.11 , p = 0.012 ) , whereas the risk among women with dm2 who had surgery at or after age 45 years was not statistically different from that for women with dm2 with intact ovaries ( hr 0.94 , 95% ci 0.481.85 , p = 0.864 and p = 0.097 for interaction ) . sex - steroid hormones levels according to dm2 and oophorectomy status are reported in table 2 . on the one hand , women with diabetes had lower shbg ( p < 0.001 ) and higher free testosterone levels ( p = 0.026 ) than women without diabetes that was independent of age and bmi . on the other hand , women with dm2 with intact ovaries had higher estrone levels than women without dm2 ( p < 0.001 ) . as expected , total testosterone ( p < 0.001 ) and free testosterone ( p < 0.001 ) levels were lower in women with bso than in women with intact ovaries . bso was associated with slightly lower shbg levels in women with dm2 but with higher levels ( p = 0.001 ) in women without dm2 . in analysis restricted to dm2 , those with bso had significantly lower levels of total testosterone and a higher percentage of free testosterone than women with dm2 and intact ovaries . however , after adjustment for age and bmi , only the variation in percentage of free testosterone by ovarian status persisted among women with dm2 . hormone levels according to diabetes and bso status among a subsample of participants ( n = 564 ) in the sof data are shown as the median ( interquartile range ) . p value for subgroup analyses restricted to only women with diabetes by bso status adjusted for age and bmi , based on ancova . after a mean follow - up of 15.1 years , 4,797 deaths occurred , with 1,638 ( 34.1% ) attributed to cvd . cvd - related mortality rates per 1,000 , according to diabetes and bso status , were no diabetes or bso , 12.9 ; bso without diabetes , 12.7 ; diabetes without bso , 25.9 ; and diabetes with bso , 31.4 . the incidence of cvd mortality was elevated in women with diabetes , with or without bso ( p < 0.001 ) ( fig . the risk of cvd mortality was elevated for those with intact ovaries ( hr 1.95 , 95% ci 1.622.35 ) and for those with bso ( hr 2.56 , 95% ci 1.793.65 ) ( table 3 ) . although cvd mortality in women with diabetes and bso was slightly higher compared with women with diabetes and intact ovaries , this difference did not meet statistical significance ( hr 1.49 , 95% ci 0.902.46 , p = 0.120 ) . overall , bso was not associated with cvd mortality ( hr 1.05 , 95% ci 0.891.23 ) . all - cause and cvd mortality according to diabetes and sbo status and age at menopause from the sof * adjusted for age , education , age at menopause , hysterectomy , parity , postmenopausal hormone use , bmi , waist - to - hip ratio , hypertension , smoking status , and physical activity . we next performed analyses stratified by age at menopause ( 45 or > 45 years ) ( table 3 ) and found that cvd mortality risk was elevated for women with diabetes regardless of age at menopause . however , in analyses restricted to dm2 , with those who had intact ovaries as referent , the adjusted risk of cvd mortality was elevated for women with dm2 who had bso before or at 45 years of age ( hr 2.75 , 95% ci 1.246.11 , p = 0.012 ) , whereas the risk among women with dm2 who had surgery at or after age 45 years was not statistically different from that for women with dm2 with intact ovaries ( hr 0.94 , 95% ci 0.481.85 , p = 0.864 and p = 0.097 for interaction ) . to our knowledge , this is the first study to assess the association between bso , serum androgens , and cvd mortality in a large population of women with or without diabetes . we found that diabetes in postmenopausal caucasian women was associated with a higher cvd mortality risk that appeared to be influenced by the age at surgical menopause . as in previous studies , androgen levels were higher in women with diabetes ( 710 ) . because androgens have been linked to cvd risk , possibly due to insulin resistance and glycemia , and to adverse effects on the coronary vasculature ( 32 ) , cardiac myocytes ( 33 ) , and carotid artery intima - media thickness ( 11,12 ) , we hypothesized that bso would actually reduce the cvd risk in women with dm2 . however , our analysis of this prospective cohort of 7,977 women aged 65 years and older does not support this hypothesis . the cvd risk among women with dm2 appeared to be greater in women who had bso before or at age 45 years . although this risk may be explained by estrogen deficiency ( 30,34 ) , bso before age 45 years did not increase cvd mortality risk in women without diabetes . on the one hand , no difference was found in bmi , waist circumference , age at menarche , or prevalence of hypertension between women with dm2 with bso and those without bso . on the other hand , parity was less among women with dm2 with bso , and shbg levels tended to be lower rather than increased as in women without diabetes . few studies ( 30,34 ) have included the reason for pelvic surgery in their analysis , but in women from olmstead county , mn ( 34 ) , elevated mortality was limited to those who were younger than age 45 years at bso and those who underwent bso for benign tumors or inflammation but not the larger group of women with endometriosis . we propose that further investigation that includes indications for bso in younger women with dm2 may provide insight into why they are at higher risk for cvd . further , we hypothesize that hysterectomies concomitant with bso may be performed in young women because of ovarian cysts in conjunction with irregular menstrual bleeding in whom the diagnosis is pcos , a high - risk group for insulin resistance , diabetes , and cvd . bso , particularly at a young age , has been associated with increased overall and cvd mortality in the general population in some studies but not in others ( 30,34 ) . the nurses health study ( 16 ) found a 17% higher risk for cvd in women with bso and a 44% higher risk among those women with bso before age 45 years . the women s health initiative ( 19 ) found no statistically significant differences in the rates of fatal and nonfatal chd , stroke , or total cvd in women with hysterectomy concomitant with bso compared with women with hysterectomy alone . the california teachers study found no elevated cvd risk or mortality with bso irrespective of hormone therapy use or age at bso ( 35 ) . in this cohort of postmenopausal women , shbg levels were lower and free testosterone levels were higher among women with diabetes independent of age and adiposity . however , results after dexamethasone suppression and acth stimulation in postmenopausal women with a history of pcos suggest that the ovary and adrenal cortex both contribute to the elevated androgen levels in these subjects ( 14 ) . in the current study , free testosterone levels among women with bso were slightly but not statistically higher in women with diabetes than in those without diabetes after accounting for the effect of age and adiposity . further , because sex hormones were measured in a small subset of only five women with diabetes and bso , we could not test whether sex steroids modify the relationship between bso and diabetes on mortality . a single measure of sex hormones at baseline was used to characterize each woman s hormonal status . our cvd findings were consistent , however , with all - cause mortality , suggesting that the influence of such misclassification on our results , if present , may be minimal . in summary , women with dm2 have a higher cvd mortality risk that is not attenuated by a history of bso . these findings imply that ovarian hyperandrogenemia may not be the primary mechanism to explain cvd mortality among postmenopausal women with dm2 . the apparent influence of age at menopause on the relation between bso and diabetes with cvd mortality raises the possibility that joint factors may influence the risk for diabetes , cvd , and the decision for bso in younger women . these results warrant further investigations among younger women with diabetes and bso to determine if this risk is due to bso or is a marker for risk factors that could be associated with pcos .

neurodegenerative diseases such as alzheimer 's ( ad ) , parkinson 's ( pd ) , and huntington 's diseases ( hd ) and amyotrophic lateral sclerosis ( als ) make up a group of pathologies characterized by separate etiologies with distinct morphological and pathophysiological features . there is a huge body of evidence that suggest that these disorders arise by multifactorial conditions such as ( a ) abnormal protein dynamics with defective protein degradation and aggregation , ( b ) oxidative stress and free radical formation , ( c ) impaired bioenergetics and mitochondrial dysfunction , and ( d ) exposure to metal toxicity and pesticides ( figure 1 ) . although a lot of research has been carried out to understand the pathophysiology of these proteinopathies , still clarity in terms of viable drug targets is elusive . however , in order to explore applications directed toward developing recent emerging therapies for these diseases , neuroscientists have exploited the understanding of the basic etiology of these diseases . although each disease has its own molecular mechanism and clinical manifestations , some general pathways might be recognized in different pathogenic cascades . they include protein misfolding and aggregation , oxidative stress and free radical formation , metal dyshomeostasis , mitochondrial dysfunction , and phosphorylation impairment , all occurring concurrently . proteins determine virtually every aspect of life by means of their highly diverse enzymatic and structural properties . at the same time , proteins are vulnerable macromolecules in the physiological environment of living cells . cellular proteins must assume and maintain their native three - dimensional conformations in order to be biochemically and functionally active . partial folding or misfolding makes protein functionally inactive , which may make the protein toxic to the cell . truncated translational polypeptide products , misfolded intermediates , and unassembled subunits of oligomeric protein complexes frequently expose hydrophobic regions that have a tendency to form an aggregation or heap [ 1 , 2 ] . due to the hydrophilic nature of the cellular medium , hydrophobic surfaces from different misfolded proteins tend to interact with each other and to form cellular aggregates . protein misfolding followed by self - association and subsequent deposition of the aggregated proteins has been observed in the brain tissues of patients affected by these disorders . the biophysical behavior of these proteins , leading to their misfolding , aggregation , and deposition , has prompted scientists to group these kinds of neurological disorders under the common name of conformational diseases or proteinopathies . although protein biogenesis , for instance , is an error - prone process , to avoid alterations in protein homeostasis , cells possess high - fidelity protein quality control ( pqc ) pathways . there are two specific lines of defense to ensure the maintenance of proteostatic equilibrium inside the cell . the first line of defense of the pqc systems consists of protein chaperones that bind to unfolded proteins , including newly synthesized proteins , and , by hydrolyzing atp , actively aid in attaining mature protein conformation . this pathway includes e1- , e2- , and e3-ubiquitin ligases , which are recruited by the chaperones themselves and tetraubiquitinate irreversibly misfolded proteins , thus targeting them for proteolysis by the 26s proteasome . the pqc system also acts on mature properly folded but metastable proteins that have a tendency to revert to a nonnative state , particularly in conditions of proteotoxic stress such as in the presence of oxidizing agents or elevated temperature [ 1 , 2 ] . a multitude of dedicated transcription factors also respond to proteotoxic stimuli by upregulating the transcription of genes that promote pqc . the pqc pathways are spatially compartmentalized according to the subcellular location of their misfolded substrates . molecular chaperones assist protein foldingand also facilitate degradation of the misfolded polypeptides by the ubiquitin - proteasome system . misfolding and aggregation are recognized as common molecular events for a large number of human diseases due to improper trafficking , premature degradation , or the appearance of toxic folds [ 7 , 8 ] . such diseases include cag - repeat / polyglutamine ( polyq ) expansion diseases including huntington 's disease ( hd ) , kennedy 's disease , and spinocerebellar ataxias , and non - cag diseases including parkinson 's disease , amyotrophic lateral sclerosis ( als ) , prion disease , and alzheimer 's disease . in eukaryotic cells , protein homeostasis depends on molecular chaperones ; these distinct functions are performed by two distinctly regulated chaperone networks . chaperones promote the folding of newly synthesized polypeptides , their translocation across membranes , and the refolding of stress - denatured substrates . chaperones also play a key role in targeting misfolded proteins for degradation as well as preventing aggregation , they are generally classified according to their molecular masses as heat shock ( hsps ) and small heat shock proteins ( smhsps ) ( e.g. , hsp100 , hsp90 , hsp70 , hsp60 , and hsp40 ) . certain atp - driven chaperones , such as hsp70 and hsp90 interact with cofactors , which directly influence their atpase activity and direct them along different folding pathways [ 10 , 11 ] . chaperones , together with the autophagy and ubiquitin proteasome system ( ups ) , clear proteins which are abundantly expressed , yet there are evidences which suggest that limitations and malfunction of the clearance machinery are risk factors in diseases of protein conformation [ 12 , 13 ] . chaperones do not prevent formation of protein inclusions ; instead of the formation of protein inclusion , chaperones are likely to inhibit the formation of toxic species by directing the misfolded species to nontoxic aggregates . chaperones have the ability to extract and refold proteins from aggregates beyond serving as a link between folding and degradation . the ring - shaped hexameric aaa - atpases , specialized class of chaperones , can extract misfolded proteins from aggregates in an atp - dependent manner . the extracted protein can then be transferred to hsp70 and hsp40 chaperones for refolding or degradation [ 1618 ] . misfolded aggregated proteins can also be degraded by a separate autophagy pathway that involves their ultimate delivery to the lysosome . autophagy is a nonspecific bulk degradation pathway that was initially described for long - lived cytoplasmic proteins and damaged organelles . this process is also a major degradation pathway for many aggregation - prone proteins associated with neurodegenerative disorders . knockdown of the autophagy genes ( e.g. , atg5 and atg7 ) leads to aggregation and neurodegeneration in certain mouse models [ 22 , 23 ] . conversely , upregulation of autophagy can play a protective role in quality control because it can promote the clearance of soluble small oligomeric aggregates , for instance , in hd models . the multidimensional functioning and the capacity of molecular chaperones and degradative clearance machinery suggest that protein quality control is an efficient process ; nevertheless the late onset of neurodegenerative diseases is indicative of the fact that these protein quality control mechanisms become overwhelmed with age and hence contribute to the disease conditions . small oligomeric aggregates and amyloid oligomers have been widely reported to permeabilize both cell and mitochondrial membranes . they are , therefore , responsible for calcium dysregulation , membrane depolarization , and impairment of mitochondrial functions , which have been identified as a further common feature of most neurodegenerative disorders . neurodegenerative diseases are a consequence of genetic mutations and/or environment factors which are strongly associated with age [ 25 , 26 ] . mitochondrial dysfunction and oxidative stress play an important role for the development of the more common neurodegenerative disorders . loss of mitochondrial function is associated with an increase in the generation of reactive oxygen intermediates and a number of human diseases . mitochondria use metabolic intermediates generated during the tricarboxylic acid ( tca ) cycle to generate adenosine triphosphate ( atp ) during oxidative phosphorylation . during the etc ( electron transport chain ) , electrons are occasionally captured by oxygen to produce superoxide anion radicals ( o ) . within the mitochondria , these superoxide radicals are converted to hydrogen peroxide by the action of manganese superoxide dismutase . hydrogen peroxide in the mitochondria is broken down to water by the action of glutathione peroxidase or peroxiredoxins . the inhibition of etc proteins can cause a subsequent increase in ros resulting in decrease in the mitochondrial membrane potential , loss of atp , and energy collapse and subsequent cell death [ 28 , 29 ] . beside the antioxidant enzymatic activities mentioned earlier , cells have nonenzymatic ( gsh , vitamin e , vitamin c , and ubiquinone ) scavengers to protect them against ros . the result of an imbalance between ros production and antioxidant action is called oxidative stress [ 30 , 31 ] . former estimates based on isolated highly energized mitochondria have suggested that as much as 24% of the oxygen consumed by mitochondria is liberated as superoxide or hydrogen peroxide , but recent studies as well as extrapolation to whole cells suggest that these early estimates are too high , and it has been estimated that mitochondria under normal physiological cellular conditions are intimately involved in the production of ros through one - electron carriers in the respiratory chain and probably produce one to two orders lower amounts of reactive oxygen species ( ros ) . neuronal tissue is particularly sensitive to oxidative stress , and imbalance in prooxidant versus antioxidant homeostasis in cns results in the production of several potentially toxic ros , including both the radical and nonradical species that participate in the initiation and/or propagation of radical chain reactions . in ad , pd , hd , and als , oxidative damage is found in every class of biological molecules within neurons , spanning from lipids to dna and proteins . however , the administration of one or few antioxidants is too simplistic , as demonstrated by the several clinical studies that have shown modest success with antioxidants in the treatment of neurodegeneration . thus , dysfunctional mitochondria , alterations in mitochondrial dynamics , increased ros , mtdna damage , and the loss of energy production are important contributors to the pathophysiology associated with several neurodegenerative diseases including alzheimer 's disease ( ad ) , parkinson 's disease ( pd ) , and huntington 's disease ( hd ) , and cancer . metals have an important role in neurodegeneration . while transition metals are essential in many biological reactions , alterations in their homeostasis result in increased free radical production , which is catalyzed by iron , copper , or other trace redox active metals . in all cases , the increase of iron in the brain associated with several neurodegenerative diseases may lead to an increased production of free radicals via the fenton reaction . the intracellular iron is usually tightly regulated , being bound by ferritin in an insoluble ferrihydrite core . the neurotoxin 6-hydroxydopamine ( 6-ohda ) releases iron from the ferritin core by reducing it to the ferrous form . in the presence of ferritin , both 6-ohda and thb strongly stimulate lipid peroxidation , an effect abolished by the addition of the iron chelator deferoxamine . it suggests that ferritin iron release contributes to free - radical - induced cell damage in vivo . iron accumulation could be an important contributor to the oxidative damage of ad while an observation by some researcher suggests that dietary fat and a systemic defect in iron metabolism may act synergistically in the process of lipid peroxidation in pd . redox - active iron is associated with the senile plaques and neurofibrillary tangles , the pathological hallmark lesions of this disease . iron associated with the lesion induces in situ oxidation and readily catalyzes an h2o2-dependent oxidation . characterization of the iron binding site suggests that binding is dependent on the available histidine residues and on protein conformation . iron can contribute to free radical damage by catalyzing the formation of the oh , inducing secondary initiation of lipid peroxidation , and promoting the oxidation of proteins . the iron chelator , deferoxamine , can limit these oxidative reactions and it scavenges the peroxynitrite independent of iron chelation because with deferoxamine the iron can be rebound to the lesions . some work has been done by the researchers to investigate the hypothesis that the exposure to pesticides could be related to central nervous system disorders . isabelle baldi et al . studied that , in 1,507 french elderly people , lower cognitive performance was observed in subjects who had been occupationally exposed to pesticides in 19921998 . in men , the relative risks of developing parkinson 's and alzheimer 's disease for occupational exposure assessed by a job exposure matrix were 5.63 ( 95% confidence interval : 1.47 , 21.58 ) and 2.39 ( 95% confidence interval : 1.02 , 5.63 ) , respectively , after the confounding factors were taken into account . no association was found with having a primary job in agriculture or with environmental pesticide exposure nor was an association found in women . these results suggest the presence of neurologic impairments in elderly persons who were exposed occupationally to pesticides . several studies have shown positive association between pesticide exposure and the development of pd . however , the specific compounds that lead to these varied effects are yet to be fully understood , and it is not very clear whether these pesticides share common molecular signatures that lead to neuronal toxicity . therefore , investigators should continue to concentrate on identifying biomarkers for the improved estimation of pesticide exposure . the availability of such extensive molecular evidence raises the issue of cell - type specificity in neuronal disorders . the issue of cell - type specificity is , therefore , an open question for those studying the pathogenesis and treatment of these illnesses . new drug candidates with disease modifying potential are now in the pipeline and have reached testing in clinical trials . stem cell therapy , gene transfer therapy , nanotechnology and medicinal chemistry based treatment , and multitarget directed ligands ( mtdls ) have emerged as promising new therapies for neurodegenerative disorders . cell replacement therapy and gene transfer to the diseased or injured brain may act as potentially powerful new therapeutic strategies for human neurological diseases . the transplantation of stem cells may provide effective treatments due to the self - renewing and multipotent nature of these cells , including delivery of therapeutic factors to provide trophic support or missing gene products , mobilization of endogenous stem cells , and replacement of lost or dysfunctional cells . in parkinson 's disease , current therapies centere on the oral administration of l - dopa and dopamine receptor agonists and on deep - brain stimulation in the subthalamic nucleus . although the pharmacological treatment is effective for some symptoms , it has some limitations because its effectiveness decreases over time and side effects develop [ 39 , 40 ] . thus , an alternative approach for restoration of the damaged dopaminergic system is the transplantation of dopaminergic - synthesizing cells . human stem cells may provide sources of cells for use in the treatment of pd . one potential approach to prevent the death of existing neurons could be to transplant human stem cells engineered to express neuroprotective molecules such as the glial - cell - line - derived neurotrophic factor ( gdnf ) . a recent strategy for treatment of hd has centered on cell therapy to protect vulnerable neuronal cell populations or to replace dysfunctional or dying cells . stem cell therapy aims to restore or preserve brain function by replacing and protecting striatal neurons . at this time , using stem cells for the delivery of trophic factors and the neuroprotection to prevent disease progression seems a more achievable clinical goal in hd than neuronal replacement . most of the recent work in stem cell therapy has been conducted in animal models of hd . the study showed that cell replacement using grafts of fetal striatal neurons promoted functional recovery , and some evidence from clinical trials indicates that this could also occur in patients . however , protocols and procedures developed from trials of fetal - derived cell transplantation in humans with hd lay the ground work to move stem cell therapy into the clinic . one of the first challenges to stem cell therapy in hd is to determine which source of stem cells is most efficacious , and many sources have been examined . in addition to human escs , stem cells derived from mesenchyme in adults have been investigated as a readily available source of stem cells in hd . although in alzheimer 's disease ( ad ) massive neuronal loss only occurs in very few brain structures , such as the hippocampal ca1 and ca2 regions , the entorhinal cortex , and the locus coeruleus , large parts of the brain are affected by pathological alterations and decreased neuronal metabolism [ 46 , 47 ] . current therapies , such as treatment with acetylcholinesterase inhibitors to enhance cholinergic function , provide only partial and temporary alleviation of symptoms . the pathological changes seen in ad offer an extremely problematic situation for cell replacement . because stem cells can be genetically modified to carry new genes and have high migratory capacity after brain transplantation , they could be used in place of fibroblasts for delivery of the nerve growth factor ( ngf ) to prevent degeneration of basal forebrain cholinergic neurons [ 49 , 50 ] . the recent breakthroughs in stem cell research might nevertheless provide possibilities for neural implantation and cell replacement therapy for patients with amyotrophic lateral sclerosis ( als ) . kim et al . showed that intrathecal injection with an optimized cell number could be a potential route for stem cell therapy in als patients . they suggested that , at this dose of 1 10 cells / ml , the average number of motor neurons was significantly higher than others , and most injected hmscs are distributed in the ventricular system and subarachnoid space . additionally , the studies suggested that successful stem cell therapy for als likely would require that the cells be combined with other drugs or treatments , such as antioxidants and/or trophic molecules . many exciting studies are taking this direction ; both in vitro and in vivo studies have shown generation of motor neurons from human escs and functional engraftment of these motor neurons after transplantation into the developing chick and adult rodent spinal cord with axonal outgrowth toward muscle . recently , a phase i clinical trial confirmed that mscs transplantation into the spinal cord of als patients is safe and that mscs might have a clinical use for future als cell - based clinical trials . recent studies have indicated that it is possible to generate motor neurons in culture from stem cells that include escs and nscs . mouse esc - derived motor neurons transplanted into motor neuron injured rat spinal cord survived and extended axons into ventral root , and human escs transplanted into cerebrospinal fluid of rats with motor neuron injury migrated into spinal cord and led to an improved motor function . however , factors that control the differentiation , survival , and maturation of stem cells in the context of a host degenerative brain must be more thoroughly understood before stem cell therapy will prove to be a robust and safe strategy that can be transferred to the clinic . nanotechnology has proven to have great potential for providing neurotherapeutic modalities to limit and reverse the neuropathology of ad and pd by supporting and promoting functional regeneration of damaged neurons , providing neuroprotection , and facilitating the delivery of neuroactives such as drugs , genes , and cells across the blood brain barrier ( bbb ) . it may contribute significantly toward the development of nano - enabled drug delivery systems for the treatment of nds , taking advantage of the nanoscale structures of neural cells . several novel approaches , inspired by recent advances in nanotechnology , are already applicable to the treatment of ad and pd . nanoscale classes of neuroactives will widen the scope of therapeutic action beyond merely modifying transmitter function to include stem cell and gene therapies that could offer a more selective mode of targeting . various potential nanostructures are employed for the treatment of neurodegenerative disorders such as polymeric nanoparticles , nanocapsules , nanospheres , polymeric nanogels , nanosuspensions , carbon nanotubes , nanofibers , polymeric nanomicelles , and polymeric nanoliposomes . pbca nanoparticles have been used to deliver drugs to cns , and gold nanoparticles have been successfully employed to destroy amyloid plaques in alzheimer 's disease . several approaches to in vivo gene therapy for neurodegenerative diseases are currently being pursued both in animal models and in early human clinical trials . gene transfer and novel approaches to in vivo gene therapy for neurodegenerative disorders were focused on ( pd ) , ( hd ) , and ( ad ) . genes can be delivered to the central nervous system ( cns ) through the use of viral and nonviral vectors . many viral vectors have been developed for this purpose , each with advantages and disadvantages . in general , viral vectors are delivered directly into the cns via a craniotomy and are locally infused into specific neuroanatomical locations . nonviral vectors such as liposomes may provide a means for delivering genes without the need for a craniotomy . the choice of vector and its mechanism of delivery will depend on the specific disease and its neurophysiology and the mechanism underlying the gene therapy . in vivo gene transfer therapy recent success in human safety - tolerability trials will likely lead to an increasing number of human efficacy trials . given the multitude of potential neuroanatomical , neurophysiologic , and genetic targets for interventions in neurodegenerative disorders , the possibilities for gene therapy are extensive . these possibilities will likely be expanded further as the techniques for viral and nonviral gene transfer to the cns are improved . many challenges exist for performing large - scale efficacy trials involving potentially risky genetic and neurosurgical interventions , including blinding and ethical considerations . medicinal - chemistry - based strategies include analogs as well as prodrug and codrug approaches . while each of these strategies may be equally promising to increase gsh levels , the codrug approach consists in linking , via a covalent chemical linkage , two different pharmacophores with similar or different pharmacological activities in order to improve the physiochemical , biopharmaceutical , and drug delivery properties of therapeutic agents . the resulting codrug has to be stable at gastrointestinal level and transported to the target site of action where it provides the two parent drugs following hydrolysis . the codrug approach has been used for the treatment of pd and ad joining antioxidant or chelating molecules with a therapeutic compound ( anti - parkinson 's or anti - alzheimer 's drugs ) . in particular , codrugs containing antioxidant molecules such as gsh , n - acetyl cysteine , methionine , and cysteinyl derivatives have been synthesized in order to permit a targeted delivery of antioxidant directly to specific groups of neurons where cellular stress is associated with pd and ad . the dual advantages of these antioxidant molecules lies in the fact that the antioxidant portion , in addition to acting as a scavenger directly or indirectly of free radicals , can be used as a carrier . in fact , gsh and cysteinyl derivatives can be used as blood brain barrier ( bbb ) shuttles for delivery of anti - parkinson 's or anti - alzheimer 's drugs since the presence of gsh transporters at the bbb is well documented . multitarget directed ligand ( mtdl ) design and discovery emerge as a possible alternative strategy for treatment of neurodegenerative disorders . mtdl may be a better candidate because it is able to hit several targets at once . such an approach has recently been applied by some research groups to the discovery of new drug candidates for the treatment of pd . curcumin and other polyphenols would appear to be useful in ad not only because of their dual function as anti - inflammatory and antioxidant agents but also because they can structurally interfere with a beta - aggregation and metal dyshomeostasis . there are numerous dietary factors that have been reported to affect brain physiology in ways that could , in theory , modify brain aging and the pathogenesis of neurodegenerative disorders . these range from amino acids such as tryptophan to caffeine and related stimulants ( to omega-3 fatty acids ) . gene array analysis of expression levels of thousands of genes in brains of young and old rats that had been maintained on control or restricted diets revealed changes in gene expression in brain cells during aging and showed that dr ( dietary restriction ) can suppress many of those changes . age - related changes in the expression of genes that encode proteins involved in innate immune responses , oxidative stress , and energy metabolism are counteracted by dr . this retardation of brain aging at the molecular level may underlie the preservation of brain function during aging in animals maintained on dr . a recently published study has reported that dr prevents neurodegenration in an experimental model via a sirtuin mediated pathway which might have implications for neurodegenerative parkinson 's disease . in light of the recent achievements in the fields of ad , pd , hd , and als , neurodegenerative diseases appear to share several common multifactorial degenerative processes that contribute to neuronal death , leading to functional impairments . because of these multifactorial aspects and complexity , gene expression analysis platforms have been extensively used to investigate altered pathways during degeneration and to identify potential biomarkers and drug targets . although many therapeutic approaches have been tested , no effective cure for these neurodegenerative diseases has been identified . therefore , high - throughput techniques like whole genome transcriptomics and microarray technology must be coupled with functional genomics and proteomics in an effort to identify specific and selective biomarkers and viable drug targets , thus allowing the successful discovery of disease - modifying therapeutic treatments .

neurodegenerative diseases are multifactorial debilitating disorders of the nervous system that affect approximately 30 millionindividuals worldwide . neurodegenerative diseases such as alzheimer 's , parkinson 's , huntington 's , and amyotrophic lateral sclerosis diseases are the consequence of misfolding and dysfunctional trafficking of proteins . beside that , mitochondrial dysfunction , oxidative stress , and/or environmental factors strongly associated with age have also been implicated in causing neurodegeneration . after years of intensive research , considerable evidence has accumulated that demonstrates an important role of these factors in the etiology of common neurodegenerative diseases . despite the extensive efforts that have attempted to define the molecular mechanisms underlying neurodegeneration , many aspects of these pathologies remain elusive . however , in order to explore the therapeutic interventions directed towards treatment of neurodegenerative diseases , neuroscientists are now fully exploiting the data obtained from studies of these basic mechanisms that have gone awry . the novelty of these mechanisms represents a challenge to the identification of viable drug targets and biomarkers for early diagnosis of the diseases . in this paper , we are reviewing various aspects associated with the disease and the recent trends that may have an application for the treatment of the neurodegenerative disorders .

1. Introduction 2. Consequences of Protein Folding and Misfolding 3. Role of Molecular Chaperones along with Autophagy in Maintaining Protein Homeostasis 4. Mitochondrial Dysfunction, ROS, and Neurodegenerative Diseases 5. Several Environmental Factors Could Be Associated with the Induction of Neurodisorders 6. Therapies for Neurodegenerative Diseases 7. Conclusions

neurodegenerative diseases such as alzheimer 's ( ad ) , parkinson 's ( pd ) , and huntington 's diseases ( hd ) and amyotrophic lateral sclerosis ( als ) make up a group of pathologies characterized by separate etiologies with distinct morphological and pathophysiological features . there is a huge body of evidence that suggest that these disorders arise by multifactorial conditions such as ( a ) abnormal protein dynamics with defective protein degradation and aggregation , ( b ) oxidative stress and free radical formation , ( c ) impaired bioenergetics and mitochondrial dysfunction , and ( d ) exposure to metal toxicity and pesticides ( figure 1 ) . although a lot of research has been carried out to understand the pathophysiology of these proteinopathies , still clarity in terms of viable drug targets is elusive . however , in order to explore applications directed toward developing recent emerging therapies for these diseases , neuroscientists have exploited the understanding of the basic etiology of these diseases . they include protein misfolding and aggregation , oxidative stress and free radical formation , metal dyshomeostasis , mitochondrial dysfunction , and phosphorylation impairment , all occurring concurrently . at the same time , proteins are vulnerable macromolecules in the physiological environment of living cells . cellular proteins must assume and maintain their native three - dimensional conformations in order to be biochemically and functionally active . partial folding or misfolding makes protein functionally inactive , which may make the protein toxic to the cell . truncated translational polypeptide products , misfolded intermediates , and unassembled subunits of oligomeric protein complexes frequently expose hydrophobic regions that have a tendency to form an aggregation or heap [ 1 , 2 ] . due to the hydrophilic nature of the cellular medium , hydrophobic surfaces from different misfolded proteins tend to interact with each other and to form cellular aggregates . protein misfolding followed by self - association and subsequent deposition of the aggregated proteins has been observed in the brain tissues of patients affected by these disorders . the biophysical behavior of these proteins , leading to their misfolding , aggregation , and deposition , has prompted scientists to group these kinds of neurological disorders under the common name of conformational diseases or proteinopathies . the first line of defense of the pqc systems consists of protein chaperones that bind to unfolded proteins , including newly synthesized proteins , and , by hydrolyzing atp , actively aid in attaining mature protein conformation . this pathway includes e1- , e2- , and e3-ubiquitin ligases , which are recruited by the chaperones themselves and tetraubiquitinate irreversibly misfolded proteins , thus targeting them for proteolysis by the 26s proteasome . the pqc system also acts on mature properly folded but metastable proteins that have a tendency to revert to a nonnative state , particularly in conditions of proteotoxic stress such as in the presence of oxidizing agents or elevated temperature [ 1 , 2 ] . the pqc pathways are spatially compartmentalized according to the subcellular location of their misfolded substrates . molecular chaperones assist protein foldingand also facilitate degradation of the misfolded polypeptides by the ubiquitin - proteasome system . misfolding and aggregation are recognized as common molecular events for a large number of human diseases due to improper trafficking , premature degradation , or the appearance of toxic folds [ 7 , 8 ] . such diseases include cag - repeat / polyglutamine ( polyq ) expansion diseases including huntington 's disease ( hd ) , kennedy 's disease , and spinocerebellar ataxias , and non - cag diseases including parkinson 's disease , amyotrophic lateral sclerosis ( als ) , prion disease , and alzheimer 's disease . chaperones promote the folding of newly synthesized polypeptides , their translocation across membranes , and the refolding of stress - denatured substrates . , hsp100 , hsp90 , hsp70 , hsp60 , and hsp40 ) . chaperones , together with the autophagy and ubiquitin proteasome system ( ups ) , clear proteins which are abundantly expressed , yet there are evidences which suggest that limitations and malfunction of the clearance machinery are risk factors in diseases of protein conformation [ 12 , 13 ] . chaperones do not prevent formation of protein inclusions ; instead of the formation of protein inclusion , chaperones are likely to inhibit the formation of toxic species by directing the misfolded species to nontoxic aggregates . misfolded aggregated proteins can also be degraded by a separate autophagy pathway that involves their ultimate delivery to the lysosome . this process is also a major degradation pathway for many aggregation - prone proteins associated with neurodegenerative disorders . knockdown of the autophagy genes ( e.g. conversely , upregulation of autophagy can play a protective role in quality control because it can promote the clearance of soluble small oligomeric aggregates , for instance , in hd models . the multidimensional functioning and the capacity of molecular chaperones and degradative clearance machinery suggest that protein quality control is an efficient process ; nevertheless the late onset of neurodegenerative diseases is indicative of the fact that these protein quality control mechanisms become overwhelmed with age and hence contribute to the disease conditions . they are , therefore , responsible for calcium dysregulation , membrane depolarization , and impairment of mitochondrial functions , which have been identified as a further common feature of most neurodegenerative disorders . neurodegenerative diseases are a consequence of genetic mutations and/or environment factors which are strongly associated with age [ 25 , 26 ] . mitochondrial dysfunction and oxidative stress play an important role for the development of the more common neurodegenerative disorders . loss of mitochondrial function is associated with an increase in the generation of reactive oxygen intermediates and a number of human diseases . the inhibition of etc proteins can cause a subsequent increase in ros resulting in decrease in the mitochondrial membrane potential , loss of atp , and energy collapse and subsequent cell death [ 28 , 29 ] . the result of an imbalance between ros production and antioxidant action is called oxidative stress [ 30 , 31 ] . former estimates based on isolated highly energized mitochondria have suggested that as much as 24% of the oxygen consumed by mitochondria is liberated as superoxide or hydrogen peroxide , but recent studies as well as extrapolation to whole cells suggest that these early estimates are too high , and it has been estimated that mitochondria under normal physiological cellular conditions are intimately involved in the production of ros through one - electron carriers in the respiratory chain and probably produce one to two orders lower amounts of reactive oxygen species ( ros ) . neuronal tissue is particularly sensitive to oxidative stress , and imbalance in prooxidant versus antioxidant homeostasis in cns results in the production of several potentially toxic ros , including both the radical and nonradical species that participate in the initiation and/or propagation of radical chain reactions . in ad , pd , hd , and als , oxidative damage is found in every class of biological molecules within neurons , spanning from lipids to dna and proteins . however , the administration of one or few antioxidants is too simplistic , as demonstrated by the several clinical studies that have shown modest success with antioxidants in the treatment of neurodegeneration . thus , dysfunctional mitochondria , alterations in mitochondrial dynamics , increased ros , mtdna damage , and the loss of energy production are important contributors to the pathophysiology associated with several neurodegenerative diseases including alzheimer 's disease ( ad ) , parkinson 's disease ( pd ) , and huntington 's disease ( hd ) , and cancer . metals have an important role in neurodegeneration . in all cases , the increase of iron in the brain associated with several neurodegenerative diseases may lead to an increased production of free radicals via the fenton reaction . the neurotoxin 6-hydroxydopamine ( 6-ohda ) releases iron from the ferritin core by reducing it to the ferrous form . in the presence of ferritin , both 6-ohda and thb strongly stimulate lipid peroxidation , an effect abolished by the addition of the iron chelator deferoxamine . iron accumulation could be an important contributor to the oxidative damage of ad while an observation by some researcher suggests that dietary fat and a systemic defect in iron metabolism may act synergistically in the process of lipid peroxidation in pd . redox - active iron is associated with the senile plaques and neurofibrillary tangles , the pathological hallmark lesions of this disease . iron associated with the lesion induces in situ oxidation and readily catalyzes an h2o2-dependent oxidation . characterization of the iron binding site suggests that binding is dependent on the available histidine residues and on protein conformation . iron can contribute to free radical damage by catalyzing the formation of the oh , inducing secondary initiation of lipid peroxidation , and promoting the oxidation of proteins . the iron chelator , deferoxamine , can limit these oxidative reactions and it scavenges the peroxynitrite independent of iron chelation because with deferoxamine the iron can be rebound to the lesions . some work has been done by the researchers to investigate the hypothesis that the exposure to pesticides could be related to central nervous system disorders . studied that , in 1,507 french elderly people , lower cognitive performance was observed in subjects who had been occupationally exposed to pesticides in 19921998 . in men , the relative risks of developing parkinson 's and alzheimer 's disease for occupational exposure assessed by a job exposure matrix were 5.63 ( 95% confidence interval : 1.47 , 21.58 ) and 2.39 ( 95% confidence interval : 1.02 , 5.63 ) , respectively , after the confounding factors were taken into account . several studies have shown positive association between pesticide exposure and the development of pd . however , the specific compounds that lead to these varied effects are yet to be fully understood , and it is not very clear whether these pesticides share common molecular signatures that lead to neuronal toxicity . therefore , investigators should continue to concentrate on identifying biomarkers for the improved estimation of pesticide exposure . the issue of cell - type specificity is , therefore , an open question for those studying the pathogenesis and treatment of these illnesses . new drug candidates with disease modifying potential are now in the pipeline and have reached testing in clinical trials . stem cell therapy , gene transfer therapy , nanotechnology and medicinal chemistry based treatment , and multitarget directed ligands ( mtdls ) have emerged as promising new therapies for neurodegenerative disorders . cell replacement therapy and gene transfer to the diseased or injured brain may act as potentially powerful new therapeutic strategies for human neurological diseases . the transplantation of stem cells may provide effective treatments due to the self - renewing and multipotent nature of these cells , including delivery of therapeutic factors to provide trophic support or missing gene products , mobilization of endogenous stem cells , and replacement of lost or dysfunctional cells . in parkinson 's disease , current therapies centere on the oral administration of l - dopa and dopamine receptor agonists and on deep - brain stimulation in the subthalamic nucleus . thus , an alternative approach for restoration of the damaged dopaminergic system is the transplantation of dopaminergic - synthesizing cells . human stem cells may provide sources of cells for use in the treatment of pd . one potential approach to prevent the death of existing neurons could be to transplant human stem cells engineered to express neuroprotective molecules such as the glial - cell - line - derived neurotrophic factor ( gdnf ) . a recent strategy for treatment of hd has centered on cell therapy to protect vulnerable neuronal cell populations or to replace dysfunctional or dying cells . at this time , using stem cells for the delivery of trophic factors and the neuroprotection to prevent disease progression seems a more achievable clinical goal in hd than neuronal replacement . most of the recent work in stem cell therapy has been conducted in animal models of hd . the study showed that cell replacement using grafts of fetal striatal neurons promoted functional recovery , and some evidence from clinical trials indicates that this could also occur in patients . however , protocols and procedures developed from trials of fetal - derived cell transplantation in humans with hd lay the ground work to move stem cell therapy into the clinic . one of the first challenges to stem cell therapy in hd is to determine which source of stem cells is most efficacious , and many sources have been examined . although in alzheimer 's disease ( ad ) massive neuronal loss only occurs in very few brain structures , such as the hippocampal ca1 and ca2 regions , the entorhinal cortex , and the locus coeruleus , large parts of the brain are affected by pathological alterations and decreased neuronal metabolism [ 46 , 47 ] . current therapies , such as treatment with acetylcholinesterase inhibitors to enhance cholinergic function , provide only partial and temporary alleviation of symptoms . because stem cells can be genetically modified to carry new genes and have high migratory capacity after brain transplantation , they could be used in place of fibroblasts for delivery of the nerve growth factor ( ngf ) to prevent degeneration of basal forebrain cholinergic neurons [ 49 , 50 ] . the recent breakthroughs in stem cell research might nevertheless provide possibilities for neural implantation and cell replacement therapy for patients with amyotrophic lateral sclerosis ( als ) . they suggested that , at this dose of 1 10 cells / ml , the average number of motor neurons was significantly higher than others , and most injected hmscs are distributed in the ventricular system and subarachnoid space . additionally , the studies suggested that successful stem cell therapy for als likely would require that the cells be combined with other drugs or treatments , such as antioxidants and/or trophic molecules . many exciting studies are taking this direction ; both in vitro and in vivo studies have shown generation of motor neurons from human escs and functional engraftment of these motor neurons after transplantation into the developing chick and adult rodent spinal cord with axonal outgrowth toward muscle . mouse esc - derived motor neurons transplanted into motor neuron injured rat spinal cord survived and extended axons into ventral root , and human escs transplanted into cerebrospinal fluid of rats with motor neuron injury migrated into spinal cord and led to an improved motor function . however , factors that control the differentiation , survival , and maturation of stem cells in the context of a host degenerative brain must be more thoroughly understood before stem cell therapy will prove to be a robust and safe strategy that can be transferred to the clinic . nanotechnology has proven to have great potential for providing neurotherapeutic modalities to limit and reverse the neuropathology of ad and pd by supporting and promoting functional regeneration of damaged neurons , providing neuroprotection , and facilitating the delivery of neuroactives such as drugs , genes , and cells across the blood brain barrier ( bbb ) . it may contribute significantly toward the development of nano - enabled drug delivery systems for the treatment of nds , taking advantage of the nanoscale structures of neural cells . several novel approaches , inspired by recent advances in nanotechnology , are already applicable to the treatment of ad and pd . various potential nanostructures are employed for the treatment of neurodegenerative disorders such as polymeric nanoparticles , nanocapsules , nanospheres , polymeric nanogels , nanosuspensions , carbon nanotubes , nanofibers , polymeric nanomicelles , and polymeric nanoliposomes . pbca nanoparticles have been used to deliver drugs to cns , and gold nanoparticles have been successfully employed to destroy amyloid plaques in alzheimer 's disease . several approaches to in vivo gene therapy for neurodegenerative diseases are currently being pursued both in animal models and in early human clinical trials . gene transfer and novel approaches to in vivo gene therapy for neurodegenerative disorders were focused on ( pd ) , ( hd ) , and ( ad ) . genes can be delivered to the central nervous system ( cns ) through the use of viral and nonviral vectors . the choice of vector and its mechanism of delivery will depend on the specific disease and its neurophysiology and the mechanism underlying the gene therapy . given the multitude of potential neuroanatomical , neurophysiologic , and genetic targets for interventions in neurodegenerative disorders , the possibilities for gene therapy are extensive . while each of these strategies may be equally promising to increase gsh levels , the codrug approach consists in linking , via a covalent chemical linkage , two different pharmacophores with similar or different pharmacological activities in order to improve the physiochemical , biopharmaceutical , and drug delivery properties of therapeutic agents . the resulting codrug has to be stable at gastrointestinal level and transported to the target site of action where it provides the two parent drugs following hydrolysis . the codrug approach has been used for the treatment of pd and ad joining antioxidant or chelating molecules with a therapeutic compound ( anti - parkinson 's or anti - alzheimer 's drugs ) . in particular , codrugs containing antioxidant molecules such as gsh , n - acetyl cysteine , methionine , and cysteinyl derivatives have been synthesized in order to permit a targeted delivery of antioxidant directly to specific groups of neurons where cellular stress is associated with pd and ad . the dual advantages of these antioxidant molecules lies in the fact that the antioxidant portion , in addition to acting as a scavenger directly or indirectly of free radicals , can be used as a carrier . in fact , gsh and cysteinyl derivatives can be used as blood brain barrier ( bbb ) shuttles for delivery of anti - parkinson 's or anti - alzheimer 's drugs since the presence of gsh transporters at the bbb is well documented . multitarget directed ligand ( mtdl ) design and discovery emerge as a possible alternative strategy for treatment of neurodegenerative disorders . such an approach has recently been applied by some research groups to the discovery of new drug candidates for the treatment of pd . there are numerous dietary factors that have been reported to affect brain physiology in ways that could , in theory , modify brain aging and the pathogenesis of neurodegenerative disorders . these range from amino acids such as tryptophan to caffeine and related stimulants ( to omega-3 fatty acids ) . age - related changes in the expression of genes that encode proteins involved in innate immune responses , oxidative stress , and energy metabolism are counteracted by dr . this retardation of brain aging at the molecular level may underlie the preservation of brain function during aging in animals maintained on dr . a recently published study has reported that dr prevents neurodegenration in an experimental model via a sirtuin mediated pathway which might have implications for neurodegenerative parkinson 's disease . in light of the recent achievements in the fields of ad , pd , hd , and als , neurodegenerative diseases appear to share several common multifactorial degenerative processes that contribute to neuronal death , leading to functional impairments . because of these multifactorial aspects and complexity , gene expression analysis platforms have been extensively used to investigate altered pathways during degeneration and to identify potential biomarkers and drug targets . therefore , high - throughput techniques like whole genome transcriptomics and microarray technology must be coupled with functional genomics and proteomics in an effort to identify specific and selective biomarkers and viable drug targets , thus allowing the successful discovery of disease - modifying therapeutic treatments .

filamentous cells represent an important advance in cellular growth and are also important devices for the colonization of land by fungi and plants . for example , filamentous growth in the form of aseptate hyphae evolved as an excellent tool for exploring dry soils . this innovation also enabled the fungi to establish close relationships with land plants , for example , through symbiotic relationships that resemble arbuscular mycorrhizal associations today . in arbuscular mycorrhiza , tip - growing fungal hyphae rapidly enter plant root tissue to establish a mutually beneficial relationship . the first tip - growing cells in land - plants arose about 450 million years ago in bryophytes , which used filamentous rhizoids for anchoring themselves to the soil . in plants today , two types of tip - growing cells dominate ; pollen tubes and root hairs . while pollen tubes provided for water - independent propagation of species , root hairs bestowed an increase in root surface area that could be used to increase nutrient uptake and better anchor plants in the soil . root hairs and pollen tubes are filamentous cells that grow uni - directionally by depositing flexible cell wall material at one site of the cell and exploiting turgor pressure as the driving force for elongation ; a process also referred to as tip growth ( figure 1 ) . root hair and pollen tube tip growth include a variety of cellular components and compounds that work in concert . the intracellular calcium concentration plays a central role in plant tip growth , and regulates actin dynamics via actin interacting proteins , such as villin and adf . these proteins are likely involved in the organization of the actin cytoskeleton at different sites in the root hair . microtubules are located net axially , i.e. along the shank , and reach into the subapical region of the root hair . their dynamic behaviour is regulated by the microtubule plus end - tracking protein ark1 and is important for maintenance of polarity and growth direction . these produce ros ( h2o2 and oh ) which mediate cell wall relaxation and peroxidase - mediated wall stiffening . ros may also partake in a positive feedback loop by activating calcium influx from the apoplast into the cytosol . the channels responsible for calcium influx are not known in root hairs but , in pollen tubes , cngc and glr channels have been identified . furthermore , calcium seems to influence extracellular ph ( possibly via h - atpases ) , which contributes to changes in cell wall structure during growth . cellulose microfibrils are short and randomly oritientated in the cell wall of the growing tip , whereas a well ordered second layer of cellulose microfibrils occurs in the shank . this organization promotes dynamic cell wall extension exclusively at the tip . in pollen tubes pmes mediate the crosslinking of pectins in the shank and remain inactive at the tip after inactivation through pmeis . pectin , pmes and pmeis are delivered from the golgi / trans golgi network to the plasma membrane of the pollen tube tip via actin - dependent vesicle trafficking . vesicles are spacially directed by the distribution of pis in the plasma membrane and are transported along the actin cytoskeleton via motor proteins . tip growth is regulated by several plasma membrane receptor - like kinases , for example , feronia and anxur . feronia is known to regulate rop signaling pathways via interaction with rop guanine nucleutide exchange factors . important rop effectors in pollen tubes are rics that directly influence tip growth by interaction with bundled and fine f - actin and modulation of intracellular calcium concentrations . abbreviations : adf , actin depolymerizing factor ; ark1 , armadillo - repeat kinesin 1 ; cngc , cyclic nucleotide - gated channel ; glr , glutamate receptor - like ; pi , inositol phosphate ; pme , pectin - methyl - esterase ; pmei , pme inhibitor ; ric , rop - interactive crib motif - containing ; rop , rho of plant gtpase ; ros , reactive oxygen species . although these tip - growing systems are very similar , they also display differences in their growth - related molecular machineries . in view of the fact that soil nutrient availability , water accessibility and soil condition are strongly affected by climate change , the study of root hair tip growth is a timely topic to help us to understand and improve plant growth and will therefore be the main focus of this review . while delicate , root hairs are excellent experimental model systems for polarized cell growth . they reach growth rates of typically 1 m min or more without losing cell wall integrity . this implies an extremely efficient system for delivering and modifying membrane and cell wall material at the tip apex . this system will therefore need to be highly coordinated , and involves a tip - derived calcium gradient , cytoskeleton dynamics , delivery of new cell wall material via vesicle trafficking , regulation of extracellular ph , and production of reactive oxygen species ( ros ) . how these players are geared to each other is only partly known , but recent studies have made great progress leading to a better understanding of the coordination . two of the major signal transducers for root hair growth and function are ca and ros ( figure 1 ) . calcium is a crucial and general regulator of plant cell growth and development , and is perhaps the most important factor for maintenance of tip growth where it is distributed as a continuous gradient in the cytosol , with the highest concentration close to the tip apex . furthermore , the cytosolic ca concentration displays periodic cycling that follows the rate of growth of the tip . this cycling appears to be coordinated with a rise and decline of both apoplastic ph and ros production . these findings suggest that changes in ca concentrations might regulate growth by the activation of ros production , and by increasing the ph , at the cell surface . the production of cell surface ros in turn activates ca influx into the cell and thereby completes a positive feedback loop . the increase in cytosolic ca is typically facilitated by membrane located ca channels . while the identity of these channels are not known in root hairs , cyclic nucleotide - gated ( cngc ) , and glutamate receptor - like ( glr ) , channels were identified as ca influx channels [ 24 ] in pollen tubes , and perhaps a similar scenario could be envisioned during root hair growth . the function of the fluctuations in intracellular ca may modulate components important for tip growth , such as cytoskeleton - organizing factors , and ros and ph homeostasis as alluded to above . however , it may also alter the apoplastic ca levels that can influence changes in the cell wall . in tip growing cells , the actin cytoskeleton is typically organized as thick actin bundles that run in parallel with the root hair shank supporting an inverse fountain flow of cytoplasmic streaming , and as fine filamentous structures at the apex of the tip that keep this region free of larger cell organelles [ 58 ] . the organization of the actin cytoskeleton is controlled by a variety of actin binding proteins ( abps ) , which aid in nucleation , depolymerisation and cross - linking of the actin cytoskeleton . for example , ca may influence actin depolymerizing factor ( adf ) in maize that can increase actin turnover by severing actin filaments , and villins that can influence the bundling of actin . the role of the actin cytoskeleton as a scaffold for polarized membrane trafficking is well established in root hairs . in particular , new membrane and cell wall material must be delivered to the tip apex to support the rapid growth . the cell wall material is engulfed in vesicles that move along the actin cytoskeleton via motor proteins . two recently characterized motor proteins of the myosin xi family that function in actin - dependent transport of cell organelles , vesicles and protein complexes , are required for normal actin dynamics and fast root hair growth . vesicle secretion can also be controlled by other factors , including rab gtpases and inositol phosphates ( pis ) . for example , pi-4p and pi-4,5p2 accumulate in the plasma membrane at the root hair apex where they might orchestrate growth by recruiting other important proteins containing pi-4p or pi-4,5p2 binding domains . one such example is the adp ribosylation factor gtpase - activating protein agd1 , which is necessary for maintenance of polarity during root hair growth . membrane secretion in root hair growth is also influenced by the exocyst complex , a rho and rop gtpase effector , that tethers vesicles to their target membranes . cortical microtubules are oriented net axially along the fringe of the root hair tube , while endoplasmic microtubules reach from the perinuclear cytoplasm towards the tip . microtubule stabilizing or destabilizing treatments using taxol or oryzalin , respectively , lead to wavy and occasionally branched root hair phenotypes . similar phenotypes were also found in ark1 mutants lacking the functional microtubule plus - end tracking protein armadillo - repeat kinesin 1 ( ark1 ) . ark1 plays a key role in root hair tip growth by promoting microtubule catastrophe events . in summary , while the actin cytoskeleton plays an important role as a dynamic scaffold for proper vesicle delivery in polarized root hair elongation , the microtubules are required for maintenance of cell polarity and directed growth . the cell wall is a dynamic structure that determines the shape of the root hair ( figure 1 ) . here , one can illustrate the cell wall as a corset that readily allows for expansion at the tip , but provides resistance against the turgor pressure along the shank . cellulose is a major load - bearing structure of the cell wall and is therefore the main contributor to the corset function . two partially redundant members of the cellulose synthase ( cesa)-like ( csl ) superfamily , csld2 and csld3 , are located at the root hair tip and are required for its growth . notably , at the growing tip of the root hair the cellulose microfibrils are short and randomly orientated , as revealed by ultrastructural studies [ 2426 ] . in contrast , the shank behind the actively growing tip contains a second layer of highly ordered cellulose microfibrils oriented transversely to the axis of expansion [ 2628 ] . lesions in enzymes that synthesize some of these matrix polymers , for example , xyloglucans , led to root hair growth defects , perhaps due to mis - alignment of cellulose fibrils . whereas the xyloglucans thus influence root hair growth , the role of pectins is not well explored . however , pectins do have a prominent role in pollen tube growth . here , pectin - methyl - esterases ( pmes ) , which de - methylesterify carboxyl residues on the pectin backbone , can affect the cell wall viscosity . through the pme action , carboxyl residues are set free to bind ca , which results in pectin cross - linking and presumably cell wall tightening at the pollen tube tip [ 3134 ] . pme and its inhibitors , pmeis , are , furthermore , spatio - temporally distributed along the pollen tube with a preferential activity of pmes at the shank and pmeis at the tip , which results in a distinct pectic composition of the pollen tube cell wall . it is possible that similar distributions also may play a role in root hair growth . in addition , extensins , cell wall structural proteins , are of crucial importance for root hair development , possibly for maintaining cell wall stability . these proteins may be post - translationally modified by o - linked glycosylation , and this modification alters cell wall properties during root hair growth . recently , pft1/med25 , a transcriptional mediator complex subunit , was revealed to orchestrate the expression of cell wall remodelling genes including a set of peroxidases and cell wall cross - linking extensins and being required for redox homeostasis in roots and normal root hair formation . these results indicate that the cell wall proteome could be a useful source to find cell wall and , therefore , tip growth modulators . however , results on the proteomic composition of root hair cell walls are scarce , not only because separating cell wall proteins from the cell wall matrix is generally challenging , but also because isolating a reasonable amount of protein from root hairs for analysis is difficult . not only calcium / pectin interactions but also surface ros and ph are thought to directly influence cell wall viscosity of the tip surface . on the other hand , h2o2 is a substrate for peroxidases that help to stiffen the wall via crosslinking of cell wall polysaccharides . this may seem like a paradox and a tight coordination between these processes is likely to occur , but how this is achieved remains unclear . surface ph may also influence the cell wall viscosity by activation of the expansins , non - hydrolytic cell wall - loosening proteins , and other cell wall modifying enzymes , which promote cell wall relaxation [ 4244 ] . two of the major signal transducers for root hair growth and function are ca and ros ( figure 1 ) . calcium is a crucial and general regulator of plant cell growth and development , and is perhaps the most important factor for maintenance of tip growth where it is distributed as a continuous gradient in the cytosol , with the highest concentration close to the tip apex . furthermore , the cytosolic ca concentration displays periodic cycling that follows the rate of growth of the tip . this cycling appears to be coordinated with a rise and decline of both apoplastic ph and ros production . these findings suggest that changes in ca concentrations might regulate growth by the activation of ros production , and by increasing the ph , at the cell surface . the production of cell surface ros in turn activates ca influx into the cell and thereby completes a positive feedback loop . the increase in cytosolic ca is typically facilitated by membrane located ca channels . while the identity of these channels are not known in root hairs , cyclic nucleotide - gated ( cngc ) , and glutamate receptor - like ( glr ) , channels were identified as ca influx channels [ 24 ] in pollen tubes , and perhaps a similar scenario could be envisioned during root hair growth . the function of the fluctuations in intracellular ca may modulate components important for tip growth , such as cytoskeleton - organizing factors , and ros and ph homeostasis as alluded to above . however , it may also alter the apoplastic ca levels that can influence changes in the cell wall . in tip growing cells , the actin cytoskeleton is typically organized as thick actin bundles that run in parallel with the root hair shank supporting an inverse fountain flow of cytoplasmic streaming , and as fine filamentous structures at the apex of the tip that keep this region free of larger cell organelles [ 58 ] . the organization of the actin cytoskeleton is controlled by a variety of actin binding proteins ( abps ) , which aid in nucleation , depolymerisation and cross - linking of the actin cytoskeleton . for example , ca may influence actin depolymerizing factor ( adf ) in maize that can increase actin turnover by severing actin filaments , and villins that can influence the bundling of actin . the role of the actin cytoskeleton as a scaffold for polarized membrane trafficking is well established in root hairs . in particular , new membrane and cell wall material must be delivered to the tip apex to support the rapid growth . the cell wall material is engulfed in vesicles that move along the actin cytoskeleton via motor proteins . two recently characterized motor proteins of the myosin xi family that function in actin - dependent transport of cell organelles , vesicles and protein complexes , are required for normal actin dynamics and fast root hair growth . vesicle secretion can also be controlled by other factors , including rab gtpases and inositol phosphates ( pis ) . for example , pi-4p and pi-4,5p2 accumulate in the plasma membrane at the root hair apex where they might orchestrate growth by recruiting other important proteins containing pi-4p or pi-4,5p2 binding domains . one such example is the adp ribosylation factor gtpase - activating protein agd1 , which is necessary for maintenance of polarity during root hair growth . membrane secretion in root hair growth is also influenced by the exocyst complex , a rho and rop gtpase effector , that tethers vesicles to their target membranes . cortical microtubules are oriented net axially along the fringe of the root hair tube , while endoplasmic microtubules reach from the perinuclear cytoplasm towards the tip . microtubule stabilizing or destabilizing treatments using taxol or oryzalin , respectively , lead to wavy and occasionally branched root hair phenotypes . similar phenotypes were also found in ark1 mutants lacking the functional microtubule plus - end tracking protein armadillo - repeat kinesin 1 ( ark1 ) . ark1 plays a key role in root hair tip growth by promoting microtubule catastrophe events . in summary , while the actin cytoskeleton plays an important role as a dynamic scaffold for proper vesicle delivery in polarized root hair elongation , the microtubules are required for maintenance of cell polarity and directed growth . the cell wall is a dynamic structure that determines the shape of the root hair ( figure 1 ) . here , one can illustrate the cell wall as a corset that readily allows for expansion at the tip , but provides resistance against the turgor pressure along the shank . cellulose is a major load - bearing structure of the cell wall and is therefore the main contributor to the corset function . two partially redundant members of the cellulose synthase ( cesa)-like ( csl ) superfamily , csld2 and csld3 , are located at the root hair tip and are required for its growth . notably , at the growing tip of the root hair the cellulose microfibrils are short and randomly orientated , as revealed by ultrastructural studies [ 2426 ] . in contrast , the shank behind the actively growing tip contains a second layer of highly ordered cellulose microfibrils oriented transversely to the axis of expansion [ 2628 ] . neighboring cellulose fibrils are interlinked with matrix polymers , such as hemicelluloses and pectins . lesions in enzymes that synthesize some of these matrix polymers , for example , xyloglucans , led to root hair growth defects , perhaps due to mis - alignment of cellulose fibrils . whereas the xyloglucans thus influence root hair growth , the role of pectins is not well explored . however , pectins do have a prominent role in pollen tube growth . here , pectin - methyl - esterases ( pmes ) , which de - methylesterify carboxyl residues on the pectin backbone , can affect the cell wall viscosity . through the pme action , carboxyl residues are set free to bind ca , which results in pectin cross - linking and presumably cell wall tightening at the pollen tube tip [ 3134 ] . pme and its inhibitors , pmeis , are , furthermore , spatio - temporally distributed along the pollen tube with a preferential activity of pmes at the shank and pmeis at the tip , which results in a distinct pectic composition of the pollen tube cell wall . it is possible that similar distributions also may play a role in root hair growth . in addition , extensins , cell wall structural proteins , are of crucial importance for root hair development , possibly for maintaining cell wall stability . these proteins may be post - translationally modified by o - linked glycosylation , and this modification alters cell wall properties during root hair growth . recently , pft1/med25 , a transcriptional mediator complex subunit , was revealed to orchestrate the expression of cell wall remodelling genes including a set of peroxidases and cell wall cross - linking extensins and being required for redox homeostasis in roots and normal root hair formation . these results indicate that the cell wall proteome could be a useful source to find cell wall and , therefore , tip growth modulators . however , results on the proteomic composition of root hair cell walls are scarce , not only because separating cell wall proteins from the cell wall matrix is generally challenging , but also because isolating a reasonable amount of protein from root hairs for analysis is difficult . not only calcium / pectin interactions but also surface ros and ph are thought to directly influence cell wall viscosity of the tip surface . hydroxyl radicals can cleave polysaccharides and thereby promote wall relaxation [ 3840 ] . on the other hand , h2o2 is a substrate for peroxidases that help to stiffen the wall via crosslinking of cell wall polysaccharides . this may seem like a paradox and a tight coordination between these processes is likely to occur , but how this is achieved remains unclear . surface ph may also influence the cell wall viscosity by activation of the expansins , non - hydrolytic cell wall - loosening proteins , and other cell wall modifying enzymes , which promote cell wall relaxation [ 4244 ] . although the molecular organization of root hair growth is described with increasing detail , how internal and environmental growth - regulating signals are sensed and integrated remains enigmatic . nevertheless , some recent reports have begun to shed some light on this topic . in plants several receptor - like kinase ( rlk ) family members are implicated in relaying information about cell wall integrity ( cwi ) to the inside of the cell ( figure 1 ) . the redundant cataranthus roseus rlk1-like subfamily members anxur1 ( anx1 ) and anx2 are required for pollen tube growth in arabidopsis . the anx1 and 2 are the closest relatives to feronia ( fer ) , which is essential for both pollen tube and root hair growth [ 4652 ] . fer can affect ros production via the regulation of rho of plant gtpases ( rops ) . rops accumulate in the apical plasma membrane of root hairs and other tip - growing cells , and impact actin dynamics and vesicle trafficking . for example , two counteracting rop - interactive crib motif - containing ( ric ) proteins control pollen tube growth by regulating f - actin dynamics . it is tempting to speculate that cell wall derived compounds may be putative ligands for the cwi related rlks . this has , for example , been shown to be the case for the wall - associated kinase family that binds pectins . furthermore , a secreted small peptide hormone , rapid alkalinisation factor ( ralf ) negatively regulates cell elongation by binding to the fer receptor as a ligand . against this backdrop , other small peptides may be considered as ligands for cwi related rlks in root hair growth . while these data are encouraging , the identification of ligands for receptors is not a trivial task . the activated receptors may subsequently cause alterations in intra- and extracellular ion homeostasis , which brings us back to the ca , ros and additional signalling compounds with which we began this review . recent undertakings have greatly improved our understanding of root hair growth ( figure 1 ) . for example , the development of highly sophisticated calcium , ph and ros sensors has revealed a tight interplay between these factors . the recent identification of several new players in the molecular synergy of root hair growth has also stimulated the field . yet , many questions remain . secondly , cell wall biosynthesis in root hair cells differs from most other cell types . thirdly , calcium , extracellular ph and ros are important regulators of tip growth , implicated by their correlated cyclic patterns . however , the mechanisms that directly link these regulators are still largely missing , as are the identities of the ion channels that contribute to the ca uptake . lastly , what are the cwi sensors that the tip growing cells use , how are they activated and what are they regulating ? these are important factors specifying external status of the root hairs but progress in this field is still in its infancy . experiments aiming at answering these questions can provide us not only with new insights into how root hair growth works but might be applicable to other tip growing systems , and to elucidate general developmental patterns during cell growth .

over the last few decades , our understanding of directed cell growth in different organisms has substantially improved . tip - growing cells in plants elongate rapidly via targeted deposition of cell wall and membrane material at the cell apex , and use turgor pressure as a driving force for expansion . this type of polar growth requires a high degree of coordination between a plethora of cellular and extracellular components and compounds , including calcium dynamics , apoplastic reactive oxygen species and ph , the cytoskeleton , and vesicular trafficking . in this review , we attempt to outline and summarize the factors that control root hair growth and how they work together as a team .

Introduction Root hair growth: from the inside to the outside Intracellular calcium and ROS Cytoskeleton and vesicles Root hair cell walls Who holds the reins? Conclusion and perspectives Disclosures

for example , filamentous growth in the form of aseptate hyphae evolved as an excellent tool for exploring dry soils . in arbuscular mycorrhiza , tip - growing fungal hyphae rapidly enter plant root tissue to establish a mutually beneficial relationship . the first tip - growing cells in land - plants arose about 450 million years ago in bryophytes , which used filamentous rhizoids for anchoring themselves to the soil . in plants today , two types of tip - growing cells dominate ; pollen tubes and root hairs . root hairs and pollen tubes are filamentous cells that grow uni - directionally by depositing flexible cell wall material at one site of the cell and exploiting turgor pressure as the driving force for elongation ; a process also referred to as tip growth ( figure 1 ) . root hair and pollen tube tip growth include a variety of cellular components and compounds that work in concert . the intracellular calcium concentration plays a central role in plant tip growth , and regulates actin dynamics via actin interacting proteins , such as villin and adf . these proteins are likely involved in the organization of the actin cytoskeleton at different sites in the root hair . along the shank , and reach into the subapical region of the root hair . these produce ros ( h2o2 and oh ) which mediate cell wall relaxation and peroxidase - mediated wall stiffening . furthermore , calcium seems to influence extracellular ph ( possibly via h - atpases ) , which contributes to changes in cell wall structure during growth . cellulose microfibrils are short and randomly oritientated in the cell wall of the growing tip , whereas a well ordered second layer of cellulose microfibrils occurs in the shank . this organization promotes dynamic cell wall extension exclusively at the tip . in pollen tubes pmes mediate the crosslinking of pectins in the shank and remain inactive at the tip after inactivation through pmeis . abbreviations : adf , actin depolymerizing factor ; ark1 , armadillo - repeat kinesin 1 ; cngc , cyclic nucleotide - gated channel ; glr , glutamate receptor - like ; pi , inositol phosphate ; pme , pectin - methyl - esterase ; pmei , pme inhibitor ; ric , rop - interactive crib motif - containing ; rop , rho of plant gtpase ; ros , reactive oxygen species . although these tip - growing systems are very similar , they also display differences in their growth - related molecular machineries . in view of the fact that soil nutrient availability , water accessibility and soil condition are strongly affected by climate change , the study of root hair tip growth is a timely topic to help us to understand and improve plant growth and will therefore be the main focus of this review . while delicate , root hairs are excellent experimental model systems for polarized cell growth . they reach growth rates of typically 1 m min or more without losing cell wall integrity . this implies an extremely efficient system for delivering and modifying membrane and cell wall material at the tip apex . this system will therefore need to be highly coordinated , and involves a tip - derived calcium gradient , cytoskeleton dynamics , delivery of new cell wall material via vesicle trafficking , regulation of extracellular ph , and production of reactive oxygen species ( ros ) . two of the major signal transducers for root hair growth and function are ca and ros ( figure 1 ) . calcium is a crucial and general regulator of plant cell growth and development , and is perhaps the most important factor for maintenance of tip growth where it is distributed as a continuous gradient in the cytosol , with the highest concentration close to the tip apex . furthermore , the cytosolic ca concentration displays periodic cycling that follows the rate of growth of the tip . these findings suggest that changes in ca concentrations might regulate growth by the activation of ros production , and by increasing the ph , at the cell surface . the production of cell surface ros in turn activates ca influx into the cell and thereby completes a positive feedback loop . while the identity of these channels are not known in root hairs , cyclic nucleotide - gated ( cngc ) , and glutamate receptor - like ( glr ) , channels were identified as ca influx channels [ 24 ] in pollen tubes , and perhaps a similar scenario could be envisioned during root hair growth . the function of the fluctuations in intracellular ca may modulate components important for tip growth , such as cytoskeleton - organizing factors , and ros and ph homeostasis as alluded to above . however , it may also alter the apoplastic ca levels that can influence changes in the cell wall . in tip growing cells , the actin cytoskeleton is typically organized as thick actin bundles that run in parallel with the root hair shank supporting an inverse fountain flow of cytoplasmic streaming , and as fine filamentous structures at the apex of the tip that keep this region free of larger cell organelles [ 58 ] . for example , ca may influence actin depolymerizing factor ( adf ) in maize that can increase actin turnover by severing actin filaments , and villins that can influence the bundling of actin . the role of the actin cytoskeleton as a scaffold for polarized membrane trafficking is well established in root hairs . in particular , new membrane and cell wall material must be delivered to the tip apex to support the rapid growth . the cell wall material is engulfed in vesicles that move along the actin cytoskeleton via motor proteins . two recently characterized motor proteins of the myosin xi family that function in actin - dependent transport of cell organelles , vesicles and protein complexes , are required for normal actin dynamics and fast root hair growth . for example , pi-4p and pi-4,5p2 accumulate in the plasma membrane at the root hair apex where they might orchestrate growth by recruiting other important proteins containing pi-4p or pi-4,5p2 binding domains . one such example is the adp ribosylation factor gtpase - activating protein agd1 , which is necessary for maintenance of polarity during root hair growth . membrane secretion in root hair growth is also influenced by the exocyst complex , a rho and rop gtpase effector , that tethers vesicles to their target membranes . microtubule stabilizing or destabilizing treatments using taxol or oryzalin , respectively , lead to wavy and occasionally branched root hair phenotypes . ark1 plays a key role in root hair tip growth by promoting microtubule catastrophe events . in summary , while the actin cytoskeleton plays an important role as a dynamic scaffold for proper vesicle delivery in polarized root hair elongation , the microtubules are required for maintenance of cell polarity and directed growth . the cell wall is a dynamic structure that determines the shape of the root hair ( figure 1 ) . here , one can illustrate the cell wall as a corset that readily allows for expansion at the tip , but provides resistance against the turgor pressure along the shank . cellulose is a major load - bearing structure of the cell wall and is therefore the main contributor to the corset function . two partially redundant members of the cellulose synthase ( cesa)-like ( csl ) superfamily , csld2 and csld3 , are located at the root hair tip and are required for its growth . notably , at the growing tip of the root hair the cellulose microfibrils are short and randomly orientated , as revealed by ultrastructural studies [ 2426 ] . lesions in enzymes that synthesize some of these matrix polymers , for example , xyloglucans , led to root hair growth defects , perhaps due to mis - alignment of cellulose fibrils . whereas the xyloglucans thus influence root hair growth , the role of pectins is not well explored . here , pectin - methyl - esterases ( pmes ) , which de - methylesterify carboxyl residues on the pectin backbone , can affect the cell wall viscosity . through the pme action , carboxyl residues are set free to bind ca , which results in pectin cross - linking and presumably cell wall tightening at the pollen tube tip [ 3134 ] . pme and its inhibitors , pmeis , are , furthermore , spatio - temporally distributed along the pollen tube with a preferential activity of pmes at the shank and pmeis at the tip , which results in a distinct pectic composition of the pollen tube cell wall . it is possible that similar distributions also may play a role in root hair growth . in addition , extensins , cell wall structural proteins , are of crucial importance for root hair development , possibly for maintaining cell wall stability . these proteins may be post - translationally modified by o - linked glycosylation , and this modification alters cell wall properties during root hair growth . recently , pft1/med25 , a transcriptional mediator complex subunit , was revealed to orchestrate the expression of cell wall remodelling genes including a set of peroxidases and cell wall cross - linking extensins and being required for redox homeostasis in roots and normal root hair formation . these results indicate that the cell wall proteome could be a useful source to find cell wall and , therefore , tip growth modulators . however , results on the proteomic composition of root hair cell walls are scarce , not only because separating cell wall proteins from the cell wall matrix is generally challenging , but also because isolating a reasonable amount of protein from root hairs for analysis is difficult . not only calcium / pectin interactions but also surface ros and ph are thought to directly influence cell wall viscosity of the tip surface . on the other hand , h2o2 is a substrate for peroxidases that help to stiffen the wall via crosslinking of cell wall polysaccharides . this may seem like a paradox and a tight coordination between these processes is likely to occur , but how this is achieved remains unclear . surface ph may also influence the cell wall viscosity by activation of the expansins , non - hydrolytic cell wall - loosening proteins , and other cell wall modifying enzymes , which promote cell wall relaxation [ 4244 ] . two of the major signal transducers for root hair growth and function are ca and ros ( figure 1 ) . calcium is a crucial and general regulator of plant cell growth and development , and is perhaps the most important factor for maintenance of tip growth where it is distributed as a continuous gradient in the cytosol , with the highest concentration close to the tip apex . furthermore , the cytosolic ca concentration displays periodic cycling that follows the rate of growth of the tip . these findings suggest that changes in ca concentrations might regulate growth by the activation of ros production , and by increasing the ph , at the cell surface . the production of cell surface ros in turn activates ca influx into the cell and thereby completes a positive feedback loop . while the identity of these channels are not known in root hairs , cyclic nucleotide - gated ( cngc ) , and glutamate receptor - like ( glr ) , channels were identified as ca influx channels [ 24 ] in pollen tubes , and perhaps a similar scenario could be envisioned during root hair growth . the function of the fluctuations in intracellular ca may modulate components important for tip growth , such as cytoskeleton - organizing factors , and ros and ph homeostasis as alluded to above . however , it may also alter the apoplastic ca levels that can influence changes in the cell wall . in tip growing cells , the actin cytoskeleton is typically organized as thick actin bundles that run in parallel with the root hair shank supporting an inverse fountain flow of cytoplasmic streaming , and as fine filamentous structures at the apex of the tip that keep this region free of larger cell organelles [ 58 ] . for example , ca may influence actin depolymerizing factor ( adf ) in maize that can increase actin turnover by severing actin filaments , and villins that can influence the bundling of actin . the role of the actin cytoskeleton as a scaffold for polarized membrane trafficking is well established in root hairs . the cell wall material is engulfed in vesicles that move along the actin cytoskeleton via motor proteins . two recently characterized motor proteins of the myosin xi family that function in actin - dependent transport of cell organelles , vesicles and protein complexes , are required for normal actin dynamics and fast root hair growth . vesicle secretion can also be controlled by other factors , including rab gtpases and inositol phosphates ( pis ) . for example , pi-4p and pi-4,5p2 accumulate in the plasma membrane at the root hair apex where they might orchestrate growth by recruiting other important proteins containing pi-4p or pi-4,5p2 binding domains . one such example is the adp ribosylation factor gtpase - activating protein agd1 , which is necessary for maintenance of polarity during root hair growth . membrane secretion in root hair growth is also influenced by the exocyst complex , a rho and rop gtpase effector , that tethers vesicles to their target membranes . microtubule stabilizing or destabilizing treatments using taxol or oryzalin , respectively , lead to wavy and occasionally branched root hair phenotypes . ark1 plays a key role in root hair tip growth by promoting microtubule catastrophe events . in summary , while the actin cytoskeleton plays an important role as a dynamic scaffold for proper vesicle delivery in polarized root hair elongation , the microtubules are required for maintenance of cell polarity and directed growth . the cell wall is a dynamic structure that determines the shape of the root hair ( figure 1 ) . here , one can illustrate the cell wall as a corset that readily allows for expansion at the tip , but provides resistance against the turgor pressure along the shank . cellulose is a major load - bearing structure of the cell wall and is therefore the main contributor to the corset function . two partially redundant members of the cellulose synthase ( cesa)-like ( csl ) superfamily , csld2 and csld3 , are located at the root hair tip and are required for its growth . notably , at the growing tip of the root hair the cellulose microfibrils are short and randomly orientated , as revealed by ultrastructural studies [ 2426 ] . in contrast , the shank behind the actively growing tip contains a second layer of highly ordered cellulose microfibrils oriented transversely to the axis of expansion [ 2628 ] . lesions in enzymes that synthesize some of these matrix polymers , for example , xyloglucans , led to root hair growth defects , perhaps due to mis - alignment of cellulose fibrils . whereas the xyloglucans thus influence root hair growth , the role of pectins is not well explored . here , pectin - methyl - esterases ( pmes ) , which de - methylesterify carboxyl residues on the pectin backbone , can affect the cell wall viscosity . through the pme action , carboxyl residues are set free to bind ca , which results in pectin cross - linking and presumably cell wall tightening at the pollen tube tip [ 3134 ] . pme and its inhibitors , pmeis , are , furthermore , spatio - temporally distributed along the pollen tube with a preferential activity of pmes at the shank and pmeis at the tip , which results in a distinct pectic composition of the pollen tube cell wall . it is possible that similar distributions also may play a role in root hair growth . in addition , extensins , cell wall structural proteins , are of crucial importance for root hair development , possibly for maintaining cell wall stability . these proteins may be post - translationally modified by o - linked glycosylation , and this modification alters cell wall properties during root hair growth . recently , pft1/med25 , a transcriptional mediator complex subunit , was revealed to orchestrate the expression of cell wall remodelling genes including a set of peroxidases and cell wall cross - linking extensins and being required for redox homeostasis in roots and normal root hair formation . these results indicate that the cell wall proteome could be a useful source to find cell wall and , therefore , tip growth modulators . however , results on the proteomic composition of root hair cell walls are scarce , not only because separating cell wall proteins from the cell wall matrix is generally challenging , but also because isolating a reasonable amount of protein from root hairs for analysis is difficult . not only calcium / pectin interactions but also surface ros and ph are thought to directly influence cell wall viscosity of the tip surface . on the other hand , h2o2 is a substrate for peroxidases that help to stiffen the wall via crosslinking of cell wall polysaccharides . surface ph may also influence the cell wall viscosity by activation of the expansins , non - hydrolytic cell wall - loosening proteins , and other cell wall modifying enzymes , which promote cell wall relaxation [ 4244 ] . although the molecular organization of root hair growth is described with increasing detail , how internal and environmental growth - regulating signals are sensed and integrated remains enigmatic . in plants several receptor - like kinase ( rlk ) family members are implicated in relaying information about cell wall integrity ( cwi ) to the inside of the cell ( figure 1 ) . the anx1 and 2 are the closest relatives to feronia ( fer ) , which is essential for both pollen tube and root hair growth [ 4652 ] . rops accumulate in the apical plasma membrane of root hairs and other tip - growing cells , and impact actin dynamics and vesicle trafficking . it is tempting to speculate that cell wall derived compounds may be putative ligands for the cwi related rlks . furthermore , a secreted small peptide hormone , rapid alkalinisation factor ( ralf ) negatively regulates cell elongation by binding to the fer receptor as a ligand . against this backdrop , other small peptides may be considered as ligands for cwi related rlks in root hair growth . while these data are encouraging , the identification of ligands for receptors is not a trivial task . the activated receptors may subsequently cause alterations in intra- and extracellular ion homeostasis , which brings us back to the ca , ros and additional signalling compounds with which we began this review . recent undertakings have greatly improved our understanding of root hair growth ( figure 1 ) . for example , the development of highly sophisticated calcium , ph and ros sensors has revealed a tight interplay between these factors . the recent identification of several new players in the molecular synergy of root hair growth has also stimulated the field . secondly , cell wall biosynthesis in root hair cells differs from most other cell types . however , the mechanisms that directly link these regulators are still largely missing , as are the identities of the ion channels that contribute to the ca uptake . lastly , what are the cwi sensors that the tip growing cells use , how are they activated and what are they regulating ? experiments aiming at answering these questions can provide us not only with new insights into how root hair growth works but might be applicable to other tip growing systems , and to elucidate general developmental patterns during cell growth .

we included serum samples from all 21 patients referred to the hammersmith hospital s infectious diseases service from november 1994 to november 2000 who had microbiologically confirmed invasive gas disease and required hospital admission . two patients who used intravenous drugs were subsequently excluded to reduce the risk for bloodborne viruses . aliquots of serum ( residual to serum required for clinical purposes ) were separated from blood drawn for clinical purposes and frozen immediately at 70c before testing for mitogens or antibodies . samples were obtained at the point of admission to hospital ( at initiation of antibiotic therapy ) and then on sequential days during treatment up to a maximum of 10 days . streptococcal isolates were cultured directly from blood or tissue , identified by the hospital diagnostic laboratory , and then cultured once in todd hewitt broth before immediate freezing in 15% glycerol and before growth for sag analysis . all 19 patients had invasive streptococcal disease ; patients with stss were identified by using standard criteria ( 13 ) ( table ) . sag , superantigen ; spe , streptococcal pyrogenic exotoxin ; sme , streptococcal mitogenic exotoxin ; stss , streptococcal toxic shock syndrome . s. pyogenes isolates from patients with and without stss were genotyped by polymerase chain reaction with sag specific primers . concentrated supernatant from the in vitro cultured isolates were analyzed for secreted sags by using western blot analysis with recombinant sag standards . smez expression was also analyzed by using the more sensitive jurkat cell assay , which has a threshold of approximately 10 pg / ml . sag expression by western blot : - , no detectable protein ; + , < 2 ng / ml ; + + , 210 ng / ml ; + + + , > 10 ng / ml . smez expression by jurkat assay : - , no detectable smez ; + , > 10 pg / ml ( 10,00020,000 cpm ) ; + + , 20,00030,000 cpm ; + + + , > 30,000 cpm . jurkat assay . human peripheral blood lymphocytes ( pbls ) were purified from blood of a healthy donor by using histopaque ficoll ( sigma chemical co. , st . pbls were incubated in 96-well , round - bottom microtiter plates at 10 cells per well with rpmi-10 ( rpmi with 10% fetal calf serum [ fcs ] ) containing varying dilutions of recombinant toxins . after 3 days , 0.1 ci [ h ] thymidine was added to each well and cells were incubated for another 24 h. cells were harvested and counted on a scintillation counter . jurkat cells ( a human t - cell line ) and lg-2 cells ( a human b - lymphoblastoid cell line , homozygous for hla - dr1 ) were harvested in log phase and resuspended in rpmi-10 . one hundred microliters of the cell suspension , containing 1x10 jurkat cells and 2x10 lg-2 cells was mixed with 100 l of s. pyogenes culture supernatant ( undiluted , 1:10 , 1:100 ) on 96-well plates . after incubating overnight at 37c , 100-l aliquots were transferred onto a fresh plate and 100 l ( 1x10 ) of sei cells ( il-2 dependent murine t - cell line ) per well was added . after incubating for 24 h , 0.1 ci [ h ] thymidine was added to each well , and cells were incubated for another 24 h. cells were harvested and counted on a scintillation counter . as a control , a dilution series of il-2 was incubated with sei cells . pbls were obtained and stimulated as described under toxin proliferation assay above , with the exception that the 10% fcs was replaced by 5% fcs plus 5% patient serum . all recombinant toxins were used at subsaturating concentrations , which were 0.05 ng / ml ( smez-2 ) , 0.1 ng / ml ( all other smez variants , spe - c , spe - i , spe - j , streptococcal superantigen [ ssa ] ) , 1 ng / ml ( spe - g ) , 2 ng / ml ( spe - a ) , and 10 ng / ml ( spe - h ) . we determined the neutralizing response by comparing the t - cell proliferation with a control test using 10% fcs instead of 5% patient serum plus 5% fcs . the relative inhibition was calculated as 1 cpm ( patient serum ) per cpm ( fcs ) . s. pyogenes isolates were grown overnight in 10 ml of brain heart infusion ( bhi ) medium ( difco laboratories , detroit , mi ) at 37c in 15-ml falcon tubes without agitation . the cells were spun down and washed , and the genomic dna was extracted as described previously ( 33 ) . the purified dna was resuspended in 50 l of tris - edta buffer and used for polymerase chain reaction ( pcr ) with specific primers for the spea , spec , speg , speh , spei , spej , ssa , and smez genes as described previously ( 3133 ) . in addition , a primer pair specific to a dna region encoding the 23s rrna ( 33 ) was used as a positive control . recombinant forms of spe - a , spe - c , spe - g , spe - h , spe - i , spe - j , ssa , smez-1 , and smez-2 were produced in escherichia coli by using the pgex-2 t expression system as described previously ( 31,33 ) . new zealand white rabbits were immunized with 50 g of recombinant protein in 1-ml phosphate - buffered saline and 1-ml incomplete freund s adjuvants ( invitrogene , san diego , ca ) followed by a booster injection 4 weeks later . smez-1 and smez-2 were injected as a 1:1 mixture to ensure generation of antibodies against a large panel of the smez variants . the medium was prepared by dialyzing 100-ml of 10x concentrated bhi medium against 1 l water . three grams of glucose , 4 g of na2hpo4 , and 10 ml of the dialysate were then added to the solution outside the tubing , which was used to grow the bacteria . bacterial cells were spun down , and the supernatant was transferred into a new tube and spun at high speed ( 10,000 rpm ) in a beckman ja20 rotor for 20 min to remove remaining cells . the proteins were then precipitated from the clear supernatant after the addition of 0.15 v of 50% tri - chloracetic acid and centrifugation at 10,000 rpm for 20 min . the precipitated proteins were resuspended in pbl to 1/50 of the original volume and mixed with an equal volume of 2x sodium dodecyl sulfate the ph was readjusted by blowing ammonia vapor onto the sample until the color changed from yellow back to blue . after boiling for 2 min , 10 l was loaded onto a 12% sds - page and run along a protein standard ( 50 ng , 25 ng , 10 ng , and 2 ng of the appropriate recombinant sag ) . the proteins were blotted onto a hybond - c extra nitrocellulose membrane ( amersham life sciences , little chalfont , uk ) by using western transfer buffer ( 10% methanol , 150 mm glycine , 25 mm tris - hcl ph 8.5 ) and a semi - phor semi - dry blotter ( hoefer scientific instruments , san francisco , ca ) . the membranes were blocked with 5% milk powder ( anchor , auckland , new zealand ) in tbst ( 120 mm nacl , 10 mm tris - hcl ph8 , 0.05% tween 20 ) for 30 min at room temperature , before incubation with the appropriate rabbit anti - sag antiserum ( 1:5000 in tbst ) overnight at 4c . the blots were developed by using the ecl western blotting analysis system ( amersham biosciences , little chalfont , uk ) we included serum samples from all 21 patients referred to the hammersmith hospital s infectious diseases service from november 1994 to november 2000 who had microbiologically confirmed invasive gas disease and required hospital admission . two patients who used intravenous drugs were subsequently excluded to reduce the risk for bloodborne viruses . aliquots of serum ( residual to serum required for clinical purposes ) were separated from blood drawn for clinical purposes and frozen immediately at 70c before testing for mitogens or antibodies . samples were obtained at the point of admission to hospital ( at initiation of antibiotic therapy ) and then on sequential days during treatment up to a maximum of 10 days . streptococcal isolates were cultured directly from blood or tissue , identified by the hospital diagnostic laboratory , and then cultured once in todd hewitt broth before immediate freezing in 15% glycerol and before growth for sag analysis . all 19 patients had invasive streptococcal disease ; patients with stss were identified by using standard criteria ( 13 ) ( table ) . sag , superantigen ; spe , streptococcal pyrogenic exotoxin ; sme , streptococcal mitogenic exotoxin ; stss , streptococcal toxic shock syndrome . s. pyogenes isolates from patients with and without stss were genotyped by polymerase chain reaction with sag specific primers . concentrated supernatant from the in vitro cultured isolates were analyzed for secreted sags by using western blot analysis with recombinant sag standards . smez expression was also analyzed by using the more sensitive jurkat cell assay , which has a threshold of approximately 10 pg / ml . sag expression by western blot : - , no detectable protein ; + , < 2 ng / ml ; + + , 210 ng / ml ; + + + , > 10 ng / ml . smez expression by jurkat assay : - , no detectable smez ; + , > 10 pg / ml ( 10,00020,000 cpm ) ; + + , 20,00030,000 cpm ; + + + , > 30,000 cpm . jurkat assay . human peripheral blood lymphocytes ( pbls ) were purified from blood of a healthy donor by using histopaque ficoll ( sigma chemical co. , st . pbls were incubated in 96-well , round - bottom microtiter plates at 10 cells per well with rpmi-10 ( rpmi with 10% fetal calf serum [ fcs ] ) containing varying dilutions of recombinant toxins . after 3 days , 0.1 ci [ h ] thymidine was added to each well and cells were incubated for another 24 h. cells were harvested and counted on a scintillation counter . jurkat cells ( a human t - cell line ) and lg-2 cells ( a human b - lymphoblastoid cell line , homozygous for hla - dr1 ) were harvested in log phase and resuspended in rpmi-10 . one hundred microliters of the cell suspension , containing 1x10 jurkat cells and 2x10 lg-2 cells was mixed with 100 l of s. pyogenes culture supernatant ( undiluted , 1:10 , 1:100 ) on 96-well plates . after incubating overnight at 37c , 100-l aliquots were transferred onto a fresh plate and 100 l ( 1x10 ) of sei cells ( il-2 dependent murine t - cell line ) per well was added . after incubating for 24 h , 0.1 ci [ h ] thymidine was added to each well , and cells were incubated for another 24 h. cells were harvested and counted on a scintillation counter . as a control , a dilution series of il-2 was incubated with sei cells . pbls were obtained and stimulated as described under toxin proliferation assay above , with the exception that the 10% fcs was replaced by 5% fcs plus 5% patient serum . all recombinant toxins were used at subsaturating concentrations , which were 0.05 ng / ml ( smez-2 ) , 0.1 ng / ml ( all other smez variants , spe - c , spe - i , spe - j , streptococcal superantigen [ ssa ] ) , 1 ng / ml ( spe - g ) , 2 ng / ml ( spe - a ) , and 10 ng / ml ( spe - h ) . we determined the neutralizing response by comparing the t - cell proliferation with a control test using 10% fcs instead of 5% patient serum plus 5% fcs . the relative inhibition was calculated as 1 cpm ( patient serum ) per cpm ( fcs ) . s. pyogenes isolates were grown overnight in 10 ml of brain heart infusion ( bhi ) medium ( difco laboratories , detroit , mi ) at 37c in 15-ml falcon tubes without agitation . the cells were spun down and washed , and the genomic dna was extracted as described previously ( 33 ) . the purified dna was resuspended in 50 l of tris - edta buffer and used for polymerase chain reaction ( pcr ) with specific primers for the spea , spec , speg , speh , spei , spej , ssa , and smez genes as described previously ( 3133 ) . in addition , a primer pair specific to a dna region encoding the 23s rrna ( 33 ) was used as a positive control . recombinant forms of spe - a , spe - c , spe - g , spe - h , spe - i , spe - j , ssa , smez-1 , and smez-2 were produced in escherichia coli by using the pgex-2 t expression system as described previously ( 31,33 ) . new zealand white rabbits were immunized with 50 g of recombinant protein in 1-ml phosphate - buffered saline and 1-ml incomplete freund s adjuvants ( invitrogene , san diego , ca ) followed by a booster injection 4 weeks later . smez-1 and smez-2 were injected as a 1:1 mixture to ensure generation of antibodies against a large panel of the smez variants . the medium was prepared by dialyzing 100-ml of 10x concentrated bhi medium against 1 l water . three grams of glucose , 4 g of na2hpo4 , and 10 ml of the dialysate were then added to the solution outside the tubing , which was used to grow the bacteria . bacterial cells were spun down , and the supernatant was transferred into a new tube and spun at high speed ( 10,000 rpm ) in a beckman ja20 rotor for 20 min to remove remaining cells . the proteins were then precipitated from the clear supernatant after the addition of 0.15 v of 50% tri - chloracetic acid and centrifugation at 10,000 rpm for 20 min . the precipitated proteins were resuspended in pbl to 1/50 of the original volume and mixed with an equal volume of 2x sodium dodecyl sulfate the ph was readjusted by blowing ammonia vapor onto the sample until the color changed from yellow back to blue . after boiling for 2 min , 10 l was loaded onto a 12% sds - page and run along a protein standard ( 50 ng , 25 ng , 10 ng , and 2 ng of the appropriate recombinant sag ) . the proteins were blotted onto a hybond - c extra nitrocellulose membrane ( amersham life sciences , little chalfont , uk ) by using western transfer buffer ( 10% methanol , 150 mm glycine , 25 mm tris - hcl ph 8.5 ) and a semi - phor semi - dry blotter ( hoefer scientific instruments , san francisco , ca ) . the membranes were blocked with 5% milk powder ( anchor , auckland , new zealand ) in tbst ( 120 mm nacl , 10 mm tris - hcl ph8 , 0.05% tween 20 ) for 30 min at room temperature , before incubation with the appropriate rabbit anti - sag antiserum ( 1:5000 in tbst ) overnight at 4c . the blots were developed by using the ecl western blotting analysis system ( amersham biosciences , little chalfont , uk ) nineteen acute - phase serum samples from patients with severe invasive streptococcal disease with stss ( n=9 ) and without stss ( n=10 ) were analyzed for mitogenic activity . serum samples 96/2 and 99/1 both induced a high proliferative response when tested in a pbl - stimulation assay , which suggests that they might contain a sag . peritonitis was diagnosed in patient 96/2 , who recovered after 2 weeks in the intensive - care unit ; tss was diagnosed in patient 99/1 , who died on the day of admission . to identify the sag responsible for the mitogenic activity , pbls were stimulated with serum 96/2 and serum 99/1 , respectively , together with rabbit antibodies against recombinant forms of spe - a , spe - c , spe - g , spe - j , or smez ( figure 1a ) . addition of anti - smez antibodies resulted in 59% and 68% inhibition of the mitogenic activity of serum samples 96/2 and 99/1 , respectively . antibodies against spe - j inhibited pbl stimulation of serum 96/2 by 51% but had no substantial effect on the serum 99/1 induced activity . spe - g antibodies did not substantially inhibit the activity in both serum samples , except for a slight inhibition ( 18% ) of serum 96/2 activity by anti inhibition of mitogenic activity in sera 96/2 and 99/1 with anti - superantigen ( sag ) antisera . a ) peripheral blood lymphocytes ( pbls ) were stimulated with 5% patient serum in the presence of 5% anti - sag antiserum or 5% fetal calf serum ( fcs ) only . after 3 days , [ h]-thymidine was added , and pbls were incubated for another 24 h , before being washed and counted . the results were blotted as percentage of inhibition with specific anti - sag serum compared to fcs . antistreptococcal pyrogenic exotoxin ( spe)-j antiserum inhibited the mitogenic activity of serum 96/2 by 51% , while antistreptococcal mitogenic exotoxin ( sme ) z antiserum inhibited the activity in 96/2 by 59% and the serum in 99/1 by 68% . b ) the specificities of the anti spe - j and anti smez sera were demonstrated by stimulating pbls with recombinant sags in the presence of 5% antiserum . spe - j and anti - smez antiserum were added to pbls stimulated with various recombinant sags ( spe - a , spe - c , spe - g , spe - h , spe - i , spe - j , smez-1 , smez-2 , and ssa ) , and results showed that anti spe - j antibodies exclusively inhibited the rspe - j activity , whereas anti - smez antibodies inhibited the activity of rsmez-1 and rsmez-2 ( figure 1b ) . to quantify the levels of sag in 96/2 and 99/1 serum samples , a comparison was made against a standard pbl proliferation response for recombinant smez-1 and rspe - j . five percent of each serum resulted in 33,00034,000 cpm after [ h]-thymidine uptake , which is equivalent to 110 pg / ml of rsmez-1 or rspe - j ( figure 2 ) . mitogenic activity of acute - phase serum samples 96/2 and 99/1 compared to recombinant superantigens ( sags ) . peripheral blood lymphocytes were stimulated for 4 d with various dilutions of recombinant sag or acute - phase serum sample 99/1 . five percent of each of the patient serum samples showed a proliferative response equal to 110 pg / ml of recombinant streptococcal pyrogenic exotoxin j or recombinant streptococcal mitogenic exotoxin 1 . sequential serum samples from stss patient 96/2 up to day 9 after admission to hospital were analyzed for clearance of mitogenic activity . figure 3 shows that the highest mitogenic activity occurred 1.5 days postadmission ( serum 96/2 - 2 ) . at day 2 postadmission , an 80% reduction of activity had occurred , and by day 3 and day 9 after admission sag activity was undetectable . peripheral blood lymphocytes were stimulated with 5% of acute - phase and sequential serum samples from patient 96/2 . the mitogenic activity reached the highest point on day 1.5 after admission to hospital and dropped sharply on day 2 . no substantial activity was found in sequential serum samples from day 3 on ( samples 96/24 to 96/210 ) . the sequential serum samples from patient 96/2 allowed us to analyze for the development of neutralizing anti - sag antibodies by using patient serum to inhibit the activity of recombinant sags in a pbl - stimulation assay ( figure 4 ) . on day 3 , patient 96/2 had neutralizing antibodies against spe - a , spe - c , spe - i , and ssa but undetectable levels of protective antibodies against smez-1 , smez-2 , spe - g , and spe - h . by day 9 after admission ( sample 96/2 - 10 ) , the serum contained high titers of neutralizing antibodies against spe - a , spe - c , spe - i , ssa , smez-1 , and smez-2 , and a moderate anti spe - h titer . no antibodies against spe - g were detected . these results show seroconversion for smez-1 and smez-2 antibodies in the stss patient 96/2 , adding further evidence that smez was the predominant sag causing stss in this patient . peripheral blood lymphocytes were stimulated with various recombinant streptococcal sags in the presence of serum 96/24 , 96/210 , or fetal calf serum only . the columns show the percentage of inhibition of recombinant sags by neutralizing antibodies in patient serum samples . the sequential serum on day 3 showed a complete lack of neutralizing antistreptococcal mitogenic exotoxin ( sme ) z antibodies , while serum 96/210 converted to a high anti - smez antibody titer . both sera enhanced the mitogenic activity of recombinant streptococcal pyrogenic exotoxin j , which suggests the presence of an unknown synergistic factor . addition of serum 96/2 - 4 or 96/2 - 10 selectively increased the mitogenicity of spe - j by twofold compared to the fcs control . this selective increase suggests that the serum might contain a substance that selectively synergizes spe - j activity , which prevented any evaluation of the anti spe - j antibody titer in the two sequential serum samples . serum 96/2 - 10 ( 10 days after admission ) was analyzed for protective antibodies against smez-1 , -2 , -3 , -4 , -5 , -7 , -8 , -9 , -13 , -16 , -20 , -21 , and -22 and found to neutralize the activity of all tested smez variants ( data not shown ) . inhibition of approximately 95% was seen with all smez variants , except smez-2 ( 88% ) , smez-16 ( 78% ) , and smez-22 ( 85% ) , indicating that challenge with a single smez variant resulted in a cross - reactive antibody response against all smez variants . matching gas isolates from all 19 patients the frequencies were 100% ( smez , speg , spej ) , 36.8% ( spea ) , 31.6% ( ssa ) , 26.3% ( speh ) , 15.8% ( spc ) , and 10.5% ( spei ) ( table ) . we observed no difference in sag gene frequencies between patients with stts and patients without stss . the smez alleles of both gas strains isolated from patients 96/2 and 99/1 were analyzed by dna sequencing and identified as smez-1 ( h297 ) and smez-16 ( h360 ) . all gas isolates were grown in liquid culture , and the supernatants were analyzed for secreted sags by western blot using rabbit antisera against individual recombinant sags ( table ) . the selectivity of each antiserum was tested with the complete panel of recombinant sags in western blots , and no cross - reactivity was observed ( data not shown ) . spe - a was expressed in substantial amounts only from isolates from patients with stss ( isolates h325 , h330 , and h366 ) . isolates from patients without stss that carry the spea gene ( h308 , h314 , and h315 ) had undetectable levels of spe - a however , patients infected with the spe - a producing strains did not show any mitogenic activity in their acute - phase serum samples . in contrast , gas isolated from the patients 96/2 and 99/1 ( h297 and h360 ) produced only small amounts of smez in vitro despite the relatively large amounts detected in the acute - phase serum samples . in vitro produced smez could not be detected in western blots , indicating a concentration of < 1 ng / ml , and could only be detected using the more sensitive jurkat cell proliferation assay that has a lower sensitivity threshold ( approximately 10 pg / ml ) . nineteen acute - phase serum samples from patients with severe invasive streptococcal disease with stss ( n=9 ) and without stss ( n=10 ) were analyzed for mitogenic activity . serum samples 96/2 and 99/1 both induced a high proliferative response when tested in a pbl - stimulation assay , which suggests that they might contain a sag . peritonitis was diagnosed in patient 96/2 , who recovered after 2 weeks in the intensive - care unit ; tss was diagnosed in patient 99/1 , who died on the day of admission . to identify the sag responsible for the mitogenic activity , pbls were stimulated with serum 96/2 and serum 99/1 , respectively , together with rabbit antibodies against recombinant forms of spe - a , spe - c , spe - g , spe - j , or smez ( figure 1a ) . addition of anti - smez antibodies resulted in 59% and 68% inhibition of the mitogenic activity of serum samples 96/2 and 99/1 , respectively . antibodies against spe - j inhibited pbl stimulation of serum 96/2 by 51% but had no substantial effect on the serum 99/1 induced activity . spe - g antibodies did not substantially inhibit the activity in both serum samples , except for a slight inhibition ( 18% ) of serum 96/2 activity by anti inhibition of mitogenic activity in sera 96/2 and 99/1 with anti - superantigen ( sag ) antisera . a ) peripheral blood lymphocytes ( pbls ) were stimulated with 5% patient serum in the presence of 5% anti - sag antiserum or 5% fetal calf serum ( fcs ) only . after 3 days , [ h]-thymidine was added , and pbls were incubated for another 24 h , before being washed and counted . the results were blotted as percentage of inhibition with specific anti - sag serum compared to fcs . antistreptococcal pyrogenic exotoxin ( spe)-j antiserum inhibited the mitogenic activity of serum 96/2 by 51% , while antistreptococcal mitogenic exotoxin ( sme ) z antiserum inhibited the activity in 96/2 by 59% and the serum in 99/1 by 68% . b ) the specificities of the anti spe - j and anti smez sera were demonstrated by stimulating pbls with recombinant sags in the presence of 5% antiserum . spe - j and anti - smez antiserum were added to pbls stimulated with various recombinant sags ( spe - a , spe - c , spe - g , spe - h , spe - i , spe - j , smez-1 , smez-2 , and ssa ) , and results showed that anti spe - j antibodies exclusively inhibited the rspe - j activity , whereas anti - smez antibodies inhibited the activity of rsmez-1 and rsmez-2 ( figure 1b ) . to quantify the levels of sag in 96/2 and 99/1 serum samples , a comparison was made against a standard pbl proliferation response for recombinant smez-1 and rspe - j . five percent of each serum resulted in 33,00034,000 cpm after [ h]-thymidine uptake , which is equivalent to 110 pg / ml of rsmez-1 or rspe - j ( figure 2 ) . mitogenic activity of acute - phase serum samples 96/2 and 99/1 compared to recombinant superantigens ( sags ) . peripheral blood lymphocytes were stimulated for 4 d with various dilutions of recombinant sag or acute - phase serum sample 99/1 . five percent of each of the patient serum samples showed a proliferative response equal to 110 pg / ml of recombinant streptococcal pyrogenic exotoxin j or recombinant streptococcal mitogenic exotoxin 1 . sequential serum samples from stss patient 96/2 up to day 9 after admission to hospital were analyzed for clearance of mitogenic activity . figure 3 shows that the highest mitogenic activity occurred 1.5 days postadmission ( serum 96/2 - 2 ) . at day 2 postadmission , an 80% reduction of activity had occurred , and by day 3 and day 9 after admission sag activity was undetectable . peripheral blood lymphocytes were stimulated with 5% of acute - phase and sequential serum samples from patient 96/2 . the mitogenic activity reached the highest point on day 1.5 after admission to hospital and dropped sharply on day 2 . no substantial activity was found in sequential serum samples from day 3 on ( samples 96/24 to 96/210 ) . the sequential serum samples from patient 96/2 allowed us to analyze for the development of neutralizing anti - sag antibodies by using patient serum to inhibit the activity of recombinant sags in a pbl - stimulation assay ( figure 4 ) . on day 3 , patient 96/2 had neutralizing antibodies against spe - a , spe - c , spe - i , and ssa but undetectable levels of protective antibodies against smez-1 , smez-2 , spe - g , and spe - h . by day 9 after admission ( sample 96/2 - 10 ) , the serum contained high titers of neutralizing antibodies against spe - a , spe - c , spe - i , ssa , smez-1 , and smez-2 , and a moderate anti spe - h titer . no antibodies against spe - g were detected . these results show seroconversion for smez-1 and smez-2 antibodies in the stss patient 96/2 , adding further evidence that smez was the predominant sag causing stss in this patient . peripheral blood lymphocytes were stimulated with various recombinant streptococcal sags in the presence of serum 96/24 , 96/210 , or fetal calf serum only . the columns show the percentage of inhibition of recombinant sags by neutralizing antibodies in patient serum samples . the sequential serum on day 3 showed a complete lack of neutralizing antistreptococcal mitogenic exotoxin ( sme ) z antibodies , while serum 96/210 converted to a high anti - smez antibody titer . both sera enhanced the mitogenic activity of recombinant streptococcal pyrogenic exotoxin j , which suggests the presence of an unknown synergistic factor . addition of serum 96/2 - 4 or 96/2 - 10 selectively increased the mitogenicity of spe - j by twofold compared to the fcs control . this selective increase suggests that the serum might contain a substance that selectively synergizes spe - j activity , which prevented any evaluation of the anti spe - j antibody titer in the two sequential serum samples . serum 96/2 - 10 ( 10 days after admission ) was analyzed for protective antibodies against smez-1 , -2 , -3 , -4 , -5 , -7 , -8 , -9 , -13 , -16 , -20 , -21 , and -22 and found to neutralize the activity of all tested smez variants ( data not shown ) . inhibition of approximately 95% was seen with all smez variants , except smez-2 ( 88% ) , smez-16 ( 78% ) , and smez-22 ( 85% ) , indicating that challenge with a single smez variant resulted in a cross - reactive antibody response against all smez variants . matching gas isolates from all 19 patients the frequencies were 100% ( smez , speg , spej ) , 36.8% ( spea ) , 31.6% ( ssa ) , 26.3% ( speh ) , 15.8% ( spc ) , and 10.5% ( spei ) ( table ) . we observed no difference in sag gene frequencies between patients with stts and patients without stss . the smez alleles of both gas strains isolated from patients 96/2 and 99/1 were analyzed by dna sequencing and identified as smez-1 ( h297 ) and smez-16 ( h360 ) . all gas isolates were grown in liquid culture , and the supernatants were analyzed for secreted sags by western blot using rabbit antisera against individual recombinant sags ( table ) . the selectivity of each antiserum was tested with the complete panel of recombinant sags in western blots , and no cross - reactivity was observed ( data not shown ) . spe - a was expressed in substantial amounts only from isolates from patients with stss ( isolates h325 , h330 , and h366 ) . isolates from patients without stss that carry the spea gene ( h308 , h314 , and h315 ) had undetectable levels of spe - a . however , patients infected with the spe - a producing strains did not show any mitogenic activity in their acute - phase serum samples . in contrast , gas isolated from the patients 96/2 and 99/1 ( h297 and h360 ) produced only small amounts of smez in vitro despite the relatively large amounts detected in the acute - phase serum samples . in vitro produced smez could not be detected in western blots , indicating a concentration of < 1 ng / ml , and could only be detected using the more sensitive jurkat cell proliferation assay that has a lower sensitivity threshold ( approximately 10 pg / ml ) . over the last 2 decades , a large increase in gas - mediated severe invasive disease has occurred ( 5,24,25 ) . evidence for sag involvement in these diseases derived from studies showing higher frequencies of spea and spec genes in severe disease isolates compared to nonsevere disease isolates ( 2,2225 ) , and reports of strong proliferative responses in the acute - phase serum samples from a patient with stss ( 28 ) and from mice infected with a sag - producing s. pyogenes strain ( 29 ) . in this study , we analyzed 19 acute - phase serum samples from patients with severe streptococcal disease with stss ( n=9 ) and without stss ( n=10 ) for mitogenic activity and detected smez in samples from 2 stss patients ( 96/2 and 99/1 ) . one of the serum samples ( 96/2 ) also contained detectable amounts of spe - j . the mitogenic activity in the two serum samples was equivalent to 110 pg / ml and 10 pg / ml of smez-1 or spe - j , which is sufficient to trigger maximal t - cell activation in pbl - stimulation assays . however , this concentration remains an estimate as inhibitory effects of serum components were not defined . the sequential serum of the surviving patient ( 96/2 ) showed no protective anti - smez antibodies on day 3 after admission ( serum 96/24 ) but substantial levels at day 9 ( serum 96/210 ) , which suggests a direct role of this toxin in the stss of this patient . challenge with smez-1 ( the smez variant produced by gas isolate h297 from patient 96/2 ) resulted in a broad neutralization response against the complete range of smez variants . we have shown previously that some variation in healthy blood donors exists in neutralizing antibodies to smez variants because of antigenic variation ( 33 ) . whether broad - spectrum anti - smez responses are a result of a few cross - reacting antibodies that were raised against a single smez variant has yet to be established , as does whether it results from infection by multiple gas strains carrying different smez alleles over a certain period . however , this response might not reflect a general response , and smez concentration in the blood might play a critical role . analysis of the 19 matching gas isolates showed no bias in sag genotype between stss isolates and non - stss isolates . furthermore , the results are consistent with the sag gene frequencies previously observed in 39 s. pyogenes isolates from new zealand patients with sore throat ( n=29 ) , wound infection ( n=4 ) , acute glomerulonephritis ( n=1 ) , otitis ( n=1 ) , endometritis ( n=2 ) , rheumatic fever ( n=1 ) , and skin ulcer ( n=1 ) ( 36 ) . the only substantial difference was the frequency of the spec gene , found in 42% of new zealand isolates and 15.8% of london isolates . these results suggest that genotyping of sag genes alone is not predictive for type or severity of streptococcal disease . we found no substantial difference in sag production in cell cultures between stss and non - stss strains , with the exception of spe - a , which was only expressed in stss strains and suggests a tight association of spe - a in some cases of stss . these data are consistent with data from other groups that showed a link between spe - a expression and severe streptococcal disease ( 17,22,23 ) . evidence points to very different regulation of sag production between in vitro culture and in vivo infection . smez-1 , for example , was undetectable in cultures of isolate h297 except by the sensitive jurkat proliferation assay , yet the patient with this gas isolate had high serum levels of smez-1 , indicating that smez production is tightly regulated . showed the in vivo induction of the spea gene by using a micropore teflon diffusion chamber implanted subcutaneously in balb / c mice ( 37 ) . broudy et al . observed an induction of spe - c after they co - cultured a spec - carrying isolate with a human pharyngeal cell line ( 35 ) . some , if not all , sags can likely be induced or upregulated by an unknown host factor . despite the fact that smez is poorly expressed in vitro while most other sags are frequently produced in culture , smez appears to be the predominant bioactive circulating sag at the time of admission . this observation is in keeping with recent findings using isogenic smez+ and smez- bacteria in a mouse model ( 29 ) . we attempted to correlate streptococcal disease and disease severity with the production of a particular sag . although the patient cohort is small , no direct correlation between disease severity and sag genotype / sag production is evident ; other factors likely contribute to stss severity . importantly , host factors may enhance the production of particular sags and , in addition , the immunogenic background of the host may contribute to the severity of sag - mediated invasive streptococcal disease , as recently reported ( 38 ) . although the overall potential role of sags in severe streptococcal disease remains elusive , our results directly implicate smez in the onset of stss in at least two patients . this presence of a particular bioreactive sag in the blood of patients with a reemerging potentially fatal disease is new and provides a platform for further investigation of the role of this potent sag in disease pathophysiology .

superantigens produced by streptococcus pyogenes have been implicated with streptococcal toxic shock syndrome ( stss ) . we analyzed 19 acute - phase serum samples for mitogenic activity from patients with severe streptococcal disease . the serum samples from two patients in the acute phase of stss showed strong proliferative activity . streptococcal mitogenic exotoxin ( sme ) z-1 and streptococcal pyrogenic exotoxin ( spe)-j were identified in one patient with peritonitis who recovered after 2 weeks in intensive care . smez-16 was found in a second patient who died on the day of admission . sequential serum samples taken on day 3 after admission from patient 1 showed clearance of mitogenic activity but absence of neutralizing anti - smez antibodies . serum samples taken on day 9 from this patient showed evidence of seroconversion with high levels of anti - smez antibodies that neutralized smez-1 and 12 other smez - variants . these results imply that a high level of smez production by group a streptococcus is a causative event in the onset and subsequent severity of stss .

Material and Methods Patient Serum Samples and Streptococcal Isolates Toxin Proliferation Assay Western Blot Analysis Results Mitogenic Activity in Samples from Patients with STSS STSS Patient 96/2 and Seroconversion to SMEZ Discussion

we included serum samples from all 21 patients referred to the hammersmith hospital s infectious diseases service from november 1994 to november 2000 who had microbiologically confirmed invasive gas disease and required hospital admission . two patients who used intravenous drugs were subsequently excluded to reduce the risk for bloodborne viruses . samples were obtained at the point of admission to hospital ( at initiation of antibiotic therapy ) and then on sequential days during treatment up to a maximum of 10 days . all 19 patients had invasive streptococcal disease ; patients with stss were identified by using standard criteria ( 13 ) ( table ) . sag , superantigen ; spe , streptococcal pyrogenic exotoxin ; sme , streptococcal mitogenic exotoxin ; stss , streptococcal toxic shock syndrome . s. pyogenes isolates from patients with and without stss were genotyped by polymerase chain reaction with sag specific primers . the blots were developed by using the ecl western blotting analysis system ( amersham biosciences , little chalfont , uk ) we included serum samples from all 21 patients referred to the hammersmith hospital s infectious diseases service from november 1994 to november 2000 who had microbiologically confirmed invasive gas disease and required hospital admission . samples were obtained at the point of admission to hospital ( at initiation of antibiotic therapy ) and then on sequential days during treatment up to a maximum of 10 days . all 19 patients had invasive streptococcal disease ; patients with stss were identified by using standard criteria ( 13 ) ( table ) . sag , superantigen ; spe , streptococcal pyrogenic exotoxin ; sme , streptococcal mitogenic exotoxin ; stss , streptococcal toxic shock syndrome . s. pyogenes isolates from patients with and without stss were genotyped by polymerase chain reaction with sag specific primers . the blots were developed by using the ecl western blotting analysis system ( amersham biosciences , little chalfont , uk ) nineteen acute - phase serum samples from patients with severe invasive streptococcal disease with stss ( n=9 ) and without stss ( n=10 ) were analyzed for mitogenic activity . serum samples 96/2 and 99/1 both induced a high proliferative response when tested in a pbl - stimulation assay , which suggests that they might contain a sag . peritonitis was diagnosed in patient 96/2 , who recovered after 2 weeks in the intensive - care unit ; tss was diagnosed in patient 99/1 , who died on the day of admission . addition of anti - smez antibodies resulted in 59% and 68% inhibition of the mitogenic activity of serum samples 96/2 and 99/1 , respectively . antibodies against spe - j inhibited pbl stimulation of serum 96/2 by 51% but had no substantial effect on the serum 99/1 induced activity . spe - g antibodies did not substantially inhibit the activity in both serum samples , except for a slight inhibition ( 18% ) of serum 96/2 activity by anti inhibition of mitogenic activity in sera 96/2 and 99/1 with anti - superantigen ( sag ) antisera . a ) peripheral blood lymphocytes ( pbls ) were stimulated with 5% patient serum in the presence of 5% anti - sag antiserum or 5% fetal calf serum ( fcs ) only . the results were blotted as percentage of inhibition with specific anti - sag serum compared to fcs . antistreptococcal pyrogenic exotoxin ( spe)-j antiserum inhibited the mitogenic activity of serum 96/2 by 51% , while antistreptococcal mitogenic exotoxin ( sme ) z antiserum inhibited the activity in 96/2 by 59% and the serum in 99/1 by 68% . spe - j and anti - smez antiserum were added to pbls stimulated with various recombinant sags ( spe - a , spe - c , spe - g , spe - h , spe - i , spe - j , smez-1 , smez-2 , and ssa ) , and results showed that anti spe - j antibodies exclusively inhibited the rspe - j activity , whereas anti - smez antibodies inhibited the activity of rsmez-1 and rsmez-2 ( figure 1b ) . to quantify the levels of sag in 96/2 and 99/1 serum samples , a comparison was made against a standard pbl proliferation response for recombinant smez-1 and rspe - j . mitogenic activity of acute - phase serum samples 96/2 and 99/1 compared to recombinant superantigens ( sags ) . peripheral blood lymphocytes were stimulated for 4 d with various dilutions of recombinant sag or acute - phase serum sample 99/1 . five percent of each of the patient serum samples showed a proliferative response equal to 110 pg / ml of recombinant streptococcal pyrogenic exotoxin j or recombinant streptococcal mitogenic exotoxin 1 . sequential serum samples from stss patient 96/2 up to day 9 after admission to hospital were analyzed for clearance of mitogenic activity . at day 2 postadmission , an 80% reduction of activity had occurred , and by day 3 and day 9 after admission sag activity was undetectable . peripheral blood lymphocytes were stimulated with 5% of acute - phase and sequential serum samples from patient 96/2 . the mitogenic activity reached the highest point on day 1.5 after admission to hospital and dropped sharply on day 2 . no substantial activity was found in sequential serum samples from day 3 on ( samples 96/24 to 96/210 ) . the sequential serum samples from patient 96/2 allowed us to analyze for the development of neutralizing anti - sag antibodies by using patient serum to inhibit the activity of recombinant sags in a pbl - stimulation assay ( figure 4 ) . on day 3 , patient 96/2 had neutralizing antibodies against spe - a , spe - c , spe - i , and ssa but undetectable levels of protective antibodies against smez-1 , smez-2 , spe - g , and spe - h . by day 9 after admission ( sample 96/2 - 10 ) , the serum contained high titers of neutralizing antibodies against spe - a , spe - c , spe - i , ssa , smez-1 , and smez-2 , and a moderate anti spe - h titer . these results show seroconversion for smez-1 and smez-2 antibodies in the stss patient 96/2 , adding further evidence that smez was the predominant sag causing stss in this patient . peripheral blood lymphocytes were stimulated with various recombinant streptococcal sags in the presence of serum 96/24 , 96/210 , or fetal calf serum only . the sequential serum on day 3 showed a complete lack of neutralizing antistreptococcal mitogenic exotoxin ( sme ) z antibodies , while serum 96/210 converted to a high anti - smez antibody titer . both sera enhanced the mitogenic activity of recombinant streptococcal pyrogenic exotoxin j , which suggests the presence of an unknown synergistic factor . this selective increase suggests that the serum might contain a substance that selectively synergizes spe - j activity , which prevented any evaluation of the anti spe - j antibody titer in the two sequential serum samples . serum 96/2 - 10 ( 10 days after admission ) was analyzed for protective antibodies against smez-1 , -2 , -3 , -4 , -5 , -7 , -8 , -9 , -13 , -16 , -20 , -21 , and -22 and found to neutralize the activity of all tested smez variants ( data not shown ) . inhibition of approximately 95% was seen with all smez variants , except smez-2 ( 88% ) , smez-16 ( 78% ) , and smez-22 ( 85% ) , indicating that challenge with a single smez variant resulted in a cross - reactive antibody response against all smez variants . the smez alleles of both gas strains isolated from patients 96/2 and 99/1 were analyzed by dna sequencing and identified as smez-1 ( h297 ) and smez-16 ( h360 ) . spe - a was expressed in substantial amounts only from isolates from patients with stss ( isolates h325 , h330 , and h366 ) . isolates from patients without stss that carry the spea gene ( h308 , h314 , and h315 ) had undetectable levels of spe - a however , patients infected with the spe - a producing strains did not show any mitogenic activity in their acute - phase serum samples . in contrast , gas isolated from the patients 96/2 and 99/1 ( h297 and h360 ) produced only small amounts of smez in vitro despite the relatively large amounts detected in the acute - phase serum samples . nineteen acute - phase serum samples from patients with severe invasive streptococcal disease with stss ( n=9 ) and without stss ( n=10 ) were analyzed for mitogenic activity . serum samples 96/2 and 99/1 both induced a high proliferative response when tested in a pbl - stimulation assay , which suggests that they might contain a sag . peritonitis was diagnosed in patient 96/2 , who recovered after 2 weeks in the intensive - care unit ; tss was diagnosed in patient 99/1 , who died on the day of admission . to identify the sag responsible for the mitogenic activity , pbls were stimulated with serum 96/2 and serum 99/1 , respectively , together with rabbit antibodies against recombinant forms of spe - a , spe - c , spe - g , spe - j , or smez ( figure 1a ) . addition of anti - smez antibodies resulted in 59% and 68% inhibition of the mitogenic activity of serum samples 96/2 and 99/1 , respectively . antibodies against spe - j inhibited pbl stimulation of serum 96/2 by 51% but had no substantial effect on the serum 99/1 induced activity . spe - g antibodies did not substantially inhibit the activity in both serum samples , except for a slight inhibition ( 18% ) of serum 96/2 activity by anti inhibition of mitogenic activity in sera 96/2 and 99/1 with anti - superantigen ( sag ) antisera . a ) peripheral blood lymphocytes ( pbls ) were stimulated with 5% patient serum in the presence of 5% anti - sag antiserum or 5% fetal calf serum ( fcs ) only . the results were blotted as percentage of inhibition with specific anti - sag serum compared to fcs . antistreptococcal pyrogenic exotoxin ( spe)-j antiserum inhibited the mitogenic activity of serum 96/2 by 51% , while antistreptococcal mitogenic exotoxin ( sme ) z antiserum inhibited the activity in 96/2 by 59% and the serum in 99/1 by 68% . b ) the specificities of the anti spe - j and anti smez sera were demonstrated by stimulating pbls with recombinant sags in the presence of 5% antiserum . spe - j and anti - smez antiserum were added to pbls stimulated with various recombinant sags ( spe - a , spe - c , spe - g , spe - h , spe - i , spe - j , smez-1 , smez-2 , and ssa ) , and results showed that anti spe - j antibodies exclusively inhibited the rspe - j activity , whereas anti - smez antibodies inhibited the activity of rsmez-1 and rsmez-2 ( figure 1b ) . to quantify the levels of sag in 96/2 and 99/1 serum samples , a comparison was made against a standard pbl proliferation response for recombinant smez-1 and rspe - j . mitogenic activity of acute - phase serum samples 96/2 and 99/1 compared to recombinant superantigens ( sags ) . peripheral blood lymphocytes were stimulated for 4 d with various dilutions of recombinant sag or acute - phase serum sample 99/1 . five percent of each of the patient serum samples showed a proliferative response equal to 110 pg / ml of recombinant streptococcal pyrogenic exotoxin j or recombinant streptococcal mitogenic exotoxin 1 . sequential serum samples from stss patient 96/2 up to day 9 after admission to hospital were analyzed for clearance of mitogenic activity . at day 2 postadmission , an 80% reduction of activity had occurred , and by day 3 and day 9 after admission sag activity was undetectable . peripheral blood lymphocytes were stimulated with 5% of acute - phase and sequential serum samples from patient 96/2 . the mitogenic activity reached the highest point on day 1.5 after admission to hospital and dropped sharply on day 2 . no substantial activity was found in sequential serum samples from day 3 on ( samples 96/24 to 96/210 ) . the sequential serum samples from patient 96/2 allowed us to analyze for the development of neutralizing anti - sag antibodies by using patient serum to inhibit the activity of recombinant sags in a pbl - stimulation assay ( figure 4 ) . on day 3 , patient 96/2 had neutralizing antibodies against spe - a , spe - c , spe - i , and ssa but undetectable levels of protective antibodies against smez-1 , smez-2 , spe - g , and spe - h . by day 9 after admission ( sample 96/2 - 10 ) , the serum contained high titers of neutralizing antibodies against spe - a , spe - c , spe - i , ssa , smez-1 , and smez-2 , and a moderate anti spe - h titer . these results show seroconversion for smez-1 and smez-2 antibodies in the stss patient 96/2 , adding further evidence that smez was the predominant sag causing stss in this patient . peripheral blood lymphocytes were stimulated with various recombinant streptococcal sags in the presence of serum 96/24 , 96/210 , or fetal calf serum only . the sequential serum on day 3 showed a complete lack of neutralizing antistreptococcal mitogenic exotoxin ( sme ) z antibodies , while serum 96/210 converted to a high anti - smez antibody titer . both sera enhanced the mitogenic activity of recombinant streptococcal pyrogenic exotoxin j , which suggests the presence of an unknown synergistic factor . this selective increase suggests that the serum might contain a substance that selectively synergizes spe - j activity , which prevented any evaluation of the anti spe - j antibody titer in the two sequential serum samples . inhibition of approximately 95% was seen with all smez variants , except smez-2 ( 88% ) , smez-16 ( 78% ) , and smez-22 ( 85% ) , indicating that challenge with a single smez variant resulted in a cross - reactive antibody response against all smez variants . spe - a was expressed in substantial amounts only from isolates from patients with stss ( isolates h325 , h330 , and h366 ) . isolates from patients without stss that carry the spea gene ( h308 , h314 , and h315 ) had undetectable levels of spe - a . however , patients infected with the spe - a producing strains did not show any mitogenic activity in their acute - phase serum samples . in contrast , gas isolated from the patients 96/2 and 99/1 ( h297 and h360 ) produced only small amounts of smez in vitro despite the relatively large amounts detected in the acute - phase serum samples . evidence for sag involvement in these diseases derived from studies showing higher frequencies of spea and spec genes in severe disease isolates compared to nonsevere disease isolates ( 2,2225 ) , and reports of strong proliferative responses in the acute - phase serum samples from a patient with stss ( 28 ) and from mice infected with a sag - producing s. pyogenes strain ( 29 ) . in this study , we analyzed 19 acute - phase serum samples from patients with severe streptococcal disease with stss ( n=9 ) and without stss ( n=10 ) for mitogenic activity and detected smez in samples from 2 stss patients ( 96/2 and 99/1 ) . one of the serum samples ( 96/2 ) also contained detectable amounts of spe - j . the mitogenic activity in the two serum samples was equivalent to 110 pg / ml and 10 pg / ml of smez-1 or spe - j , which is sufficient to trigger maximal t - cell activation in pbl - stimulation assays . the sequential serum of the surviving patient ( 96/2 ) showed no protective anti - smez antibodies on day 3 after admission ( serum 96/24 ) but substantial levels at day 9 ( serum 96/210 ) , which suggests a direct role of this toxin in the stss of this patient . challenge with smez-1 ( the smez variant produced by gas isolate h297 from patient 96/2 ) resulted in a broad neutralization response against the complete range of smez variants . whether broad - spectrum anti - smez responses are a result of a few cross - reacting antibodies that were raised against a single smez variant has yet to be established , as does whether it results from infection by multiple gas strains carrying different smez alleles over a certain period . however , this response might not reflect a general response , and smez concentration in the blood might play a critical role . the only substantial difference was the frequency of the spec gene , found in 42% of new zealand isolates and 15.8% of london isolates . these results suggest that genotyping of sag genes alone is not predictive for type or severity of streptococcal disease . we found no substantial difference in sag production in cell cultures between stss and non - stss strains , with the exception of spe - a , which was only expressed in stss strains and suggests a tight association of spe - a in some cases of stss . these data are consistent with data from other groups that showed a link between spe - a expression and severe streptococcal disease ( 17,22,23 ) . smez-1 , for example , was undetectable in cultures of isolate h297 except by the sensitive jurkat proliferation assay , yet the patient with this gas isolate had high serum levels of smez-1 , indicating that smez production is tightly regulated . this observation is in keeping with recent findings using isogenic smez+ and smez- bacteria in a mouse model ( 29 ) . we attempted to correlate streptococcal disease and disease severity with the production of a particular sag . importantly , host factors may enhance the production of particular sags and , in addition , the immunogenic background of the host may contribute to the severity of sag - mediated invasive streptococcal disease , as recently reported ( 38 ) . although the overall potential role of sags in severe streptococcal disease remains elusive , our results directly implicate smez in the onset of stss in at least two patients . this presence of a particular bioreactive sag in the blood of patients with a reemerging potentially fatal disease is new and provides a platform for further investigation of the role of this potent sag in disease pathophysiology .

syncope is common in the general population and is perceived as an important clinical problem with adverse outcomes from associated physical trauma , negative impact on quality of life , and an increased cardiovascular risk in many patients . the first european society of cardiology ( esc ) guidelines for diagnosis and treatment of syncope were published in 2001 and were revised in 2004 and 2009 . the 2009 guidelines differ from earlier versions not only in their specific recommendations but also in their transatlantic endorsement ; the guidelines were developed in collaboration with the heart failure association ( hfa ) and heart rhythm society ( hrs ) and were endorsed by a large number of national societies on both sides of the atlantic . this range of specialties reflects the fact that syncope is a transient and often un - witnessed symptom with many possible underlying causes , leading to difficulties in standardizing diagnostic procedures . the updated versions of the guidelines highlight the use of implantable loop recorders ( ilrs ) and recommend their early use in the diagnostic work - up . in the majority of patients with a syncopal event , the gold standard of diagnosis remains the correlation of a spontaneous event with a specific ecg finding , but as the occurrence of syncope tends to be unpredictable , observers are unlikely to have the opportunity to record ecgs at the time of an event . the ability of ilrs to continuously record ecgs over long periods , with newer versions having longevity up to 3 years , makes them powerful diagnostic tools in patients with syncope secondary to an arrhythmia . the picture ( place of reveal in the care pathway and treatment of patients with unexplained recurrent syncope ) registry aimed to collect information on the use of the reveal ilr ( medtronic inc . ) in the patient care pathway and to investigate the effectiveness of reveal in the diagnosis of unexplained recurrent syncope and pre - syncope in everyday clinical practice . data were gathered after the publication of the 2004 esc guidelines and before the new 2009 version became available . the picture was a prospective , multicentre , observational post - marketing study conducted from november 2006 until october 2009 in 11 countries ( austria , the czech republic , denmark , finland , france , germany , israel , the netherlands , the slovak republic , sweden , and switzerland ) . all investigators were required to follow the policies and procedures set forth by their governing institutional review board ( irb)/ethics committee in compliance with local requirements . the main objectives of the picture study were to describe the standard care pathway in unexplained syncope , evaluate the burden of diagnostic tests , determine the diagnostic yield of the ilr , evaluate the time to diagnosis in relation to the timing of the ilr implant , determine the ratio between cardiac - related and non - cardiac - related diagnoses , determine the relationship between the number of preceding syncopal events and the time to diagnosis , and evaluate the relationship between diagnostic test results and ilr - documented diagnosis . patients were eligible if they had recurrent unexplained syncope or pre - syncope , estimated after the event and not separated in the analysis . unexplained was not defined in the protocol but the application of the term to a patient and the subsequent decision to implant an ilr and the programming of the device were left to the investigator 's discretion . investigators were to indicate if they implanted an ilr in the initial phase of the diagnostic process ( in the guidelines of 2004 consisting of a thorough medical history , physical examination , orthostatic blood pressure measurements , or echocardiogram ) or after a full diagnostic work - up ( as interpreted by the respective investigator ) . patients were followed up until the first recurrence of a syncopal event leading to a diagnosis or for at least 1 year . at the time of enrolment , the following was recorded : history of syncope in the previous 2 years , total number of syncopal events during life time of patient , clinical characteristics of syncope , number of syncopal episodes with severe trauma ( defined as fractures or injury with bleeding ) , clinical history , number of specialists seen in relation to syncope , specialty of physician referring for implant , number of admissions to er and/or hospitalizations , diagnostic tests performed before implant of an ilr , and suspected diagnosis guided by the diagnostic tests before implant . follow - up was per normal clinical practice and subjects were advised to contact the treating physician promptly in the case of a suspected event . at follow - up visits , clinical characteristics of syncope , occurrence of severe trauma , admissions to er and/or hospitalizations , the role of the ilr in establishing a diagnosis , ilr - guided diagnosis , other tests undertaken , and treatment administered for syncope were recorded . all enrolled subjects received a reveal plus , dx or xt ilr . for a patient to be included in the final analysis , an implant / discharge visit was required together with a recurrence visit or a 1-year follow - up visit . for patients with more than one follow - up visit , the first visit after an event was included in the analysis . if patients had no event , the last visit that took place during the pre - specified follow - up period was included in the analysis . the mean and standard deviation , or median and interquartile ( iq ) range were calculated as appropriate . for qualitative variables , time - to - event outcomes were described using the kaplan meier curves , with day of implant as time zero . the diagnostic yield was defined as reveal - guided diagnosis : a syncopal episode for which reveal played a major role in determining whether the cause of syncope was either cardiac or non - cardiac . further , investigators could specify the role of reveal in a diagnosis as primary , confirmatory , or additional to other confirmatory tests . all enrolled subjects received a reveal plus , dx or xt ilr . for a patient to be included in the final analysis , an implant / discharge visit was required together with a recurrence visit or a 1-year follow - up visit . for patients with more than one follow - up visit , the first visit after an event was included in the analysis . if patients had no event , the last visit that took place during the pre - specified follow - up period was included in the analysis . descriptive statistics were used for the analyses . for quantitative variables such as age , the mean and standard deviation , or median and interquartile ( iq ) range time - to - event outcomes were described using the kaplan meier curves , with day of implant as time zero . the diagnostic yield was defined as reveal - guided diagnosis : a syncopal episode for which reveal played a major role in determining whether the cause of syncope was either cardiac or non - cardiac . further , investigators could specify the role of reveal in a diagnosis as primary , confirmatory , or additional to other confirmatory tests . of a total 650 enrolled subjects , follow - up visit data ( with or without events ) were available for 570 subjects who were included in the analysed population . fifty - three patients had to wait a median of 3 days ( iq range 114 ) from enrolment to implant . the follow - up dates were missing for three patients and implant date for one patient . these patients were included in the follow - up analysis but excluded from the survival analysis . seven per cent of patients were enrolled based on unexplained pre - syncope ; 91% had unexplained syncope and the classification was missing for 2% of patients . patients with syncope had experienced a median of 4 reported events prior to enrolment , of which three were within the 2 years prior to enrolment . reveal versions dx / xt were used in 316 patients ( 55% ) ; the remaining 45% received reveal plus . baseline characteristics for the 80 patients without complete follow - up did not differ from those in the overall population . table 1general characteristics of patients at study enrolmenttotal recruitment570 ( 100%)women306 ( 54%)age sd61 17primary indication unexplained syncope517 ( 91% ) unexplained pre - syncope42 ( 7% ) other11 ( 2%)history hypertension277 ( 49% ) coronary artery disease84 ( 15% ) valvular heart disease30 ( 5% ) cardiomyopathy18 ( 3% ) stroke / tia57 ( 10%)diagnostic work - up before ilr implant in an initial phase of diagnostic work - up of syncope128 ( 22% ) after full evaluation of mechanism of syncope386 ( 68%)device implanted reveal dx264 ( 46% ) reveal xt52 ( 9% ) reveal plus254 ( 45%)age at first syncope55 20previous syncopes median ( iq range)4 ( 26)syncopes in the last 2 years median ( iq range)3 ( 24)syncopal episodes per year median ( iq range)2 ( 13.5)median interval between first and last episode years ( iq range)2 ( 04)any previous hospitalization because of syncope399 ( 70%)any syncopal episodes without prodromes339 ( 59%)any syncopal episodes with severe trauma204 ( 36%)any syncopal episodes suggestive of vasovagal origin86 ( 15%)any situational syncope39 ( 7%)characteristics of last syncope after effort28 ( 5% ) during effort144 ( 25% ) at rest294 ( 52% ) unknown97 ( 17% ) missing7 ( 1%)symptoms muscle spasms ( one sided)8 ( 1% ) muscle spasms ( two sided)19 ( 3% ) grand mal10 ( 2% ) other muscle spasms14 ( 2% ) transpiration73 ( 13% ) cyanosis19 ( 3% ) angina pectoris23 ( 4% ) palpitations76 ( 13% ) dizziness163 ( 29% ) dyspnoea33 ( 6% ) fatigue95 ( 17% ) other80 ( 14% ) none266 ( 47% ) general characteristics of patients at study enrolment the first specialist consulted for syncope in almost 23% of patients was an emergency medicine consultant . cardiologists were the first specialists consulted in 43% of cases and neurologists in 11% ( figure 1 ) . the last specialist consulted before the referral for implant of the ilr was a cardiologist in 72% of cases , with no other specialties represented in more than 10% of cases ( figure 1 ) . cardiologists were the most frequently consulted specialists , with general practitioners second - most consulted physicians overall . forty - seven per cent of the study population had consulted a neurologist at some point . overall , patients had seen an average of three different specialists for management of their syncope . most patients ( 70% ) had been hospitalized at least once for syncope and one - third ( 36% ) had experienced significant trauma in association with a syncopal episode . blue , red , and green bars indicate specialists seen at the hospital for the latest syncope episode ; shaded bars last specialist consulted before ilr implant ; open bars all specialists reported by patients as being consulted in relation to syncope in the past . blue , red , and green bars indicate specialists seen at the hospital for the latest syncope episode ; shaded bars last specialist consulted before ilr implant ; open bars all specialists reported by patients as being consulted in relation to syncope in the past . the median number of tests performed per patient in the total study population was 13 ( iq range 920 ; table 2 ) . the tests performed most frequently were echocardiography , ecg , ambulatory ecg monitoring , in - hospital ecg monitoring , exercise testing , and orthostatic blood pressure measurements . about half the patient population had undergone an mri / ct scan ( 47% ) , neurological or psychiatric evaluation ( 47% ) , or electroencephalography ( eeg ; 39% ) . in contrast , carotid sinus massage or tilt tests were only undertaken in one - third of subjects . the ilr was implanted during the initial phase of the diagnostic work - up ( up to four diagnostic tests ) in 128 patients ( 22% ) . table 2history of diagnostic tests performed before ilr implanttotal recruitment570 ( 100%)standard ecg556 ( 98%)echocardiography490 ( 86%)basic laboratory tests488 ( 86%)ambulatory ecg monitoring382 ( 67%)in - hospital ecg monitoring311 ( 55%)exercise testing297 ( 52%)orthostatic blood pressure measurements275 ( 48%)mri / ct scan267 ( 47%)neurological or psychiatric evaluation270 ( 47%)eeg222 ( 39%)carotid sinus massage205 ( 36%)tilt test201 ( 35%)electrophysiology testing144 ( 25%)coronary angiography133 ( 23%)external loop recording67 ( 12%)atp test15 ( 3%)other tests52 ( 9%)no tests performed1 ( 0% ) history of diagnostic tests performed before ilr implant during follow - up , a total of 218 patients ( 38% of the population ) experienced an episode of syncope , 149 ( 26% of patients or 68% of episodes ) with prodromal symptoms . the percentages of patients who had a recurrence of syncope were 19 , 26 , and 36% after 3 , 6 , and 12 months , respectively ( figure 2 ) . ten patients with an episode during follow - up ( 5.2% of the population with an event ) had associated severe trauma . figure 2the kaplan meier estimates of time to syncopal episode ( green line ) and time to syncopal episode where reveal played a role in the diagnosis ( red line ) . the kaplan meier estimates of time to syncopal episode ( green line ) and time to syncopal episode where reveal played a role in the diagnosis ( red line ) . time to a syncopal event where reveal played a role in the diagnosis showed the recurrence of diagnosis within 180 days as 20% , within 365 days as 30% , and within 462 days as 38% ( figure 2 ) . the estimated rate of syncope after 30 days of follow - up was 10% and the estimated rate of diagnosis where reveal played a role at this time of follow - up was 9% . of the 218 events , 23 diagnoses were reported as not guided by reveal data and for 12 patients , data were inconclusive . the diagnosis was reported as guided by reveal in 78% of cases or 170 patients ( figure 3 ) ; the role in 13 diagnoses was inconclusive . figure 3patient flow chart and diagnostic yield of the ilr in picture . patient flow chart and diagnostic yield of the ilr in picture . of the 170 reveal - guided diagnoses , 75% , or 128 cases , were cardiac . reveal - guided diagnosis led to pacemaker implants in 86 patients , 51% of diagnosed cases . antiarrhythmic drug therapy ( 7% ) , implantable cardioverter defibrillators ( 6% ) and ablation ( 5% ) were also utilized as treatment . therapies were similar in the total population with an event ( n = 218 ) but more patients in this population remained untreated upon diagnosis . in the 48 patients with an episode but without reveal - guided diagnosis , 12% received dual - chamber pacemakers , 4% anti - arrhythmic drug therapy , there were no icd implants or catheter ablations in the 48 patients with an episode but without reveal - guided diagnosis . there were no icd implants or catheter ablations in the 48 patients with an episode but without reveal - guided diagnosis . of a total 650 enrolled subjects , follow - up visit data ( with or without events ) were available for 570 subjects who were included in the analysed population . fifty - three patients had to wait a median of 3 days ( iq range 114 ) from enrolment to implant . the follow - up dates were missing for three patients and implant date for one patient . these patients were included in the follow - up analysis but excluded from the survival analysis . seven per cent of patients were enrolled based on unexplained pre - syncope ; 91% had unexplained syncope and the classification was missing for 2% of patients . patients with syncope had experienced a median of 4 reported events prior to enrolment , of which three were within the 2 years prior to enrolment . reveal versions dx / xt were used in 316 patients ( 55% ) ; the remaining 45% received reveal plus . baseline characteristics for the 80 patients without complete follow - up did not differ from those in the overall population . table 1general characteristics of patients at study enrolmenttotal recruitment570 ( 100%)women306 ( 54%)age sd61 17primary indication unexplained syncope517 ( 91% ) unexplained pre - syncope42 ( 7% ) other11 ( 2%)history hypertension277 ( 49% ) coronary artery disease84 ( 15% ) valvular heart disease30 ( 5% ) cardiomyopathy18 ( 3% ) stroke / tia57 ( 10%)diagnostic work - up before ilr implant in an initial phase of diagnostic work - up of syncope128 ( 22% ) after full evaluation of mechanism of syncope386 ( 68%)device implanted reveal dx264 ( 46% ) reveal xt52 ( 9% ) reveal plus254 ( 45%)age at first syncope55 20previous syncopes median ( iq range)4 ( 26)syncopes in the last 2 years median ( iq range)3 ( 24)syncopal episodes per year median ( iq range)2 ( 13.5)median interval between first and last episode years ( iq range)2 ( 04)any previous hospitalization because of syncope399 ( 70%)any syncopal episodes without prodromes339 ( 59%)any syncopal episodes with severe trauma204 ( 36%)any syncopal episodes suggestive of vasovagal origin86 ( 15%)any situational syncope39 ( 7%)characteristics of last syncope after effort28 ( 5% ) during effort144 ( 25% ) at rest294 ( 52% ) unknown97 ( 17% ) missing7 ( 1%)symptoms muscle spasms ( one sided)8 ( 1% ) muscle spasms ( two sided)19 ( 3% ) grand mal10 ( 2% ) other muscle spasms14 ( 2% ) transpiration73 ( 13% ) cyanosis19 ( 3% ) angina pectoris23 ( 4% ) palpitations76 ( 13% ) dizziness163 ( 29% ) dyspnoea33 ( 6% ) fatigue95 ( 17% ) other80 ( 14% ) none266 ( 47% ) general characteristics of patients at study enrolment the first specialist consulted for syncope in almost 23% of patients was an emergency medicine consultant . cardiologists were the first specialists consulted in 43% of cases and neurologists in 11% ( figure 1 ) . the last specialist consulted before the referral for implant of the ilr was a cardiologist in 72% of cases , with no other specialties represented in more than 10% of cases ( figure 1 ) . cardiologists were the most frequently consulted specialists , with general practitioners second - most consulted physicians overall . forty - seven per cent of the study population had consulted a neurologist at some point . overall , patients had seen an average of three different specialists for management of their syncope . most patients ( 70% ) had been hospitalized at least once for syncope and one - third ( 36% ) had experienced significant trauma in association with a syncopal episode . blue , red , and green bars indicate specialists seen at the hospital for the latest syncope episode ; shaded bars last specialist consulted before ilr implant ; open bars all specialists reported by patients as being consulted in relation to syncope in the past . blue , red , and green bars indicate specialists seen at the hospital for the latest syncope episode ; shaded bars last specialist consulted before ilr implant ; open bars all specialists reported by patients as being consulted in relation to syncope in the past . the median number of tests performed per patient in the total study population was 13 ( iq range 920 ; table 2 ) . the tests performed most frequently were echocardiography , ecg , ambulatory ecg monitoring , in - hospital ecg monitoring , exercise testing , and orthostatic blood pressure measurements . about half the patient population had undergone an mri / ct scan ( 47% ) , neurological or psychiatric evaluation ( 47% ) , or electroencephalography ( eeg ; 39% ) . in contrast , carotid sinus massage or tilt tests were only undertaken in one - third of subjects . the ilr was implanted during the initial phase of the diagnostic work - up ( up to four diagnostic tests ) in 128 patients ( 22% ) . table 2history of diagnostic tests performed before ilr implanttotal recruitment570 ( 100%)standard ecg556 ( 98%)echocardiography490 ( 86%)basic laboratory tests488 ( 86%)ambulatory ecg monitoring382 ( 67%)in - hospital ecg monitoring311 ( 55%)exercise testing297 ( 52%)orthostatic blood pressure measurements275 ( 48%)mri / ct scan267 ( 47%)neurological or psychiatric evaluation270 ( 47%)eeg222 ( 39%)carotid sinus massage205 ( 36%)tilt test201 ( 35%)electrophysiology testing144 ( 25%)coronary angiography133 ( 23%)external loop recording67 ( 12%)atp test15 ( 3%)other tests52 ( 9%)no tests performed1 ( 0% ) history of diagnostic tests performed before ilr implant during follow - up , a total of 218 patients ( 38% of the population ) experienced an episode of syncope , 149 ( 26% of patients or 68% of episodes ) with prodromal symptoms . the percentages of patients who had a recurrence of syncope were 19 , 26 , and 36% after 3 , 6 , and 12 months , respectively ( figure 2 ) . ten patients with an episode during follow - up ( 5.2% of the population with an event ) had associated severe trauma . figure 2the kaplan meier estimates of time to syncopal episode ( green line ) and time to syncopal episode where reveal played a role in the diagnosis ( red line ) . the kaplan meier estimates of time to syncopal episode ( green line ) and time to syncopal episode where reveal played a role in the diagnosis ( red line ) . time to a syncopal event where reveal played a role in the diagnosis showed the recurrence of diagnosis within 180 days as 20% , within 365 days as 30% , and within 462 days as 38% ( figure 2 ) . the estimated rate of syncope after 30 days of follow - up was 10% and the estimated rate of diagnosis where reveal played a role at this time of follow - up was 9% . of the 218 events , 23 diagnoses were reported as not guided by reveal data and for 12 patients , data were inconclusive . figure 3patient flow chart and diagnostic yield of the ilr in picture . patient flow chart and diagnostic yield of the ilr in picture . of the 170 reveal - guided diagnoses , 75% , or 128 cases , were cardiac . reveal - guided diagnosis led to pacemaker implants in 86 patients , 51% of diagnosed cases . antiarrhythmic drug therapy ( 7% ) , implantable cardioverter defibrillators ( 6% ) and ablation ( 5% ) were also utilized as treatment . therapies were similar in the total population with an event ( n = 218 ) but more patients in this population remained untreated upon diagnosis . in the 48 patients with an episode but without reveal - guided diagnosis , 12% received dual - chamber pacemakers , 4% anti - arrhythmic drug therapy , there were no icd implants or catheter ablations in the 48 patients with an episode but without reveal - guided diagnosis . there were no icd implants or catheter ablations in the 48 patients with an episode but without reveal - guided diagnosis . the results of the picture study provide new and important insights into the discrepancy between real - life care and the recommendations in the esc guidelines at the time . the results underline that more efforts are needed if better adherence to guidelines are to be obtained . two important findings were the large number of diagnostic tests that patients underwent before an ilr implant and the high diagnostic yield from the use of an ilr in the overall population with unexplained syncope . together , these findings imply that if an ilr is implanted early , as emphasized in the 2009 esc guidelines , a reduced number of tests might be needed . the picture is the largest observational study to date to evaluate the usage and diagnostic effectiveness of ilrs in the everyday clinical diagnostic work - up of patients with unexplained syncope . the registry included european countries and israel , but the findings may have wider relevance . as in europe , implementation of guidelines in the usa has been patchy with several different recommendations published . the 2009 guidelines are based on transatlantic consensus and might be better prepared to improve adherence but at present , there is an important gap between what guidelines prescribe and how the real - life care of patients with unexplained syncope is carried out . the population in picture was slightly younger and healthier than those in other syncope studies such as easyas , egsys 2 , and issue 2 . the number of syncopal events before patients were considered for ilr implantation was close to those reported from easyas and egsys 2 . in comparison with these and other reports , the incidence of severe trauma was high in picture , particularly considering that the first syncope occurred only a few years before the implant . however , the study protocol did not include a specific term for mild traumas and the rates may be increased by mild injuries being reported as severe . the picture study did not ascertain how familiar investigators were with the 2004 guidelines which recommended ilrs only as a last resort when other tests have failed to reach a diagnosis . nevertheless , a median of 13 tests per patient before considering an implant seems unnecessarily high . the large number of investigations may well have been due to the number of different specialists that patients met . the frequent use of eeg has been observed in other evaluations of diagnostic pathways for patients with syncope . in picture , the eeg rates are most likely explained by the high percentage of referrals to neurologists . the clinical picture of syncope frequently includes neurological symptoms and is often mistaken for epilepsy , which probably explains why neurologists are frequently consulted . conversely , epilepsy may be mistaken as syncope , but in such patients , an ilr may be a faster way to arrive at the correct diagnosis , if an ilr recording can be obtained during a typical episode . furthermore , almost half the patient population had undergone an mri / ct scan , neurological evaluation , or psychiatric evaluation . in the egsys 1 population , reflecting the clinical context in 2001 , the rates of mri and/or ct scan were 20% . there were , however , simple diagnostic tests that could have been used more widely and would have been included in the initial phase of evaluation , e.g. tilt test , carotid sinus massage , and orthostatic blood pressure measurements . carotid sinus massage or tilt tests were performed on about one - third of the picture subjects and since multiple tests were allowed , it is likely that more than two - thirds of the population did not undergo these tests . in theory , the differentiation between various forms of loss of consciousness and seizures may appear reasonably easy , but in the real world , the diagnosis most often has to be based on the retrospective observations of bystanders and little information from the patient , who has frequently no memory of the event . thus , the patient or relatives do not know which specialist to see first , and the first physician may not be the most appropriate to reach a diagnosis . however , in picture , the final diagnosis was most often a cardiac event and almost always an arrhythmic event . also , cardiologists were often seen , both as the first and the last specialist before implant . these observations must lead to the question how to make the first contact with a cardiologist more useful , since the final diagnosis is most often to be found in his / her area of competence . still , syncope is a very heterogeneous condition , and although the majority of events can be effectively handled , some diagnoses will be difficult . in those cases , a multidisciplinary approach is of great importance and access to physicians in other specialties needs to be considered . the picture found a high diagnostic yield with ilrs , which guided the diagnosis in 78% of patients with recurrent syncope and provided useful information in another 6% . observational studies reflect clinical practice and without end point adjudication , site monitoring , etc . on the other hand , it can be argued that clinically relevant diagnostic yield measurements have to be obtained from studies in clinical practice with all that implies as to compliance , methodological differences between practices , etc . diagnostic rates above 50% with the use of an ilr have been reported in other , smaller studies with one early study claiming > 90% . one report by farwell and sulke noted markedly enhanced diagnosis rates with an increased use of automatic recording of events . half the population in picture received the newer reveal device models in which the automatic recording mode is always active . in the other half of the patients , when automatic detection was not the default and could be switched off at the discretion of the investigator , we can not exclude that the yield would have been improved if the automatic detection had always been enabled . there were 25 symptomatic recurrences without an ilr recording , implying that the rhythm during the event was sinus rhythm or , possibly , that automatic detection was switched off and the patient forgot or was unable to activate the device . in the reveal plus device , used in 254 subjects , automatic capture can be switched off , an option not available in the newer devices . an obvious action would be to advise clinicians that automatic detection should always be activated . subjects were advised to contact the treating physician promptly in the case of a suspected event ; hence , it is unlikely that any symptomatic event went unrecorded from loss of memory . ideally , a device - based diagnosis should lead to a specific treatment , and this was very often the case in picture . a cardiac cause of syncope was found in 59% of the patients with recurrent syncope , leading to symptomatic treatment with pacemakers ( at rates corresponding to reported rates of bradycardia in syncope patients ) , ablation and medication as well as life - saving treatment with implantable defibrillators . in less than 20% of patients with a reveal - guided diagnosis was there a decision to undertake no specific treatment . in this group of patients , the benefit of knowing the cause of syncope would have reduced the need for further diagnostic tests . this finding implies that many tests undertaken prior to implant were unnecessary in that they had a low probability of reaching a diagnosis . the main advantage of ilrs is to provide continuous monitoring , increasing the chances of obtaining such findings . meier estimates showed that most picture patients had their first syncopal recurrence after more than 30 days from implant ( figure 2 ) . recurrence rates during the longer follow - up period of 10 6 months were 38% . this is within the range of what has been reported in other studies with similar study populations . farwell et al . found a 1-year recurrence rate of 66% , whereas solano et al . reported 21% yearly recurrence rates . differences between the groups of patients are probably responsible for these discrepancies . in picture , the 50% of patients who received a reveal plus device with a battery life of around 14 months may not have had sufficient time to experience a syncopal event . the reveal dx / xt models that became available during the course of the conduct of picture have a battery life of 3 years and given the recurrence rates increasing with time in patients with follow - up visit data beyond 12 months ( figure 2 ) , it seems reasonable that within this time span the majority of the study population would experience an event . however , such data complement those of randomized , controlled studies , and may better describe the real - world situation . the fact that 12% of implanted patients did not have follow - up visit data is a limitation . follow - up rates may well be improved in the future by newer technologies for remote monitoring and automatic data delivery . patients with pre - syncope only were admitted into the registry , and they have been analysed and reported together with patients with syncope , since the subgroup was small . however , such data complement those of randomized , controlled studies , and may better describe the real - world situation . the fact that 12% of implanted patients did not have follow - up visit data is a limitation . follow - up rates may well be improved in the future by newer technologies for remote monitoring and automatic data delivery . patients with pre - syncope only were admitted into the registry , and they have been analysed and reported together with patients with syncope , since the subgroup was small . in patients with unexplained syncope and a recurrence during follow - up , the ilr revealed or contributed to establishing the mechanism of syncope in the vast majority . the picture study found a great diversity and number of physicians consulted , plus a large number of tests performed . the proportion of patients with an ilr - guided diagnosis increased over time and was still growing at the end of follow - up , implying that more patients would get a diagnosis before the end of battery life . the findings support the recommendation in current guidelines that an ilr should be implanted early rather than late in the evaluation of unexplained syncope . the best way to disseminate this information to physicians and increase the adherence to guidelines remains a challenge . nils edvardsson , md , phd , rob van mechelen , md , jean - luc pasqui , md , phd , rodolfo ventura , md , and nicholas j. linker , md . m. ait said , clinique ambroise par , neuilly sur seine , france ; p. ammann , kantonsspital st gallen , st gallen , switzerland ; t. aronsson , vxj medicinkliniken centralsjukhuset , vxj , sweden ; a. bauer , universittsklinikum heidelberg , heidelberg , germany ; w. benzer , landeskrankenhaus feldkirch , feldkirch , austria ; v. bernt , nsch , bratislava , slovak republic ; d. bcker , st marien - hospital hamm , hamm , germany ; a. brandes , odense university hospital , odense , denmark ; p. breuls , merwede ziekenhuis dordrecht , dordrecht , the netherlands ; s. buffler , centre hospitalier hagueneau , hagueneau , france ; h. ebert , kardiologische gemeinschaftspraxis , riesa , germany ; a. ebrahimi , mlndal / kunglv , mlndal , sweden ; o. eschen , aalborg hospital , aalborg , denmark ; t. fhraeus , universitetssjukhuset lund , lund , sweden ; g. falck , bollnssjukhus , bollns , sweden ; w. fehske , st vinzenz - hospital , kln , germany ; r. frank , aphp hospital pitie salpetriere , paris , france ; v. frykman , danderyds sjukhus , stockholm , sweden ; f. gadler , karolinska sjukhuset solna & huddinge , stockholm , sweden ; g. gehling , katholische krankenhaus hagen gem . gmbh , st - johannes - hospital , hagen , germany ; m. geist , edith wolfson hospital , holon , israel ; j. gnther , kardiologisches centrum and der klinik rotes kreuz am zoo , frankfurt , germany ; m. gutmann , kantonsspital liestal , liestal , switzerland ; h. hartog , diakonessenhuis utrecht , utrecht , netherlands ; h. hartog , diakonessenhuis zeist , zeist , the netherlands ; s. jensen , norrlands universitetssjukhus i ume , ume , sweden ; w. kainz , hanusch krankenhaus , vienna , austria ; j. kautzner , institut klinick a experimentln medicny , prague , czech republic ; w. kiowski , klinik i m park ag , zrich , switzerland ; b. kjellman , sdersjukhuset , stockholm , sweden ; h. klomps , st jans ziekenhuis , weert , the netherlands ; h. krappinger , landeskrankenhaus villach , villach , austria ; p. lercher , landeskrankenhaus graz - medizinische universittsklinik , graz , austria ; j. lindstrm , centralsjukhuset i karlstad , karlstad , sweden ; m. lukat , kliniken essen nordwest , philippusstift krankenhaus , essen , germany ; z. machov , vsch , koice , slovak republic ; c. magnusson , mlarsjukhuset , eskilstuna , sweden ; f. maru , rnskldsvikssjukhus , rnskldsvik , sweden ; j. melichercik , herzzentrum lahr / baden , lahr , germany ; a. militianu , carmel medical center , haifa , israel ; t. minak , fakultn nemocnice , ostrava , ostrava , czech republic ; p. mitro , faculty hospital l. pasteura , koice , slovak republic ; a. mohii - oskarsson , st grans sjukhus , stockholm , sweden ; h. mlgaard , skejby hospital , aarhus , denmark ; c. nimeth , krankenhaus der barmherzigen schwestern ried , ried i m innkreis , austria ; t. nordt , klinikum stuttgart katharinenhospital , stuttgart , germany ; m. novk , fakultn nemocnice u sv . anny v brn , brno , czech republic ; k. nyman , keski - suomen keskussairaala , jyvskyl , finland ; j .- pasqui , chu arnaud de villeneuve , montpellier , france ; j. plomp , tergooi ziekenhuizen lok . hilversum , hilversum , the netherlands ; a. podczeck - schweighofer , sozialmedizinisches zentrum sd kaiser - franz - josef - spital , vienna , austria ; h. ramanna , medisch centrum haaglanden , den haag , netherlands ; j.m . rigollaud , hpital d'instruction des armees robert picque , bordeaux - armees , france ; c. rorsman , sjukhuset varberg , varberg , sweden ; a. rtzer , klinikum ludwigsburg , ludwigsburg , germany ; t. salo , tyks raision sairaala , raision , finland ; n. samnieh , bnai zion medical center , haifa , israel ; f. schwertfeger , spreewaldklinik lbben , lbben , germany ; g. strupp , klinikum fulda , fulda , germany ; h. sunthorn , hpital cantonal universitaire , genve , switzerland ; s. trinks , krankenhaus hietzing , vienna , austria ; i.c . van gelder , university medical center groningen , groningen , the netherlands ; r. van mechelen , st franciscus hospital rotterdam , rotterdam , the netherlands ; r. ventura , uke hamburg , hamburg , germany ; e. vester , evangelisches krankenhaus dsseldorf , dsseldorf , germany ; s. viskin , tel aviv medical center , tel aviv , israel ; p. visman , beatrix ziekenhuis , gorinchem , the netherlands ; j. vlanov , fakultn nemocnice brno , brno , czech republic ; i. westbom , sahlgrenska . winkler , krankenanstalt der stadt wien rudolfstiftung , vienna , austria ; j. woltmann , st vincenz - krankenhaus , menden , germany .

aimsto collect information on the use of the reveal implantable loop recorder ( ilr ) in the patient care pathway and to investigate its effectiveness in the diagnosis of unexplained recurrent syncope in everyday clinical practice.methods and resultsprospective , multicentre , observational study conducted in 20062009 in 10 european countries and israel . eligible patients had recurrent unexplained syncope or pre - syncope . subjects received a reveal plus , dx or xt . follow up was until the first recurrence of a syncopal event leading to a diagnosis or for 1 year . in the course of the study , patients were evaluated by an average of three different specialists for management of their syncope and underwent a median of 13 tests ( range 920 ) . significant physical trauma had been experienced in association with a syncopal episode by 36% of patients . average follow - up time after ilr implant was 10 6 months . follow - up visit data were available for 570 subjects . the percentages of patients with recurrence of syncope were 19 , 26 , and 36% after 3 , 6 , and 12 months , respectively . of 218 events within the study , ilr - guided diagnosis was obtained in 170 cases ( 78% ) , of which 128 ( 75% ) were cardiac.conclusiona large number of diagnostic tests were undertaken in patients with unexplained syncope without providing conclusive data . in contrast , the ilr revealed or contributed to establishing the mechanism of syncope in the vast majority of patients . the findings support the recommendation in current guidelines that an ilr should be implanted early rather than late in the evaluation of unexplained syncope .

Introduction Methods Statistical analysis Results Patients and follow-up Patient characteristics at baseline Physicians consulted and diagnostic tests performed before ILR implant Diagnostic yield Specific treatment based on diagnosis made by ILR Discussion Limitations Conclusions Funding Steering committee PICTURE participating investigators

the updated versions of the guidelines highlight the use of implantable loop recorders ( ilrs ) and recommend their early use in the diagnostic work - up . in the majority of patients with a syncopal event , the gold standard of diagnosis remains the correlation of a spontaneous event with a specific ecg finding , but as the occurrence of syncope tends to be unpredictable , observers are unlikely to have the opportunity to record ecgs at the time of an event . the picture ( place of reveal in the care pathway and treatment of patients with unexplained recurrent syncope ) registry aimed to collect information on the use of the reveal ilr ( medtronic inc . ) in the patient care pathway and to investigate the effectiveness of reveal in the diagnosis of unexplained recurrent syncope and pre - syncope in everyday clinical practice . the picture was a prospective , multicentre , observational post - marketing study conducted from november 2006 until october 2009 in 11 countries ( austria , the czech republic , denmark , finland , france , germany , israel , the netherlands , the slovak republic , sweden , and switzerland ) . the main objectives of the picture study were to describe the standard care pathway in unexplained syncope , evaluate the burden of diagnostic tests , determine the diagnostic yield of the ilr , evaluate the time to diagnosis in relation to the timing of the ilr implant , determine the ratio between cardiac - related and non - cardiac - related diagnoses , determine the relationship between the number of preceding syncopal events and the time to diagnosis , and evaluate the relationship between diagnostic test results and ilr - documented diagnosis . patients were eligible if they had recurrent unexplained syncope or pre - syncope , estimated after the event and not separated in the analysis . unexplained was not defined in the protocol but the application of the term to a patient and the subsequent decision to implant an ilr and the programming of the device were left to the investigator 's discretion . investigators were to indicate if they implanted an ilr in the initial phase of the diagnostic process ( in the guidelines of 2004 consisting of a thorough medical history , physical examination , orthostatic blood pressure measurements , or echocardiogram ) or after a full diagnostic work - up ( as interpreted by the respective investigator ) . patients were followed up until the first recurrence of a syncopal event leading to a diagnosis or for at least 1 year . at the time of enrolment , the following was recorded : history of syncope in the previous 2 years , total number of syncopal events during life time of patient , clinical characteristics of syncope , number of syncopal episodes with severe trauma ( defined as fractures or injury with bleeding ) , clinical history , number of specialists seen in relation to syncope , specialty of physician referring for implant , number of admissions to er and/or hospitalizations , diagnostic tests performed before implant of an ilr , and suspected diagnosis guided by the diagnostic tests before implant . follow - up was per normal clinical practice and subjects were advised to contact the treating physician promptly in the case of a suspected event . at follow - up visits , clinical characteristics of syncope , occurrence of severe trauma , admissions to er and/or hospitalizations , the role of the ilr in establishing a diagnosis , ilr - guided diagnosis , other tests undertaken , and treatment administered for syncope were recorded . all enrolled subjects received a reveal plus , dx or xt ilr . for a patient to be included in the final analysis , an implant / discharge visit was required together with a recurrence visit or a 1-year follow - up visit . for patients with more than one follow - up visit , the first visit after an event was included in the analysis . if patients had no event , the last visit that took place during the pre - specified follow - up period was included in the analysis . the diagnostic yield was defined as reveal - guided diagnosis : a syncopal episode for which reveal played a major role in determining whether the cause of syncope was either cardiac or non - cardiac . all enrolled subjects received a reveal plus , dx or xt ilr . for a patient to be included in the final analysis , an implant / discharge visit was required together with a recurrence visit or a 1-year follow - up visit . for patients with more than one follow - up visit , the first visit after an event was included in the analysis . if patients had no event , the last visit that took place during the pre - specified follow - up period was included in the analysis . the diagnostic yield was defined as reveal - guided diagnosis : a syncopal episode for which reveal played a major role in determining whether the cause of syncope was either cardiac or non - cardiac . of a total 650 enrolled subjects , follow - up visit data ( with or without events ) were available for 570 subjects who were included in the analysed population . these patients were included in the follow - up analysis but excluded from the survival analysis . seven per cent of patients were enrolled based on unexplained pre - syncope ; 91% had unexplained syncope and the classification was missing for 2% of patients . patients with syncope had experienced a median of 4 reported events prior to enrolment , of which three were within the 2 years prior to enrolment . table 1general characteristics of patients at study enrolmenttotal recruitment570 ( 100%)women306 ( 54%)age sd61 17primary indication unexplained syncope517 ( 91% ) unexplained pre - syncope42 ( 7% ) other11 ( 2%)history hypertension277 ( 49% ) coronary artery disease84 ( 15% ) valvular heart disease30 ( 5% ) cardiomyopathy18 ( 3% ) stroke / tia57 ( 10%)diagnostic work - up before ilr implant in an initial phase of diagnostic work - up of syncope128 ( 22% ) after full evaluation of mechanism of syncope386 ( 68%)device implanted reveal dx264 ( 46% ) reveal xt52 ( 9% ) reveal plus254 ( 45%)age at first syncope55 20previous syncopes median ( iq range)4 ( 26)syncopes in the last 2 years median ( iq range)3 ( 24)syncopal episodes per year median ( iq range)2 ( 13.5)median interval between first and last episode years ( iq range)2 ( 04)any previous hospitalization because of syncope399 ( 70%)any syncopal episodes without prodromes339 ( 59%)any syncopal episodes with severe trauma204 ( 36%)any syncopal episodes suggestive of vasovagal origin86 ( 15%)any situational syncope39 ( 7%)characteristics of last syncope after effort28 ( 5% ) during effort144 ( 25% ) at rest294 ( 52% ) unknown97 ( 17% ) missing7 ( 1%)symptoms muscle spasms ( one sided)8 ( 1% ) muscle spasms ( two sided)19 ( 3% ) grand mal10 ( 2% ) other muscle spasms14 ( 2% ) transpiration73 ( 13% ) cyanosis19 ( 3% ) angina pectoris23 ( 4% ) palpitations76 ( 13% ) dizziness163 ( 29% ) dyspnoea33 ( 6% ) fatigue95 ( 17% ) other80 ( 14% ) none266 ( 47% ) general characteristics of patients at study enrolment the first specialist consulted for syncope in almost 23% of patients was an emergency medicine consultant . overall , patients had seen an average of three different specialists for management of their syncope . most patients ( 70% ) had been hospitalized at least once for syncope and one - third ( 36% ) had experienced significant trauma in association with a syncopal episode . blue , red , and green bars indicate specialists seen at the hospital for the latest syncope episode ; shaded bars last specialist consulted before ilr implant ; open bars all specialists reported by patients as being consulted in relation to syncope in the past . blue , red , and green bars indicate specialists seen at the hospital for the latest syncope episode ; shaded bars last specialist consulted before ilr implant ; open bars all specialists reported by patients as being consulted in relation to syncope in the past . in contrast , carotid sinus massage or tilt tests were only undertaken in one - third of subjects . the ilr was implanted during the initial phase of the diagnostic work - up ( up to four diagnostic tests ) in 128 patients ( 22% ) . table 2history of diagnostic tests performed before ilr implanttotal recruitment570 ( 100%)standard ecg556 ( 98%)echocardiography490 ( 86%)basic laboratory tests488 ( 86%)ambulatory ecg monitoring382 ( 67%)in - hospital ecg monitoring311 ( 55%)exercise testing297 ( 52%)orthostatic blood pressure measurements275 ( 48%)mri / ct scan267 ( 47%)neurological or psychiatric evaluation270 ( 47%)eeg222 ( 39%)carotid sinus massage205 ( 36%)tilt test201 ( 35%)electrophysiology testing144 ( 25%)coronary angiography133 ( 23%)external loop recording67 ( 12%)atp test15 ( 3%)other tests52 ( 9%)no tests performed1 ( 0% ) history of diagnostic tests performed before ilr implant during follow - up , a total of 218 patients ( 38% of the population ) experienced an episode of syncope , 149 ( 26% of patients or 68% of episodes ) with prodromal symptoms . the percentages of patients who had a recurrence of syncope were 19 , 26 , and 36% after 3 , 6 , and 12 months , respectively ( figure 2 ) . ten patients with an episode during follow - up ( 5.2% of the population with an event ) had associated severe trauma . time to a syncopal event where reveal played a role in the diagnosis showed the recurrence of diagnosis within 180 days as 20% , within 365 days as 30% , and within 462 days as 38% ( figure 2 ) . the estimated rate of syncope after 30 days of follow - up was 10% and the estimated rate of diagnosis where reveal played a role at this time of follow - up was 9% . in the 48 patients with an episode but without reveal - guided diagnosis , 12% received dual - chamber pacemakers , 4% anti - arrhythmic drug therapy , there were no icd implants or catheter ablations in the 48 patients with an episode but without reveal - guided diagnosis . of a total 650 enrolled subjects , follow - up visit data ( with or without events ) were available for 570 subjects who were included in the analysed population . these patients were included in the follow - up analysis but excluded from the survival analysis . seven per cent of patients were enrolled based on unexplained pre - syncope ; 91% had unexplained syncope and the classification was missing for 2% of patients . patients with syncope had experienced a median of 4 reported events prior to enrolment , of which three were within the 2 years prior to enrolment . table 1general characteristics of patients at study enrolmenttotal recruitment570 ( 100%)women306 ( 54%)age sd61 17primary indication unexplained syncope517 ( 91% ) unexplained pre - syncope42 ( 7% ) other11 ( 2%)history hypertension277 ( 49% ) coronary artery disease84 ( 15% ) valvular heart disease30 ( 5% ) cardiomyopathy18 ( 3% ) stroke / tia57 ( 10%)diagnostic work - up before ilr implant in an initial phase of diagnostic work - up of syncope128 ( 22% ) after full evaluation of mechanism of syncope386 ( 68%)device implanted reveal dx264 ( 46% ) reveal xt52 ( 9% ) reveal plus254 ( 45%)age at first syncope55 20previous syncopes median ( iq range)4 ( 26)syncopes in the last 2 years median ( iq range)3 ( 24)syncopal episodes per year median ( iq range)2 ( 13.5)median interval between first and last episode years ( iq range)2 ( 04)any previous hospitalization because of syncope399 ( 70%)any syncopal episodes without prodromes339 ( 59%)any syncopal episodes with severe trauma204 ( 36%)any syncopal episodes suggestive of vasovagal origin86 ( 15%)any situational syncope39 ( 7%)characteristics of last syncope after effort28 ( 5% ) during effort144 ( 25% ) at rest294 ( 52% ) unknown97 ( 17% ) missing7 ( 1%)symptoms muscle spasms ( one sided)8 ( 1% ) muscle spasms ( two sided)19 ( 3% ) grand mal10 ( 2% ) other muscle spasms14 ( 2% ) transpiration73 ( 13% ) cyanosis19 ( 3% ) angina pectoris23 ( 4% ) palpitations76 ( 13% ) dizziness163 ( 29% ) dyspnoea33 ( 6% ) fatigue95 ( 17% ) other80 ( 14% ) none266 ( 47% ) general characteristics of patients at study enrolment the first specialist consulted for syncope in almost 23% of patients was an emergency medicine consultant . overall , patients had seen an average of three different specialists for management of their syncope . most patients ( 70% ) had been hospitalized at least once for syncope and one - third ( 36% ) had experienced significant trauma in association with a syncopal episode . blue , red , and green bars indicate specialists seen at the hospital for the latest syncope episode ; shaded bars last specialist consulted before ilr implant ; open bars all specialists reported by patients as being consulted in relation to syncope in the past . blue , red , and green bars indicate specialists seen at the hospital for the latest syncope episode ; shaded bars last specialist consulted before ilr implant ; open bars all specialists reported by patients as being consulted in relation to syncope in the past . in contrast , carotid sinus massage or tilt tests were only undertaken in one - third of subjects . the ilr was implanted during the initial phase of the diagnostic work - up ( up to four diagnostic tests ) in 128 patients ( 22% ) . table 2history of diagnostic tests performed before ilr implanttotal recruitment570 ( 100%)standard ecg556 ( 98%)echocardiography490 ( 86%)basic laboratory tests488 ( 86%)ambulatory ecg monitoring382 ( 67%)in - hospital ecg monitoring311 ( 55%)exercise testing297 ( 52%)orthostatic blood pressure measurements275 ( 48%)mri / ct scan267 ( 47%)neurological or psychiatric evaluation270 ( 47%)eeg222 ( 39%)carotid sinus massage205 ( 36%)tilt test201 ( 35%)electrophysiology testing144 ( 25%)coronary angiography133 ( 23%)external loop recording67 ( 12%)atp test15 ( 3%)other tests52 ( 9%)no tests performed1 ( 0% ) history of diagnostic tests performed before ilr implant during follow - up , a total of 218 patients ( 38% of the population ) experienced an episode of syncope , 149 ( 26% of patients or 68% of episodes ) with prodromal symptoms . the percentages of patients who had a recurrence of syncope were 19 , 26 , and 36% after 3 , 6 , and 12 months , respectively ( figure 2 ) . ten patients with an episode during follow - up ( 5.2% of the population with an event ) had associated severe trauma . time to a syncopal event where reveal played a role in the diagnosis showed the recurrence of diagnosis within 180 days as 20% , within 365 days as 30% , and within 462 days as 38% ( figure 2 ) . the estimated rate of syncope after 30 days of follow - up was 10% and the estimated rate of diagnosis where reveal played a role at this time of follow - up was 9% . in the 48 patients with an episode but without reveal - guided diagnosis , 12% received dual - chamber pacemakers , 4% anti - arrhythmic drug therapy , there were no icd implants or catheter ablations in the 48 patients with an episode but without reveal - guided diagnosis . two important findings were the large number of diagnostic tests that patients underwent before an ilr implant and the high diagnostic yield from the use of an ilr in the overall population with unexplained syncope . together , these findings imply that if an ilr is implanted early , as emphasized in the 2009 esc guidelines , a reduced number of tests might be needed . the picture is the largest observational study to date to evaluate the usage and diagnostic effectiveness of ilrs in the everyday clinical diagnostic work - up of patients with unexplained syncope . the registry included european countries and israel , but the findings may have wider relevance . the 2009 guidelines are based on transatlantic consensus and might be better prepared to improve adherence but at present , there is an important gap between what guidelines prescribe and how the real - life care of patients with unexplained syncope is carried out . in theory , the differentiation between various forms of loss of consciousness and seizures may appear reasonably easy , but in the real world , the diagnosis most often has to be based on the retrospective observations of bystanders and little information from the patient , who has frequently no memory of the event . thus , the patient or relatives do not know which specialist to see first , and the first physician may not be the most appropriate to reach a diagnosis . the picture found a high diagnostic yield with ilrs , which guided the diagnosis in 78% of patients with recurrent syncope and provided useful information in another 6% . a cardiac cause of syncope was found in 59% of the patients with recurrent syncope , leading to symptomatic treatment with pacemakers ( at rates corresponding to reported rates of bradycardia in syncope patients ) , ablation and medication as well as life - saving treatment with implantable defibrillators . in less than 20% of patients with a reveal - guided diagnosis was there a decision to undertake no specific treatment . in this group of patients , the benefit of knowing the cause of syncope would have reduced the need for further diagnostic tests . recurrence rates during the longer follow - up period of 10 6 months were 38% . in picture , the 50% of patients who received a reveal plus device with a battery life of around 14 months may not have had sufficient time to experience a syncopal event . the reveal dx / xt models that became available during the course of the conduct of picture have a battery life of 3 years and given the recurrence rates increasing with time in patients with follow - up visit data beyond 12 months ( figure 2 ) , it seems reasonable that within this time span the majority of the study population would experience an event . patients with pre - syncope only were admitted into the registry , and they have been analysed and reported together with patients with syncope , since the subgroup was small . in patients with unexplained syncope and a recurrence during follow - up , the ilr revealed or contributed to establishing the mechanism of syncope in the vast majority . the proportion of patients with an ilr - guided diagnosis increased over time and was still growing at the end of follow - up , implying that more patients would get a diagnosis before the end of battery life . the findings support the recommendation in current guidelines that an ilr should be implanted early rather than late in the evaluation of unexplained syncope .

medical ultrasound imaging is considered to be one of the edge technologies in noninvasive diagnose procedures . despite its great advantages , as cost - benefit , accessibility , portability , and safety this is the result of the attenuations , refractions , nonlinearities , frequency selection , or probe properties . as a result signal processing methods offer a reasonable approach for resolution improvement . from this point of view the most important methods for reconstruction are superresolution and deconvolution . if superresolution methods seem to be impractical , the deconvolution ones are more practical . supperresolution is a complex problem because of the difficulties in aproximation of reconstruction operators ( e.g. , motion , degradation , and subsampling operators ) and the use of multiple frames which puzzles also the implementation . this was conducted on the proposition of multiple deconvolution approaches for ultrasound imaging , like methods used in system identification or bayesian statistics based ones . from these algorithms , the methods based on bayesian approach , especially maximum a posteriori ( map ) seem to offer the most interesting results [ 410 ] . in these methods the point spread function ( psf ) is estimated and then the information is reconstructed in a nonblind way using a priori information about tissue reflectivity function . as the psf estimation is an important problem that is complex , a lot of methods were advanced to propose an acceptable solution . primary studies have considered a measured radio - frequency ( rf ) psf [ 4 , 11 ] . however , the use of only one rf psf to deconvolve the entire image is not feasible due to the nonstationarity of the psf along the rf line which results from the attenuations , reflections , refractions , and phase aberrations phenomena . a common solution is to estimate the psf locally by supposing that it is a slow variant in time . this needs to divide the image in segments where one may consider that the psf is constant and can be estimated for each segment . based on the local estimation of the psf , a certain number of methods were proposed . an approach based on a 1d implementation was proposed to estimate the rf psf using high order statistics . introduced a method of rf psf estimation using the cepstrum and homomorphic deconvolution [ 6 , 1214 ] . in this approach it is considered that the psf spectrum is a function smoother than the reflectivity function . this approach was developed for the cases when the interrogated tissues are composed by high reflectors superimposed by speckle noise , that is , the reflectivity function has laplacian distribution . for that , it was proposed a complex homomorphic procedure , where for the elimination of the spectrum of reflectivity function a multilevel decomposition denoising technique was used . this denoising technique was improved with an outlier resistant denoising procedure and the phase was estimated using minimum phase assumption . from the point of view of nonblind algorithms used for deconvolution , the proposed methods supposed that the reflectivity function has a gaussian or a laplacian probability of distribution function . for that it was frequently used the wiener filter ( or l2-norm regularization ) [ 46 , 1114 ] or l1-norm regularization [ 7 , 9 ] . if the wiener filtering seems to smooth the information and to offer a resulted image with a small resolution improvement and speckle noise suppression , the methods based on laplacian distribution showed a better improvement in terms of contrast and speckle noise reduction . the authors proposed an expectation - maximization algorithm that solved the problem iteratively , by alternating between wiener filtering and wavelet - based denoising . in , 2-steps deconvolution algorithm was proposed , where in first step the psf is estimated using the cepstrum technique and for deconvolution a two steps iterative shrinkage / thresholding ( twist ) is used . however , all the previous methods suffer of difficulties in the phase approximation of the rf psf in the algorithm robustness . to overcome these difficulties recent works were focused to extract the reflectivity function using the envelope of rf data [ 810 , 18 ] . the most important part of algorithms based on rf envelope intends to extract the tissue reflectivity function using the idea of the inverse filtering . the first contribution concerns the combination of three main steps that are the envelope detection based on hilbert transform , the psf estimation based on a general homomorphic deconvolution approach , and the deconvolution algorithm based on greedy implementation . knowing that each method taken alone is not novel , this combination of the above three steps constitutes a novelty . the second contribution concerns the application 's field since it is the first time when it is used in ultrasound medical imaging . being a blind algorithm firstly , the psf is estimated for each sequence composing the ultrasound image , and , secondly the reflectivity function is obtained using proposed algorithm with the a priori assumption that reflectivity function is a sparse signal ; that is , it has a laplacian probability of density function ( pdf ) . the proposed approach is an iterative algorithm based on the matching pursuit principle that avoids the difficult inverse problem in signal reconstruction . finally , to take into account that psf can be variant with depth , note that the proposed method can be used on short time subsequences derived from the analyzed sequence at different depths . in the following , the paper is organized as follows . section 2 describes the problem reconstruction for ultrasound imaging , section 3 presents the proposed method , section 4 shows the experimental results , and section 5 provides several comments and concludes the current study . in ultrasound imaging the obtained a - mode and b - mode images suppose the interaction between the acoustic beam , generated by the transducer and the scanned tissues . usually , the phenomena are not linear but for computations simplicity the greatest part of the methods proposed in the literature suppose that the acquired signal is a quasi - linear combination between the reflectivity function and the rf pulse . this supposes that the ultrasound sequences are divided in segments and each segment is processed individually . for the sake of simplicity we reduce the analysis to singular segment . the mathematical formulation of the measured signal y(n ) can be described as follows : ( 1)y(n)=h(n)x(n)+u(n ) , where is the convolution operator , h(n ) is the psf , x(n ) is reflectivity function , u(n ) is a gaussian white noise , and n is the samples index . in the frequency domain , ( 1 ) can be written as ( 2)y()=h()x()+u( ) , where the upper case letters represent the fourier transform ( ft ) of the components from ( 1 ) and is the angular frequency . as previously mentioned in section 1 , the purpose of the blind reconstruction methods is to obtain the true signal x(n ) starting from the acquired signal y(n ) . a natural solution from ( 2 ) is to obtain x( ) by inverting h( ) , which is the ft of the psf . the main problem is that the small values of h( ) will amplify by its inversion the high frequencies and implicitly the noise . the most used solutions on this problem are the regularization , according to pdf of the reconstructed signals . in ultrasound imaging a part of the generated pulse is reflected when it finds an interface between two tissues with different physical properties . therefore , we classically suppose that the reflectivity function has a laplacian pdf [ 7 , 9 , 10 , 15 ] . the acquired signal can be described like an amplitude modulated signal , where the carrier is the wave generated by the transducer and the information is located in its envelope . based on this assumption , the proposed method starts from the acquired rf signals . from these signals , we extract the envelope and afterwards , this envelope is used in two steps blind deconvolution algorithm : in the first step , the psf is extracted ; then it is used in the greedy noninverse deconvolution algorithm . in the following sections we detail the methods implemented in this paper : hilbert transform for envelope detection;homomorphic deconvolution and soft - thresholding denoising for psf estimation;noninverse greedy deconvolution . hilbert transform for envelope detection ; homomorphic deconvolution and soft - thresholding denoising for psf estimation ; noninverse greedy deconvolution . the most popular methods for envelope detection are hilbert transform or low - pass filtering to separate the useful information contained in envelope from the sinusoidal rf carrier wave . since the design of low - pass filter may be critical due to the unclear signals spectrum specifications , in this work the hilbert transform has been preferred . the envelope y(n ) can be extracted by applying the absolute value operator at the analytic signal as follows : ( 4)y(n)=|ya(n)| , where the ya(n ) means the analytic signal , y(n ) means the obtained envelope , and || is the absolute value operator . the analytic signal is generated using hilbert transform as follows : ( 5)ya(n)=yrf(n)+j{yrf(n ) } , where ya(n ) is the analytic signal , yrf(n ) is the original rf signal , and {yrf(n ) } is the hilbert transform of yrf(n ) . in ultrasound imaging it was widely assumed in many works that the psf is a much smoother function than the tissue reflectivity function and that two composing signals of the measured signal spectrum can be separated using homomorphic deconvolution and the denoising procedure as in . the greatest advantage of this kind of homomorphic filters is that they may accept as input a signal composed of two components and return a signal with one of them removed . the proposed estimation is a three steps algorithm ; in the first step we assume that the noise level in ( 2 ) is quite small and we may ignore it . in this case it can be rewitten as follows : ( 6)lny()=lnh()+lnx( ) , where ln is the natural logarithm . in this way the output signal is split into two parts : a part which comes from psf and another one , which occurs from the input signal . this linear transformation helps us to make a distinction between the signals , under the above presented assumptions that psf is a smoother function . the main idea of this technique is the use of a denoising method in the frequency domain by applying a wavelet soft thresholding and an outlier resistant denoising algorithm . the threshold was calculated as follows : ( 7)t=2lnn , where n is the length of the array and is the noise variance . the parameter is automatically estimated by = mx/0.6745 , where mx was the median absolute value of the finest decomposition level . having obtained lnh( ) , the final step of the homomorphic deconvolution is to get the psf h(n ) by using the inverse fourier transform ( ift ) of the logarithm spectrum of the psf , as follows : ( 8)h(n)=ift{exp[ln(h())]}. in our implementation , the fourier transform was evaluated using discrete fourier transform ( dft ) . it is a greedy algorithm analogous with matching pursuit algorithm . before describing the computational method , the ultrasound imaging is a technique based on the physical properties of acoustical wave reflection when it finds an interface of two different regions with different densities along its propagation . this allows the consideration of the acquired signal as a collection of rf echoes with different amplitude size . using the above presented considerations , one can say that the useful information , that is , the topological function of scanned tissues can be simulated as a sparse signal superimposed by white gaussian noise . here , the high amplitude pulses simulate the strongest reflectors and correspond to edges / details of imaging target . the white gaussian noise will correspond to the speckle noise , which according to the final objective of ultrasound sequences processing must be eliminated , reduced , or preserved . using the sparsity constraint , we propose an algorithm which is able to reconstruct the original signal without inverting the psf . this helps us to avoid the inverse problem , which has been known as one of the difficult problems in signal processing . within this approach each subproblem has the objective to eliminate the influence of the most important reflector . in this way , it extracts iteratively from the envelope of the measured signal the influence of the most important blurred scatter ; then replaces it with a unit pulse in the output signal , at the same position . at the beginning it provides a locally optimal choice to solve the subproblem , in the hope that at the end , the final solution is optimal . for the implementation of the proposed deconvolution algorithm see algorithm 1 . here , r(n ) is so called the residual signal , ri(ni ) is the value of maximum amplitude at the position ni at iteration i , and is the convolution operator . for this study it was fixed that k = 0 , because in this way the algorithm extracts the maximum number of possible reflectors . according to the condition of positiveness for the reflectivity function , the proposed algorithm iterations have sense , while the residual signal has values greater than zero . also , the envelope of the psf being a positive function , it results that the subtraction of a positive function from another positive one will generate a new residual function , at the iteration i + 1 which always satisfy the inequality ri+1(n ) < this condition is enough to prove that the algorithm will always reach the exit condition . the number of iterations corresponds with the sparsity coefficient , where sparsity coefficient means the number of nonzero elements in the final result . the psf amplitude is normalized to preserve the same amplitude as in the envelope signal for the resulted sparse signal . the experiments with simulated signals are motivated by the allowance of quantitative evaluations under controlled conditions . then the algorithms were applied to real data to test the feasibility of algorithms in clinical applications where the original topology of tissues is unknown . in the following , these two directions of evaluation will be presented as follows : section 4.1 presents the results for simulated data and section 4.2 shows the results for real data . the so called reflectivity function , which simulates the tissues topology , was generated using laplacian pdf assumption . the length of the signals was 512 points , the sampling frequency was 20 mhz , and the central transducer frequency was 3.2 mhz . this corresponded to a sequence of 160 s and an approximately 3.94 cm deep scanning ( for a standard ultrasound velocity c = 1540 m / s ) . during the experiments the above mentioned added gaussian white noise was generated according to different snr values . with this noise we intended to simulate different types of tissues . for example , we find more speckle noise and weak scatters in the soft tissues , like abdominal tissues . this reflectivity function must be transformed into an rf signal to simulate the acquired signal of the ultrasound probe . according to ( 1 ) it can be obtained if the reflectivity function is convolved with a simulated radio - frequency psf . for current studies the rf psf was generated using the formula : ( 9)psf = aexp[(tn)2]sint , where a means the psf amplitude , exp is the exponential function , is the angular frequency , t symbolizes time , and n is the number of the periods of the sinusoidal wave of the psf . the use of this formula is motivated by its capability to control the number of oscillations in the simulated rf pulse . from the experiments figure 1 presents an rf simulated signal example as follows : figure 1(a ) represents simulated tissue reflectivity function , figure 1(b ) represents the generated rf psf , and figure 1(c ) represents the rf obtained after convolution and its envelope . wavelet decomposition and denoising were performed using wavelab toolbox , downloaded from http://statweb.stanford.edu/~wavelab/. for the parameter in ( 7 ) , it was observed experimentally that using 5 levels of decomposition was enough for a good elimination of the noise . also , the estimation of the psf was made under assumption of minimum phase . the current algorithm , described in section 3.3 was compared also with different state of the art methods used in deconvolution as follows : regularized least square using l1-norm , wiener filter ( or l2-norm regularization ) , and total variation [ 3 , 25 ] . the lagrangian parameter , , for comparative methods was fixed empirically to obtain the best results as follows : for l1-norm = 0.2 , for wiener filter ( also known as tikhonov regularization ) = 0.08 , and for tv - norm = 0.14 . the results were presented in terms of visual and quantitative evaluation . for quantitative measurements , we assessed the execution time for each method and we computed the normalized mean square error ( nmse ) and also resolution gain ( rg ) parameter . rg parameter is based on the ratio between normalized autocorrelation function of the original envelope and the resulted signal higher than 3 db . the nmse is defined as follows : ( 10)nmse = e[||x^x||22||x||22 ] , where e is the statistical expectation , x is the original reflectivity function , and x^ is the resulted reflectivity function . it contains in figure 2(a ) the original rf signal envelope , which was used in all deconvolution methods as input signal ; then the obtained results as follows : figure 2(b ) : results were obtained with our algorithm , figure 2(c ) : results obtained with l1-norm , figure 2(d ) : results obtained with wiener filter , and figure 2(e ) : results obtained with tv - norm . for a better evaluation of the results , after computations , all results were normalized and then displayed to have the same dynamic ranges . it could be seen that our algorithm outperforms the comparative methods in terms of amplitude and scatters estimation . l1-norm method offered also a sparse solution for the final result , but it could be seen that the final result was more contaminated with noise , which limits the approach for clinical investigations . the last two methods , wiener filtering and tv - norm , offered smooth solutions which did not always offer well distinct or well contoured reflectors . to complete the qualitative evaluation , the results were assessed using some numerical criteria . table 1 summarizes the results for the nmse according to ( 10 ) and resolution gain . the displayed values are the results of trade off over 100 independently generated signals for all snr values . in terms of nmse it could be observed that the best results were offered by l1-norm followed closely by our method , but wiener filter and tv - norm were outperformed and in terms of resolution gain the best results are offered by our method followed by l1-norm . wiener filter and tv - norm had an insignificant resolution improvement . also , an important feature of the proposed algorithm was its execution time . in table 2 this was the logical consequence of the fact that it worked directly in time domain and with the most important operation being vector subtraction . it must be mentioned that for execution time , deconvolution algorithms without psf estimation were evaluated . table 3 showed the results of a statistical evaluation for scatters detection for proposed algorithms ( our method , wiener filter , and l1-norm ) . the evaluations were made using simulated signals using different levels of speckle noise and different number of scatters . it must be said that to make the same evaluation for wiener filter result , we use a signal where we keep only all local maximums . the objective of this simulation was to evaluate the detection capability for each algorithm in different conditions . it could be observed that the proposed algorithm and l1-norm offer similar results and they have a bigger detection capacity than wiener filter . this is normal because wiener filters smooth the information and a part of small details was lost in the reconstruction process . from the point of view of real scatters discovery , we can observe that the more the number of scatters increased , the more the number of detected ones decreased . this can be explained if we refer to rayleigh condition of superresolution . in the case of a high number of scatters a part of them can not be recovered , the scatters that are closer to /2 , where is the wavelength of the emitted psf . this fact is visible also in figure 2 ( at the samples 250 ) where a part of them is not recovered . as a next step , the proposed deconvolution algorithms were compared using real ultrasound sequences composing ultrasound images . as shown in synthetic signals evaluations , the ultrasound sequences can be done in the same procedure . the envelope could be obtained using hilbert transform ; then psf was estimated for each sequence and finally the reflectivity function was estimated . the first experiment on this section is focused on testing real independently measured signals . in the figure 3 are shown the real measured signal in the subplot figure 3(a ) and then , the results of reconstruction for used algorithms , in the same order as in figure 2 . because of no a priori information about the original reflectivity function it was impossible to evaluate the nmse parameter and also to make the superimposition of the obtained results over the original reflectivity function . for that , the qualitative evaluation is completed using just the rg parameter . in table 4 it can be seen that our method outperforms the wiener filter and tv - norm and offers similar results with l1-norm . then , in our experiments we used multiple images obtained in our laboratory . figure 4 is a log - compressed b - mode image of the skin obtained by an ultrasound scanner developed in - house called ecoderm . the probe used with this imaging device is a 128 elements linear array working at 20 mhz center frequency with 87% relative bandwidth . the linear scan is performed by the scanner through an emission aperture composed of 15 elements having focalization delays set up for 8 mm in soft tissues . for computation constraint all the sequences were zero - padded until the next 2 value . in terms of visual evaluation the reflectors are more visible and the speckle noise , which reduces the image quality , was almost suppressed . moreover , the contours were more visible and the regions without reflectors were better distinguished . the proposed algorithm assumes that the signal to be recovered is sparse ; that is , it has a laplacian distribution . for that it and l1-norm are more adapted to reconstruct ultrasound images of tissues with a small number of scatters . this means that in the final result the important details are furthermore revealed and the smaller details ( i.e. , the speckle noise ) are reduced or eliminated . such similar behavior is observed also in the synthetic signals and real sequences . as expected , in some cases , the sparse reflectivity sequence is difficult to be interpreted directly because of speckle noise suppression . some possible improvement can be made for a more realistic interpretation like convolution with an ideal psf or superimposition of the sparse data over b - mode image . the present paper addresses the problem of blind deconvolution for ultrasound sequences in medical imaging by formulating a solution that is able to extract the reflectivity function avoiding the hard problem of inverse filtering . the proposed algorithm is a time domain blind deconvolution that works as a greedy algorithm . the solution estimates in a blind way the psf , and then , it extracts iteratively the tissue reflectivity function using the estimated psf . being a blind technique , it was assumed a priori that the reflectivity function had a sparse shape ( i.e. , it follows the laplacian distribution law ) . , it can be seen that the greedy algorithm method outperforms the most used methods in the domain . also the algorithm works using the envelope of the acquired rf signals , which avoid the problem of the acoustic wave central frequency estimation . it is well known that in its moving along the propagation direction the psf shape is changing according to attenuation / nonlinear effects in the tissues . generally , for perfect results the state of the art approaches divide the image in sections and then the psf is calculated locally for each section . in reconstruction , for each section , it is used the locally estimated psf with the same deconvolution algorithm . this means that the deconvolution algorithms work identically , but the results change because of the different used psfs . the purpose of this research is to prove their feasibility for ultrasound sequences ; therefore we only considered the nonvariant case in the experimentations . from the simulations it resulted as well that scatters were well identified and the speckle noise was almost suppressed . however , in some conditions the results were too sparse and this could create some difficulties in information interpretation . first , the next step will be to analyze the proposed method for different types of tissues in a clinical investigation . also , we can try to improve the algorithm by imposing supplementary constraints for a better interfaces detection in the situations when they are very close . as discussed before , the sparsity constraint is not always well suitable and this could be improved by making a convolution of the resulted sparse signal with a psf as in . choosing the width for psf can be an interesting study and can offer different solutions according to desired application .

the blind deconvolution of ultrasound sequences in medical ultrasound technique is still a major problem despite the efforts made . this paper presents a blind noninverse deconvolution algorithm to eliminate the blurring effect , using the envelope of the acquired radio - frequency sequences and a priori laplacian distribution for deconvolved signal . the algorithm is executed in two steps . firstly , the point spread function is automatically estimated from the measured data . secondly , the data are reconstructed in a nonblind way using proposed algorithm . the algorithm is a nonlinear blind deconvolution which works as a greedy algorithm . the results on simulated signals and real images are compared with different state of the art methods deconvolution . our method shows good results for scatters detection , speckle noise suppression , and execution time .

1. Introduction 2. Problem Formulation in Ultrasound Medical Images Restoration 3. Envelope Based Blind Deconvolution 4. Results 5. Discussion and Conclusion

medical ultrasound imaging is considered to be one of the edge technologies in noninvasive diagnose procedures . despite its great advantages , as cost - benefit , accessibility , portability , and safety this is the result of the attenuations , refractions , nonlinearities , frequency selection , or probe properties . as a result signal processing methods offer a reasonable approach for resolution improvement . if superresolution methods seem to be impractical , the deconvolution ones are more practical . supperresolution is a complex problem because of the difficulties in aproximation of reconstruction operators ( e.g. , motion , degradation , and subsampling operators ) and the use of multiple frames which puzzles also the implementation . from these algorithms , the methods based on bayesian approach , especially maximum a posteriori ( map ) seem to offer the most interesting results [ 410 ] . in these methods the point spread function ( psf ) is estimated and then the information is reconstructed in a nonblind way using a priori information about tissue reflectivity function . primary studies have considered a measured radio - frequency ( rf ) psf [ 4 , 11 ] . however , the use of only one rf psf to deconvolve the entire image is not feasible due to the nonstationarity of the psf along the rf line which results from the attenuations , reflections , refractions , and phase aberrations phenomena . a common solution is to estimate the psf locally by supposing that it is a slow variant in time . based on the local estimation of the psf , a certain number of methods were proposed . in this approach it is considered that the psf spectrum is a function smoother than the reflectivity function . this approach was developed for the cases when the interrogated tissues are composed by high reflectors superimposed by speckle noise , that is , the reflectivity function has laplacian distribution . for that , it was proposed a complex homomorphic procedure , where for the elimination of the spectrum of reflectivity function a multilevel decomposition denoising technique was used . from the point of view of nonblind algorithms used for deconvolution , the proposed methods supposed that the reflectivity function has a gaussian or a laplacian probability of distribution function . if the wiener filtering seems to smooth the information and to offer a resulted image with a small resolution improvement and speckle noise suppression , the methods based on laplacian distribution showed a better improvement in terms of contrast and speckle noise reduction . in , 2-steps deconvolution algorithm was proposed , where in first step the psf is estimated using the cepstrum technique and for deconvolution a two steps iterative shrinkage / thresholding ( twist ) is used . however , all the previous methods suffer of difficulties in the phase approximation of the rf psf in the algorithm robustness . to overcome these difficulties recent works were focused to extract the reflectivity function using the envelope of rf data [ 810 , 18 ] . the most important part of algorithms based on rf envelope intends to extract the tissue reflectivity function using the idea of the inverse filtering . the first contribution concerns the combination of three main steps that are the envelope detection based on hilbert transform , the psf estimation based on a general homomorphic deconvolution approach , and the deconvolution algorithm based on greedy implementation . being a blind algorithm firstly , the psf is estimated for each sequence composing the ultrasound image , and , secondly the reflectivity function is obtained using proposed algorithm with the a priori assumption that reflectivity function is a sparse signal ; that is , it has a laplacian probability of density function ( pdf ) . section 2 describes the problem reconstruction for ultrasound imaging , section 3 presents the proposed method , section 4 shows the experimental results , and section 5 provides several comments and concludes the current study . usually , the phenomena are not linear but for computations simplicity the greatest part of the methods proposed in the literature suppose that the acquired signal is a quasi - linear combination between the reflectivity function and the rf pulse . this supposes that the ultrasound sequences are divided in segments and each segment is processed individually . the mathematical formulation of the measured signal y(n ) can be described as follows : ( 1)y(n)=h(n)x(n)+u(n ) , where is the convolution operator , h(n ) is the psf , x(n ) is reflectivity function , u(n ) is a gaussian white noise , and n is the samples index . in the frequency domain , ( 1 ) can be written as ( 2)y()=h()x()+u( ) , where the upper case letters represent the fourier transform ( ft ) of the components from ( 1 ) and is the angular frequency . as previously mentioned in section 1 , the purpose of the blind reconstruction methods is to obtain the true signal x(n ) starting from the acquired signal y(n ) . a natural solution from ( 2 ) is to obtain x( ) by inverting h( ) , which is the ft of the psf . the most used solutions on this problem are the regularization , according to pdf of the reconstructed signals . in ultrasound imaging a part of the generated pulse is reflected when it finds an interface between two tissues with different physical properties . the acquired signal can be described like an amplitude modulated signal , where the carrier is the wave generated by the transducer and the information is located in its envelope . based on this assumption , the proposed method starts from the acquired rf signals . from these signals , we extract the envelope and afterwards , this envelope is used in two steps blind deconvolution algorithm : in the first step , the psf is extracted ; then it is used in the greedy noninverse deconvolution algorithm . in the following sections we detail the methods implemented in this paper : hilbert transform for envelope detection;homomorphic deconvolution and soft - thresholding denoising for psf estimation;noninverse greedy deconvolution . the most popular methods for envelope detection are hilbert transform or low - pass filtering to separate the useful information contained in envelope from the sinusoidal rf carrier wave . the envelope y(n ) can be extracted by applying the absolute value operator at the analytic signal as follows : ( 4)y(n)=|ya(n)| , where the ya(n ) means the analytic signal , y(n ) means the obtained envelope , and || is the absolute value operator . the analytic signal is generated using hilbert transform as follows : ( 5)ya(n)=yrf(n)+j{yrf(n ) } , where ya(n ) is the analytic signal , yrf(n ) is the original rf signal , and {yrf(n ) } is the hilbert transform of yrf(n ) . in ultrasound imaging it was widely assumed in many works that the psf is a much smoother function than the tissue reflectivity function and that two composing signals of the measured signal spectrum can be separated using homomorphic deconvolution and the denoising procedure as in . the proposed estimation is a three steps algorithm ; in the first step we assume that the noise level in ( 2 ) is quite small and we may ignore it . in this way the output signal is split into two parts : a part which comes from psf and another one , which occurs from the input signal . this linear transformation helps us to make a distinction between the signals , under the above presented assumptions that psf is a smoother function . the main idea of this technique is the use of a denoising method in the frequency domain by applying a wavelet soft thresholding and an outlier resistant denoising algorithm . the threshold was calculated as follows : ( 7)t=2lnn , where n is the length of the array and is the noise variance . the parameter is automatically estimated by = mx/0.6745 , where mx was the median absolute value of the finest decomposition level . having obtained lnh( ) , the final step of the homomorphic deconvolution is to get the psf h(n ) by using the inverse fourier transform ( ift ) of the logarithm spectrum of the psf , as follows : ( 8)h(n)=ift{exp[ln(h())]}. in our implementation , the fourier transform was evaluated using discrete fourier transform ( dft ) . it is a greedy algorithm analogous with matching pursuit algorithm . before describing the computational method , the ultrasound imaging is a technique based on the physical properties of acoustical wave reflection when it finds an interface of two different regions with different densities along its propagation . this allows the consideration of the acquired signal as a collection of rf echoes with different amplitude size . using the above presented considerations , one can say that the useful information , that is , the topological function of scanned tissues can be simulated as a sparse signal superimposed by white gaussian noise . here , the high amplitude pulses simulate the strongest reflectors and correspond to edges / details of imaging target . the white gaussian noise will correspond to the speckle noise , which according to the final objective of ultrasound sequences processing must be eliminated , reduced , or preserved . using the sparsity constraint , we propose an algorithm which is able to reconstruct the original signal without inverting the psf . this helps us to avoid the inverse problem , which has been known as one of the difficult problems in signal processing . within this approach each subproblem has the objective to eliminate the influence of the most important reflector . in this way , it extracts iteratively from the envelope of the measured signal the influence of the most important blurred scatter ; then replaces it with a unit pulse in the output signal , at the same position . at the beginning it provides a locally optimal choice to solve the subproblem , in the hope that at the end , the final solution is optimal . for the implementation of the proposed deconvolution algorithm see algorithm 1 . here , r(n ) is so called the residual signal , ri(ni ) is the value of maximum amplitude at the position ni at iteration i , and is the convolution operator . for this study it was fixed that k = 0 , because in this way the algorithm extracts the maximum number of possible reflectors . according to the condition of positiveness for the reflectivity function , the proposed algorithm iterations have sense , while the residual signal has values greater than zero . also , the envelope of the psf being a positive function , it results that the subtraction of a positive function from another positive one will generate a new residual function , at the iteration i + 1 which always satisfy the inequality ri+1(n ) < this condition is enough to prove that the algorithm will always reach the exit condition . the experiments with simulated signals are motivated by the allowance of quantitative evaluations under controlled conditions . in the following , these two directions of evaluation will be presented as follows : section 4.1 presents the results for simulated data and section 4.2 shows the results for real data . the length of the signals was 512 points , the sampling frequency was 20 mhz , and the central transducer frequency was 3.2 mhz . for example , we find more speckle noise and weak scatters in the soft tissues , like abdominal tissues . this reflectivity function must be transformed into an rf signal to simulate the acquired signal of the ultrasound probe . according to ( 1 ) it can be obtained if the reflectivity function is convolved with a simulated radio - frequency psf . for current studies the rf psf was generated using the formula : ( 9)psf = aexp[(tn)2]sint , where a means the psf amplitude , exp is the exponential function , is the angular frequency , t symbolizes time , and n is the number of the periods of the sinusoidal wave of the psf . from the experiments figure 1 presents an rf simulated signal example as follows : figure 1(a ) represents simulated tissue reflectivity function , figure 1(b ) represents the generated rf psf , and figure 1(c ) represents the rf obtained after convolution and its envelope . for the parameter in ( 7 ) , it was observed experimentally that using 5 levels of decomposition was enough for a good elimination of the noise . also , the estimation of the psf was made under assumption of minimum phase . the current algorithm , described in section 3.3 was compared also with different state of the art methods used in deconvolution as follows : regularized least square using l1-norm , wiener filter ( or l2-norm regularization ) , and total variation [ 3 , 25 ] . the lagrangian parameter , , for comparative methods was fixed empirically to obtain the best results as follows : for l1-norm = 0.2 , for wiener filter ( also known as tikhonov regularization ) = 0.08 , and for tv - norm = 0.14 . the results were presented in terms of visual and quantitative evaluation . for quantitative measurements , we assessed the execution time for each method and we computed the normalized mean square error ( nmse ) and also resolution gain ( rg ) parameter . rg parameter is based on the ratio between normalized autocorrelation function of the original envelope and the resulted signal higher than 3 db . the nmse is defined as follows : ( 10)nmse = e[||x^x||22||x||22 ] , where e is the statistical expectation , x is the original reflectivity function , and x^ is the resulted reflectivity function . it contains in figure 2(a ) the original rf signal envelope , which was used in all deconvolution methods as input signal ; then the obtained results as follows : figure 2(b ) : results were obtained with our algorithm , figure 2(c ) : results obtained with l1-norm , figure 2(d ) : results obtained with wiener filter , and figure 2(e ) : results obtained with tv - norm . for a better evaluation of the results , after computations , all results were normalized and then displayed to have the same dynamic ranges . to complete the qualitative evaluation , the results were assessed using some numerical criteria . table 1 summarizes the results for the nmse according to ( 10 ) and resolution gain . in terms of nmse it could be observed that the best results were offered by l1-norm followed closely by our method , but wiener filter and tv - norm were outperformed and in terms of resolution gain the best results are offered by our method followed by l1-norm . also , an important feature of the proposed algorithm was its execution time . in table 2 this was the logical consequence of the fact that it worked directly in time domain and with the most important operation being vector subtraction . it must be mentioned that for execution time , deconvolution algorithms without psf estimation were evaluated . table 3 showed the results of a statistical evaluation for scatters detection for proposed algorithms ( our method , wiener filter , and l1-norm ) . the evaluations were made using simulated signals using different levels of speckle noise and different number of scatters . this is normal because wiener filters smooth the information and a part of small details was lost in the reconstruction process . from the point of view of real scatters discovery , we can observe that the more the number of scatters increased , the more the number of detected ones decreased . in the case of a high number of scatters a part of them can not be recovered , the scatters that are closer to /2 , where is the wavelength of the emitted psf . as a next step , the proposed deconvolution algorithms were compared using real ultrasound sequences composing ultrasound images . as shown in synthetic signals evaluations , the ultrasound sequences can be done in the same procedure . the envelope could be obtained using hilbert transform ; then psf was estimated for each sequence and finally the reflectivity function was estimated . in the figure 3 are shown the real measured signal in the subplot figure 3(a ) and then , the results of reconstruction for used algorithms , in the same order as in figure 2 . because of no a priori information about the original reflectivity function it was impossible to evaluate the nmse parameter and also to make the superimposition of the obtained results over the original reflectivity function . for that , the qualitative evaluation is completed using just the rg parameter . in table 4 it can be seen that our method outperforms the wiener filter and tv - norm and offers similar results with l1-norm . figure 4 is a log - compressed b - mode image of the skin obtained by an ultrasound scanner developed in - house called ecoderm . the probe used with this imaging device is a 128 elements linear array working at 20 mhz center frequency with 87% relative bandwidth . in terms of visual evaluation the reflectors are more visible and the speckle noise , which reduces the image quality , was almost suppressed . moreover , the contours were more visible and the regions without reflectors were better distinguished . the proposed algorithm assumes that the signal to be recovered is sparse ; that is , it has a laplacian distribution . , the speckle noise ) are reduced or eliminated . such similar behavior is observed also in the synthetic signals and real sequences . as expected , in some cases , the sparse reflectivity sequence is difficult to be interpreted directly because of speckle noise suppression . some possible improvement can be made for a more realistic interpretation like convolution with an ideal psf or superimposition of the sparse data over b - mode image . the present paper addresses the problem of blind deconvolution for ultrasound sequences in medical imaging by formulating a solution that is able to extract the reflectivity function avoiding the hard problem of inverse filtering . the proposed algorithm is a time domain blind deconvolution that works as a greedy algorithm . the solution estimates in a blind way the psf , and then , it extracts iteratively the tissue reflectivity function using the estimated psf . being a blind technique , it was assumed a priori that the reflectivity function had a sparse shape ( i.e. , it follows the laplacian distribution law ) . , it can be seen that the greedy algorithm method outperforms the most used methods in the domain . also the algorithm works using the envelope of the acquired rf signals , which avoid the problem of the acoustic wave central frequency estimation . generally , for perfect results the state of the art approaches divide the image in sections and then the psf is calculated locally for each section . in reconstruction , for each section , it is used the locally estimated psf with the same deconvolution algorithm . this means that the deconvolution algorithms work identically , but the results change because of the different used psfs . the purpose of this research is to prove their feasibility for ultrasound sequences ; therefore we only considered the nonvariant case in the experimentations . from the simulations it resulted as well that scatters were well identified and the speckle noise was almost suppressed . however , in some conditions the results were too sparse and this could create some difficulties in information interpretation . first , the next step will be to analyze the proposed method for different types of tissues in a clinical investigation . also , we can try to improve the algorithm by imposing supplementary constraints for a better interfaces detection in the situations when they are very close . as discussed before , the sparsity constraint is not always well suitable and this could be improved by making a convolution of the resulted sparse signal with a psf as in .

sepsis is one of the most serious conditions related to high mortality in approximately 0.15 per 100 cases admitted to the hospital , and it also accounts for 515 percent of cases with overall infections . in 2007 , there were 201 ( 5.8% ) cases diagnosed as sepsis from 3,451 patients admitted to the medical wards in the siriraj hospital , of which 38.8% developed septic shock . the mortality rate of patients with sepsis and septic shock was as high as 34.3% and 52.6% , respectively . two important factors on antimicrobial therapy pertaining to adverse events and death in septic patients were the initiation of inappropriate antimicrobial therapy and the delay of appropriate antimicrobial therapy . inappropriate empiric antimicrobial therapy was attributed to 46.5% of cases , with 35% overall mortality . the elapsed time to appropriate antimicrobial therapy was crucial for the mortality in patients with severe sepsis and septic shock . the surviving sepsis campaign 's 2008 international guidelines for the management of severe sepsis and septic shock recommended that appropriate antimicrobial therapy should be administered within 1 hour of severe sepsis or septic shock recognition [ 3 , 5 ] . hence , we aimed to assess the prevalence and impact of inappropriate first - dose antimicrobial therapy and delayed antimicrobial administration on the mortality of patients with sepsis . also , the risk factors associated with inappropriate antimicrobial therapy and high mortality rate in these patients were determined . a retrospective cohort study was conducted during january december 2009 at the medical wards of the siriraj hospital . we enrolled the patients by looking at their positive blood cultures first and then searched for those who did have clinical sepsis by looking at the patients ' charts . patients diagnosed as sepsis , severe sepsis , or septic shock , with positive hemoculture on the day of diagnosis , were included . we excluded the patients with second episode of sepsis or more likely with bacteremia in the same admission , polymicrobial infection , and organisms other than bacteria ( e.g. , fungus ) . all cases with positive hemoculture result were determined to meet the specific criteria for sepsis , severe sepsis , and septic shock according to the american college of chest physicians / society of critical care medicine ( accp / sccm ) consensus conference definition . data were collected from inpatient - recording and drug - prescribing charts by the first author , using standardized case record forms . all data were collected from inpatient - recording charts and drug - prescribing chart by first researcher using a standardized case record forms . data collection included patients ' demographics , co - existing conditions , diagnosis of sepsis ( severe sepsis or septic shock ) , acute physiology and chronic health evaluation ( apache ) ii score , site of infection , causative bacteria , antimicrobial usage , timing of blood culture collection , actual time of initial parenteral antimicrobial administration , clinical course , and treatment outcome . timing of first - dose and 24-hour antimicrobial therapy was retrieved from drug - prescribing charts . the definitions of clinical infection , systemic inflammatory response syndrome ( sirs ) , sepsis , severe sepsis , and septic shock were adapted from previous recommendations and studies [ 6 , 811 ] . bacteremia was defined as the presence of viable bacteria in blood , detected by positive hemoculture . severity of diseases was classified as sepsis , severe sepsis , and septic shock according to previous definitions and apache ii scores , determined by the highest abnormal data within 24 hours of sepsis onset . community - acquired and nosocomial infections were defined as previous studies . healthcare - associated infection was defined as an infection in a patient being hospitalized for more than 2 days in the preceding 90 days or a resident in a nursing home or extended care facility , with home infusion therapy , chronic dialysis treatment within 30 days , or home wound care . timing of the diagnosis of sepsis was defined as the time when positive blood culture was performed , and then signs and symptoms of sepsis of these patients must be present when their clinical status were retrieved from the patient record chart at that time point . hence , the patients with subsequent positive blood cultures and presence of clinical criteria of sepsis , severe sepsis , or septic shock were enrolled in our study . timing of first and 24-hour dose antimicrobial therapy , defined as duration from diagnosis of sepsis to antimicrobials administration , retrieved from drug - prescribing charts . drug - resistant gram - positive bacteria included methicillin - resistant staphylococcus aureus ( mrsa ) and enterococcus spp .. drug - resistant gram - negative bacteria were extended - spectrum -lactamase ( esbl)-producing enterobacteriaclae , acinetobacter spp . , and pseudomonas aeruginosa . appropriate antimicrobial therapy was defined as the isolated bacteria being susceptible to at least one of the antimicrobials empirically administered as the first dose or 24 hours later . in the absence of specific sensitivity testing , the followings were considered as appropriate therapy : ( a ) group a , b , and g streptococcus treated with all beta - lactams ; ( b ) all gram - positive bacteria except enterococci treated with vancomycin . meanwhile , in the absence of specific sensitivity testing , the enterococci treated with all cephalosporin and trimethoprim / sulfamethoxazole were considered as inappropriate therapy [ 2 , 11 , 13 ] . the primary objectives were to determine the prevalence of first - dose and 24-hour inappropriate empiric antimicrobial therapy in septic patients with bacteremia and its impact on 28-day and overall mortality rates . the secondary objectives were to define the appropriate timing of antimicrobial administration and factors associated with inappropriate antimicrobial therapy and high mortality rate in bacteremic patients with sepsis . descriptive statistics were implemented to summarize the patients ' characteristics as mean ( sd ) or proportions . chi - square or fisher 's exact test for the categorical data and student 's t - test or mann - whitney u test for the continuous data were employed to compare data between different groups . multiple logistic regression analysis of independent factors associated with 28-day and overall mortality rates and factors related to inappropriate empirical antimicrobial treatment of bacteremia was performed . the mean ( sd ) age of septic patients was 63.5 ( 17.2 ) years , with 49.8% male patients . the common comorbidities were diabetes mellitus ( 31.0% ) , immunosuppressive therapy ( 29.3% ) , reduced mobility ( 29.7% ) , liver failure ( 21.8% ) , congestive heart failure ( 21.8% ) , chronic kidney disease ( 18.3% ) , and hematologic malignancy ( 18.2% ) ( table 1 ) . types of infection were community - acquired ( 27.5% ) , healthcare - associated ( 37.1% ) , and nosocomial infections ( 35.4% ) . septic shock and severe sepsis were present in 61.1% and 25.3% of the enrolled patients . the common sites of infection were respiratory tract ( 32.8% ) , gastrointestinal tract ( 23.6% ) , and genitourinary tract ( 20.5% ) ( table 2 ) . the most commonly identified bloodstream pathogens were gram - negative bacteria ( 72.5% ) as follows : escherichia coli ( 28.2% ) , klebsiella pneumoniae ( 12.7% ) , and acinetobacter spp . gram - positive bacteria were found in 27.5% of cases , including methicillin - susceptible s. aureus ( mssa ) ( 8.7% ) , methicillin - resistant s. aureus ( mrsa ) ( 4.8% ) , and streptococcus group d ( 3.5% ) . drug - resistant gram - positive bacteria , gram - negative bacteria , esbl - producing organism , and eskape organism were observed in 7.4% , 32.3% , 15.7% , and 73.8% of the isolates , respectively ( table 3 ) . antimicrobials frequently administered were cephalosporin ( 57.6% ) , carbapenem ( 23.1% ) , beta - lactam / beta - lactamase inhibitor ( 12.2% ) , vancomycin ( 11.4% ) , aminoglycosides ( 7.4% ) , fluoroquinolones ( 8.3% ) , and colistin ( 4.8% ) . only colistin significantly associated with appropriate first - dose antimicrobial therapy ( 6.8% versus 0% , p < 0.05 ) ( table 4 ) . among 229 patients , 143 and 161 cases died within 28 days and during the hospitalization , subsequently . in general , the 28-day and overall mortality rates were 62.4% and 70.3% , respectively . inappropriate first - dose and 24-hour empiric antimicrobial therapies were consequently noted in 68 cases ( 29.7% ) and 58 cases ( 25.3% ) of the patients . at 24 hours , 58 cases in the appropriate group and 19 cases in the inappropriate group had their empiric antimicrobials adapted . at 24 hours of diagnosis , one case in the appropriate group and 8 cases in the inappropriate group were still treated with inappropriate antimicrobials ( figure 1 ) . when compared to appropriate antimicrobial therapy , inappropriate first - dose antimicrobial therapy tended to have higher 28-day and overall mortality rates ( 67.6% versus 60.2% ( p = 0.301 ) and 75.0% versus 68.3% ( p = 0.345 ) , resp . ) . additionally , inappropriate 24-hour empirical antimicrobials were also likely to have higher 28-day and overall mortality rates ( 65.5 versus 61.4% ( p = 0.64 ) and 74.1 versus 69.0% ( p = 0.51 ) , resp . ) . univariate analysis of factors associated with 28-day and overall hospital mortalities in all sepsis patients was shown in table 5 . there were significant variations in the appropriateness of initial antimicrobial therapy between the groups of clinical infections and isolated organisms . nosocomial infection ( 37.0% ) appeared to have significantly higher inappropriate antimicrobials than community - acquired ( 14.3% ) and healthcare - associated infection ( 22.3% , p = 0.004 ) . drug - resistant gram - positive bacteria ( mrsa and enterococci ) and drug - resistant gram - negative bacteria ( acinetobacter spp . , pseudomonas aeruginosa , and esbl - producing organisms ) were significantly related to inappropriate first - dose empirical antimicrobials ( 64.7% versus 35.3% , p = 0.002 and 62.2% versus 37.8% , p = 0.001 , resp . ) . subgroup analyses of drug - resistant organisms also showed a significant relation with the inappropriateness of first - dose antimicrobial therapies , such as acinetobacter spp . ( 60.7% versus 39.3% , p < 0.001 ) , eskape organism ( enterococcus faecium , staphylococcus aureus , klebsiella pneumoniae , acinetobacter baumannii , pseudomonas aeruginosa , enterobacter spp . ( 65.1% versus 34.9% , p < 0.001 ) , and extended - spectrum -lactamase ( esbl)-producing organisms ( 77.8% versus 22.2% , p < 0.001 ) . moreover , both drug - resistant gram - positive and gram - negative bacteria significantly had higher inappropriate antimicrobial rates ( 64.7% versus 35.3% ; p = 0.002 and 62.2% versus 37.8% ; p = 0.001 , resp . ) . patients with genitourinary tract and skin and soft tissue infections appeared to have lower rate of inappropriate antimicrobial therapy than those with infections at other sites ( 57.4% versus 42.6% ; p = 0.048 and 94.4% versus 5.6% ; p = 0.016 , resp . ) . from multivariate analysis , factors significantly associated with the inappropriateness of first - dose empirical antimicrobials included drug - resistant gram - positive organism ( or 6.03 , 95% ci 2.3515.43 ) , drug - resistant gram - negative organism ( or 10.76 , 95% ci 3.9829.09 ) , leukopenia ( or 2.45 , 95% ci 1.185.08 ) , neutropenia ( or 2.94 , 95% ci 1.167.14 ) , and platelet < 100,000 per mm ( or 2.42 , 95% ci 1.204.88 ) ( table 6 ) . at hospital discharge , patients with inappropriate initial empiric antimicrobial therapy had a trend towards higher mortality rate than those with appropriate empiric antimicrobial therapy ( 75.0% versus 68.3% ; p = 0.345 ) . in addition , the first - dose and 24-hour inappropriate antimicrobial therapy resulted in shorter median survival duration than the appropriate antimicrobial therapy administration ( 214.7 hours versus 301.1 hours , p = 0.86 and 180.2 hours versus 301.1 hours , p = 0.85 , resp . ) . multivariate analysis of factors potentially associated with fatal outcome revealed that the inappropriateness of the first - dose antimicrobial therapy administration remained the most strongly correlated with high mortality ( or 2.52 ; 95% ci 1.016.32 ; p = 0.049 ) among all the factors assessed . other factors related to overall mortality included the timing from diagnosis until the first - dose antimicrobial therapy , septic shock status , presence of congestive heart failure , age > 65 years , apache ii score > 25 , platelet count < 100,000 per mm , and serum albumin < 3.2 g / dl ( table 7 ) . antimicrobials were initiated within 1 hour and 6 hours after onset of sepsis in 20.1% and 64.2% of cases , respectively . nevertheless , 9.6% of the patients received antimicrobial therapy after 24 hours of sepsis onset . the mean ( sd ) length of time from onset of diagnosis to the first - dose antimicrobial therapy was 9.57 ( 25.33 ) hours in all cases and 7.00 ( 16.61 ) hours in those with appropriate antimicrobial therapy . the patients with appropriate antimicrobial therapy were significantly administered to receive antimicrobials earlier than those treated with inappropriate antimicrobials ( 7.00 ( 16.60 ) versus 15.66 ( 38.37 ) hours , p = 0.018 ) . all patients and those with appropriate antimicrobial therapy in the 28-day and overall hospital mortality group were likely to have delayed first - dose antimicrobials . in all patients , the mean ( sd ) duration from onset of sepsis to appropriate antimicrobial therapy of the 28-day and overall mortality group was longer than the 28-day and overall survival group ( 7.09 ( 18.61 ) versus 6.85 ( 13.15 ) hours , p = 0.931 and 7.14 ( 18.48 ) versus 6.68 ( 11.73 ) hours , p = 0.931 , resp . ) . in patients with appropriate antimicrobials , the mean ( sd ) duration from onset of sepsis to appropriate antimicrobial therapy in the 28-day and overall mortality group was also more prolonged than the 28-day and overall survival group ( 9.89 ( 27.69 ) versus 9.03 ( 20.99 ) hours , p = 0.806 and 10.27 ( 27.30 ) versus 7.91 ( 19.99 ) hours , p = 0.522 , resp . ) . there was significant relationship between the delayed timing from diagnosis to the administration of first - dose antimicrobials and the overall mortality . the mortality rates in patients with antimicrobials less than 1 hour , from 1 hour to 6 hours , and more than 6 hours after the diagnosis of sepsis were 63.0% , 65.3% , and 80.5% , respectively ( p = 0.04 ) . also , higher overall mortality was demonstrated after the delayed antimicrobial initiation over 1 hour ( 72.1 versus 48.7% , p = 0.30 ) . however , significantly higher overall mortality was observed in those with antimicrobial therapy more than 3 hours after the diagnosis ( or 1.92 , 95% ci 1.083.42 ) . our data strongly supported the notion that the inappropriate antimicrobials and the delayed administration of appropriate antimicrobial therapy could substantially increase the mortality rate in septic patients with bacteremia . we reported herein that the inappropriate empirical antimicrobial therapy was frequent among bacteremic patients with high overall mortality and a tendency towards higher mortality rate than the appropriate antimicrobial therapy . this similar high percentages of inappropriate antimicrobial therapy , ranging from 20% to 46.5% , were also described by several previous reports , which resulted in high mortality rate from 35% to 70% [ 1 , 2 , 1727 ] . however , the overall mortality rate in our study was higher than the one conducted in the same hospital during 2007 . the difference in mortality rates between these two studies could be explained by distinctive population groups , higher percentages of healthcare - related and nosocomial infections , and more severity of infections in the latter study . the study conducted in 2007 enrolled patients with clinical entities of sepsis , severe sepsis , or septic shock regardless of whether they were bacteremic or not at the entry point . meanwhile , the study in 2009 recruited patients with bacteremia and clinical sepsis , severe sepsis , or septic shock . the population study groups in 2007 consisted of sepsis patients with bacteremia ( 40.8% ) , community - acquired infection ( 61.2% ) , and septic shock ( 38.8% ) . on the contrary , our study populations were bacteremic patients with healthcare - related infection ( 37.1% ) , nosocomial infection ( 35.4% ) , and septic shock ( 61.1% ) . hence , there was more severity of infections in our study population groups that led to higher mortality rate . other factors pertaining to higher mortality rate included microbiological factors such as the presence of frequent healthcare - associated and nosocomial infections , and drug resistant organisms . despite multiple risk factors for high mortality in sepsis patients , the inappropriate first - dose empirical antimicrobial therapy still significantly increased 6.7% of mortality rate . , demonstrating higher mortality among septic shock patients with inappropriate empirical antimicrobials ( 89.7% versus 48.0% ) . nevertheless , the impact of inappropriate empirical antimicrobials in patients with less comorbidities and severity of sepsis remained to be elucidated . there were also significant variations in the inappropriateness of initial antimicrobial therapy among infection sites , of which genitourinary and skin and soft tissue infections seemed to have appreciably better empirical antimicrobial therapies . inappropriate empiric antimicrobial therapy was notably higher in the gram - negative bacteria group than the gram - positive bacteria group , resulting in the increasing of mortality in gram - negative bacteremia , especially drug - resistant organisms as reported by kang et al . that inappropriate antimicrobial therapy in gram - negative septicemia had higher 30-day mortality rate ( 38 versus 27% , p < 0.05 ) . one of the major causes of inappropriate initial antimicrobial therapy was the underrecognition of the infections with antimicrobial - resistant organisms [ 2 , 21 , 24 , 2931 ] , of which drug - resistant gram - negative bacteria significantly associated with inappropriateness empirical antimicrobials . especially , esbl - producing escherichia coli ( e. coli ) and klebsiella pneumonia ( k. pneumoniae ) rapidly spread worldwide and became a serious healthcare problem . the thai prevalence of esbl - producing e. coli and esbl - producing k. pneumoniae in healthcare - associated infections was 13.2% and 12.7% , respectively . from our study , esbl - producing enterobacteriacae accounted for 15.7% of bacteremia , which considerably associated with both first - dose and 24-hour inappropriate empirical antimicrobials . e. coli was the most commonly conducting to bacteremia in our study , of which 42.4% were esbl - positive organisms that led to more inappropriate antimicrobials than esbl - negative organisms . consequently , the patients ' risk factors for harboring esbl - producing resistant organisms , including length of hospital or icu stay , central venous or arterial catheters , emergency intra - abdominal surgery , gastrostomy or jejunostomy tube , colonization of esbl - producing organisms in the gastrointestinal tract [ 34 , 35 ] , prior administration of any antibiotics , prior long - term care facility stay , severity of diseases , urinary catheter , ventilatory assistance , and hemodialysis , should be considered in order that treatment with carbapenem antimicrobial in producing the better outcomes of survival and bacteriologic clearance [ 38 , 39 ] would be more implemented . , multidrug - resistant strains as common causes of nosocomial infection and important antimicrobial - resistant organism [ 32 , 41 , 42 ] , was 12.2% frequently found and significantly associated with inappropriate antimicrobials . therefore , risk factors for colonization or infection with multidrug - resistant strains of acinetobacter spp . should be recognized , including icu admission , previous colonization with methicillin - resistant staphylococcus aureus ( mrsa ) , beta - lactam , beta - lactam / beta - lactamase inhibitor and carbapenem antibiotics use , bedridden status , previous intensive care admission , central venous catheter , surgery , mechanical ventilation , hemodialysis , and malignancy [ 4346 ] . for infections caused by multidrug - resistant acinetobacter spp . , antibiotic choices were usually limited . nevertheless , it showed in our study that colistin was only significantly associated with appropriate antimicrobial therapy . this was due to the fact that broad - spectrum antibacterial activity of colistin should be against most gram - negative bacteria whether they were pathogens of either community- or hospital - acquired infections . other antimicrobials did not have adequate broad - spectrum antibacterial activity compared to colistin , especially when the mdr gram - negative bacteria were the causative agents . however , pseudomonas aeruginosa did not beget inappropriate antimicrobial problems in our study . drug resistant gram - positive bacteria , such as the most frequent mrsa , also significantly associated with the inappropriateness of empirical antimicrobials . paul et al . demonstrated that inappropriate antimicrobials in mrsa septicemia obviously increased a risk for mortality . risk factors for infections with mrsa , including previous antibiotic use , prolonged hospitalization , intensive care , invasive devices , hemodialysis , mrsa colonization , and proximity to others with mrsa colonization or infection , and hiv infection should be concerned in order to select the better appropriate empirical antimicrobials [ 5355 ] . nevertheless , our study showed no benefits of combination antimicrobial therapy on the appropriateness of antimicrobials and lower mortality , which contrasted to the studies by kumar et al . [ 11 , 57 ] . therefore , risk factors for drug - resistant organisms as well as local patterns of antimicrobial susceptibility should be of utmost concern in selecting the empiric regimen [ 2931 , 5860 ] . particularly , in the cases of critically ill patients , septic shock , high apache ii score , leukopenia , respiratory or intra - abdominal infections , and nosocomial infection , physicians should be alert with particular attention on choosing appropriate initial empiric antimicrobials due to high mortality in these subgroups . the education program and antimicrobial treatment guideline should emphasize the knowledge of appropriate antimicrobial therapy administration . otherwise , better empiric coverage of antimicrobial therapy could be obtained through immediate consultation with infectious disease specialists , such as i d fast track ( like stroke or st elevation myocardial infarction fast track ) or antibiotic management teams in some institutions [ 6365 ] . as shown in our study , the adjustment of 24-hour antimicrobial therapy significantly resulted in a lower rate of inappropriateness than the first dose of antimicrobial therapy . nevertheless , there were the controversies of the impact on early and appropriate antimicrobial therapy on lower mortality in some studies [ 6668 ] . the delayed initiation of effective antimicrobial therapy following the onset of sepsis also increased mortality . the surviving sepsis campaign 's guideline recommended that the appropriate antimicrobial therapy should be administered within one hour of severe sepsis or septic shock recognition . the recent study confirmed the significantly higher mortality associated with duration from triage or qualification for early goal - directed therapy more than one hour . our study showed the association between higher mortality and delay in giving antimicrobial therapy over one hour after the sepsis onset . however , there was statistical difference in mortality if the first - dose antimicrobial therapy was delayed more than three hours . to this reason , the appropriate antimicrobial therapy should be immediately initiated in patients with diagnosis of sepsis to achieve lower mortality . for this purpose , the antimicrobial guideline for sepsis patients , development of strategies such as an immediately available antibiotics in the emergency department and critical wards , immediate sepsis fast track consultants , and antibiotic management teams should be accomplished to eliminate the delayed timing for antimicrobial medications and thus to ensure the timely administration of appropriate empiric antimicrobial therapy in sepsis patients in order to reduce the morbidity and mortality . the limitations of this study were retrospective methodology and the inclusion of patients only from medical wards of an academic referral center / tertiary medical center , which may not reflect the entire population in other departments or hospitals . inappropriate empirical antimicrobial therapy frequently occurs in about 29.7% of septic bacteremic patients , and it is associated with 75% mortality rate . important risk factors of inappropriate antimicrobials including nosocomial infection , leukopenia , and drug - resistant organisms should be concerned to achieve better appropriateness . furthermore , the mortality rate could be significantly increased by the delayed timing of first - dose antimicrobial therapy more than 3 hours after the diagnosis of sepsis . thus , more efforts must be employed to increase the appropriateness of initial antimicrobial therapy and decrease timing from sepsis diagnosis to initiation of antimicrobials in a bid to reduce the mortality in patients with sepsis .

background . inappropriate empiric antimicrobials could be a major cause of unfavorable mortality rates in co - morbid patients . this study aimed to assess the prevalence and impact of first - dose and 24-hour inappropriate antimicrobials on mortality rates of bacteremic septic patients . methods . a retrospective cohort study was employed . case record forms of patients diagnosed as sepsis , severe sepsis , or septic shock with positive hemoculture during 2009 were retrieved from the medical wards , siriraj hospital . demographic data , antimicrobial use , types of bacteria isolated from blood and susceptibilities , patients ' comorbidities , 28-day and overall mortality rates were collected and analyzed . results . there were 229 cases , mean age ( sd ) of 63.5 ( 17.2 ) years and mean ( sd ) apache ii score of 24.7 ( 6.8 ) . the prevalence of first - dose and 24-hour inappropriate antimicrobials was 29.7% and 25.3% , respectively . the 28-day and overall mortality rates between first - dose inappropriate and appropriate antimicrobial were 67.6% versus 60.2% ( p = 0.301 ) and 75.0% versus 68.3% ( p = 0.345 ) , consequently . patients with septic shock and inappropriate first - dose antimicrobials significantly had higher 28-day mortality rate ( 61.6% versus 41.9% ; p = 0.017 ) . conclusion . higher mortality rates in bacteremic septic patients were substantially associated with inappropriate first - dose antimicrobials and 3-hour delayed antimicrobial administration after sepsis diagnosis .

1. Background 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

in 2007 , there were 201 ( 5.8% ) cases diagnosed as sepsis from 3,451 patients admitted to the medical wards in the siriraj hospital , of which 38.8% developed septic shock . the mortality rate of patients with sepsis and septic shock was as high as 34.3% and 52.6% , respectively . two important factors on antimicrobial therapy pertaining to adverse events and death in septic patients were the initiation of inappropriate antimicrobial therapy and the delay of appropriate antimicrobial therapy . inappropriate empiric antimicrobial therapy was attributed to 46.5% of cases , with 35% overall mortality . the elapsed time to appropriate antimicrobial therapy was crucial for the mortality in patients with severe sepsis and septic shock . the surviving sepsis campaign 's 2008 international guidelines for the management of severe sepsis and septic shock recommended that appropriate antimicrobial therapy should be administered within 1 hour of severe sepsis or septic shock recognition [ 3 , 5 ] . hence , we aimed to assess the prevalence and impact of inappropriate first - dose antimicrobial therapy and delayed antimicrobial administration on the mortality of patients with sepsis . also , the risk factors associated with inappropriate antimicrobial therapy and high mortality rate in these patients were determined . a retrospective cohort study was conducted during january december 2009 at the medical wards of the siriraj hospital . patients diagnosed as sepsis , severe sepsis , or septic shock , with positive hemoculture on the day of diagnosis , were included . all cases with positive hemoculture result were determined to meet the specific criteria for sepsis , severe sepsis , and septic shock according to the american college of chest physicians / society of critical care medicine ( accp / sccm ) consensus conference definition . data were collected from inpatient - recording and drug - prescribing charts by the first author , using standardized case record forms . all data were collected from inpatient - recording charts and drug - prescribing chart by first researcher using a standardized case record forms . data collection included patients ' demographics , co - existing conditions , diagnosis of sepsis ( severe sepsis or septic shock ) , acute physiology and chronic health evaluation ( apache ) ii score , site of infection , causative bacteria , antimicrobial usage , timing of blood culture collection , actual time of initial parenteral antimicrobial administration , clinical course , and treatment outcome . timing of first - dose and 24-hour antimicrobial therapy was retrieved from drug - prescribing charts . the definitions of clinical infection , systemic inflammatory response syndrome ( sirs ) , sepsis , severe sepsis , and septic shock were adapted from previous recommendations and studies [ 6 , 811 ] . severity of diseases was classified as sepsis , severe sepsis , and septic shock according to previous definitions and apache ii scores , determined by the highest abnormal data within 24 hours of sepsis onset . timing of the diagnosis of sepsis was defined as the time when positive blood culture was performed , and then signs and symptoms of sepsis of these patients must be present when their clinical status were retrieved from the patient record chart at that time point . hence , the patients with subsequent positive blood cultures and presence of clinical criteria of sepsis , severe sepsis , or septic shock were enrolled in our study . timing of first and 24-hour dose antimicrobial therapy , defined as duration from diagnosis of sepsis to antimicrobials administration , retrieved from drug - prescribing charts . appropriate antimicrobial therapy was defined as the isolated bacteria being susceptible to at least one of the antimicrobials empirically administered as the first dose or 24 hours later . the primary objectives were to determine the prevalence of first - dose and 24-hour inappropriate empiric antimicrobial therapy in septic patients with bacteremia and its impact on 28-day and overall mortality rates . the secondary objectives were to define the appropriate timing of antimicrobial administration and factors associated with inappropriate antimicrobial therapy and high mortality rate in bacteremic patients with sepsis . descriptive statistics were implemented to summarize the patients ' characteristics as mean ( sd ) or proportions . multiple logistic regression analysis of independent factors associated with 28-day and overall mortality rates and factors related to inappropriate empirical antimicrobial treatment of bacteremia was performed . the mean ( sd ) age of septic patients was 63.5 ( 17.2 ) years , with 49.8% male patients . the common comorbidities were diabetes mellitus ( 31.0% ) , immunosuppressive therapy ( 29.3% ) , reduced mobility ( 29.7% ) , liver failure ( 21.8% ) , congestive heart failure ( 21.8% ) , chronic kidney disease ( 18.3% ) , and hematologic malignancy ( 18.2% ) ( table 1 ) . types of infection were community - acquired ( 27.5% ) , healthcare - associated ( 37.1% ) , and nosocomial infections ( 35.4% ) . septic shock and severe sepsis were present in 61.1% and 25.3% of the enrolled patients . the common sites of infection were respiratory tract ( 32.8% ) , gastrointestinal tract ( 23.6% ) , and genitourinary tract ( 20.5% ) ( table 2 ) . the most commonly identified bloodstream pathogens were gram - negative bacteria ( 72.5% ) as follows : escherichia coli ( 28.2% ) , klebsiella pneumoniae ( 12.7% ) , and acinetobacter spp . gram - positive bacteria were found in 27.5% of cases , including methicillin - susceptible s. aureus ( mssa ) ( 8.7% ) , methicillin - resistant s. aureus ( mrsa ) ( 4.8% ) , and streptococcus group d ( 3.5% ) . drug - resistant gram - positive bacteria , gram - negative bacteria , esbl - producing organism , and eskape organism were observed in 7.4% , 32.3% , 15.7% , and 73.8% of the isolates , respectively ( table 3 ) . antimicrobials frequently administered were cephalosporin ( 57.6% ) , carbapenem ( 23.1% ) , beta - lactam / beta - lactamase inhibitor ( 12.2% ) , vancomycin ( 11.4% ) , aminoglycosides ( 7.4% ) , fluoroquinolones ( 8.3% ) , and colistin ( 4.8% ) . only colistin significantly associated with appropriate first - dose antimicrobial therapy ( 6.8% versus 0% , p < 0.05 ) ( table 4 ) . in general , the 28-day and overall mortality rates were 62.4% and 70.3% , respectively . inappropriate first - dose and 24-hour empiric antimicrobial therapies were consequently noted in 68 cases ( 29.7% ) and 58 cases ( 25.3% ) of the patients . at 24 hours of diagnosis , one case in the appropriate group and 8 cases in the inappropriate group were still treated with inappropriate antimicrobials ( figure 1 ) . when compared to appropriate antimicrobial therapy , inappropriate first - dose antimicrobial therapy tended to have higher 28-day and overall mortality rates ( 67.6% versus 60.2% ( p = 0.301 ) and 75.0% versus 68.3% ( p = 0.345 ) , resp . ) additionally , inappropriate 24-hour empirical antimicrobials were also likely to have higher 28-day and overall mortality rates ( 65.5 versus 61.4% ( p = 0.64 ) and 74.1 versus 69.0% ( p = 0.51 ) , resp . ) univariate analysis of factors associated with 28-day and overall hospital mortalities in all sepsis patients was shown in table 5 . nosocomial infection ( 37.0% ) appeared to have significantly higher inappropriate antimicrobials than community - acquired ( 14.3% ) and healthcare - associated infection ( 22.3% , p = 0.004 ) . , pseudomonas aeruginosa , and esbl - producing organisms ) were significantly related to inappropriate first - dose empirical antimicrobials ( 64.7% versus 35.3% , p = 0.002 and 62.2% versus 37.8% , p = 0.001 , resp . ) subgroup analyses of drug - resistant organisms also showed a significant relation with the inappropriateness of first - dose antimicrobial therapies , such as acinetobacter spp . ( 60.7% versus 39.3% , p < 0.001 ) , eskape organism ( enterococcus faecium , staphylococcus aureus , klebsiella pneumoniae , acinetobacter baumannii , pseudomonas aeruginosa , enterobacter spp . moreover , both drug - resistant gram - positive and gram - negative bacteria significantly had higher inappropriate antimicrobial rates ( 64.7% versus 35.3% ; p = 0.002 and 62.2% versus 37.8% ; p = 0.001 , resp . ) patients with genitourinary tract and skin and soft tissue infections appeared to have lower rate of inappropriate antimicrobial therapy than those with infections at other sites ( 57.4% versus 42.6% ; p = 0.048 and 94.4% versus 5.6% ; p = 0.016 , resp . ) from multivariate analysis , factors significantly associated with the inappropriateness of first - dose empirical antimicrobials included drug - resistant gram - positive organism ( or 6.03 , 95% ci 2.3515.43 ) , drug - resistant gram - negative organism ( or 10.76 , 95% ci 3.9829.09 ) , leukopenia ( or 2.45 , 95% ci 1.185.08 ) , neutropenia ( or 2.94 , 95% ci 1.167.14 ) , and platelet < 100,000 per mm ( or 2.42 , 95% ci 1.204.88 ) ( table 6 ) . at hospital discharge , patients with inappropriate initial empiric antimicrobial therapy had a trend towards higher mortality rate than those with appropriate empiric antimicrobial therapy ( 75.0% versus 68.3% ; p = 0.345 ) . in addition , the first - dose and 24-hour inappropriate antimicrobial therapy resulted in shorter median survival duration than the appropriate antimicrobial therapy administration ( 214.7 hours versus 301.1 hours , p = 0.86 and 180.2 hours versus 301.1 hours , p = 0.85 , resp . ) multivariate analysis of factors potentially associated with fatal outcome revealed that the inappropriateness of the first - dose antimicrobial therapy administration remained the most strongly correlated with high mortality ( or 2.52 ; 95% ci 1.016.32 ; p = 0.049 ) among all the factors assessed . other factors related to overall mortality included the timing from diagnosis until the first - dose antimicrobial therapy , septic shock status , presence of congestive heart failure , age > 65 years , apache ii score > 25 , platelet count < 100,000 per mm , and serum albumin < 3.2 g / dl ( table 7 ) . antimicrobials were initiated within 1 hour and 6 hours after onset of sepsis in 20.1% and 64.2% of cases , respectively . the mean ( sd ) length of time from onset of diagnosis to the first - dose antimicrobial therapy was 9.57 ( 25.33 ) hours in all cases and 7.00 ( 16.61 ) hours in those with appropriate antimicrobial therapy . the patients with appropriate antimicrobial therapy were significantly administered to receive antimicrobials earlier than those treated with inappropriate antimicrobials ( 7.00 ( 16.60 ) versus 15.66 ( 38.37 ) hours , p = 0.018 ) . all patients and those with appropriate antimicrobial therapy in the 28-day and overall hospital mortality group were likely to have delayed first - dose antimicrobials . in all patients , the mean ( sd ) duration from onset of sepsis to appropriate antimicrobial therapy of the 28-day and overall mortality group was longer than the 28-day and overall survival group ( 7.09 ( 18.61 ) versus 6.85 ( 13.15 ) hours , p = 0.931 and 7.14 ( 18.48 ) versus 6.68 ( 11.73 ) hours , p = 0.931 , resp . ) in patients with appropriate antimicrobials , the mean ( sd ) duration from onset of sepsis to appropriate antimicrobial therapy in the 28-day and overall mortality group was also more prolonged than the 28-day and overall survival group ( 9.89 ( 27.69 ) versus 9.03 ( 20.99 ) hours , p = 0.806 and 10.27 ( 27.30 ) versus 7.91 ( 19.99 ) hours , p = 0.522 , resp . ) there was significant relationship between the delayed timing from diagnosis to the administration of first - dose antimicrobials and the overall mortality . the mortality rates in patients with antimicrobials less than 1 hour , from 1 hour to 6 hours , and more than 6 hours after the diagnosis of sepsis were 63.0% , 65.3% , and 80.5% , respectively ( p = 0.04 ) . also , higher overall mortality was demonstrated after the delayed antimicrobial initiation over 1 hour ( 72.1 versus 48.7% , p = 0.30 ) . our data strongly supported the notion that the inappropriate antimicrobials and the delayed administration of appropriate antimicrobial therapy could substantially increase the mortality rate in septic patients with bacteremia . we reported herein that the inappropriate empirical antimicrobial therapy was frequent among bacteremic patients with high overall mortality and a tendency towards higher mortality rate than the appropriate antimicrobial therapy . this similar high percentages of inappropriate antimicrobial therapy , ranging from 20% to 46.5% , were also described by several previous reports , which resulted in high mortality rate from 35% to 70% [ 1 , 2 , 1727 ] . however , the overall mortality rate in our study was higher than the one conducted in the same hospital during 2007 . the difference in mortality rates between these two studies could be explained by distinctive population groups , higher percentages of healthcare - related and nosocomial infections , and more severity of infections in the latter study . the study conducted in 2007 enrolled patients with clinical entities of sepsis , severe sepsis , or septic shock regardless of whether they were bacteremic or not at the entry point . meanwhile , the study in 2009 recruited patients with bacteremia and clinical sepsis , severe sepsis , or septic shock . the population study groups in 2007 consisted of sepsis patients with bacteremia ( 40.8% ) , community - acquired infection ( 61.2% ) , and septic shock ( 38.8% ) . on the contrary , our study populations were bacteremic patients with healthcare - related infection ( 37.1% ) , nosocomial infection ( 35.4% ) , and septic shock ( 61.1% ) . hence , there was more severity of infections in our study population groups that led to higher mortality rate . other factors pertaining to higher mortality rate included microbiological factors such as the presence of frequent healthcare - associated and nosocomial infections , and drug resistant organisms . despite multiple risk factors for high mortality in sepsis patients , the inappropriate first - dose empirical antimicrobial therapy still significantly increased 6.7% of mortality rate . , demonstrating higher mortality among septic shock patients with inappropriate empirical antimicrobials ( 89.7% versus 48.0% ) . nevertheless , the impact of inappropriate empirical antimicrobials in patients with less comorbidities and severity of sepsis remained to be elucidated . there were also significant variations in the inappropriateness of initial antimicrobial therapy among infection sites , of which genitourinary and skin and soft tissue infections seemed to have appreciably better empirical antimicrobial therapies . that inappropriate antimicrobial therapy in gram - negative septicemia had higher 30-day mortality rate ( 38 versus 27% , p < 0.05 ) . the thai prevalence of esbl - producing e. coli and esbl - producing k. pneumoniae in healthcare - associated infections was 13.2% and 12.7% , respectively . from our study , esbl - producing enterobacteriacae accounted for 15.7% of bacteremia , which considerably associated with both first - dose and 24-hour inappropriate empirical antimicrobials . , multidrug - resistant strains as common causes of nosocomial infection and important antimicrobial - resistant organism [ 32 , 41 , 42 ] , was 12.2% frequently found and significantly associated with inappropriate antimicrobials . should be recognized , including icu admission , previous colonization with methicillin - resistant staphylococcus aureus ( mrsa ) , beta - lactam , beta - lactam / beta - lactamase inhibitor and carbapenem antibiotics use , bedridden status , previous intensive care admission , central venous catheter , surgery , mechanical ventilation , hemodialysis , and malignancy [ 4346 ] . nevertheless , it showed in our study that colistin was only significantly associated with appropriate antimicrobial therapy . particularly , in the cases of critically ill patients , septic shock , high apache ii score , leukopenia , respiratory or intra - abdominal infections , and nosocomial infection , physicians should be alert with particular attention on choosing appropriate initial empiric antimicrobials due to high mortality in these subgroups . otherwise , better empiric coverage of antimicrobial therapy could be obtained through immediate consultation with infectious disease specialists , such as i d fast track ( like stroke or st elevation myocardial infarction fast track ) or antibiotic management teams in some institutions [ 6365 ] . nevertheless , there were the controversies of the impact on early and appropriate antimicrobial therapy on lower mortality in some studies [ 6668 ] . the surviving sepsis campaign 's guideline recommended that the appropriate antimicrobial therapy should be administered within one hour of severe sepsis or septic shock recognition . the recent study confirmed the significantly higher mortality associated with duration from triage or qualification for early goal - directed therapy more than one hour . however , there was statistical difference in mortality if the first - dose antimicrobial therapy was delayed more than three hours . to this reason , the appropriate antimicrobial therapy should be immediately initiated in patients with diagnosis of sepsis to achieve lower mortality . the limitations of this study were retrospective methodology and the inclusion of patients only from medical wards of an academic referral center / tertiary medical center , which may not reflect the entire population in other departments or hospitals . inappropriate empirical antimicrobial therapy frequently occurs in about 29.7% of septic bacteremic patients , and it is associated with 75% mortality rate . furthermore , the mortality rate could be significantly increased by the delayed timing of first - dose antimicrobial therapy more than 3 hours after the diagnosis of sepsis . thus , more efforts must be employed to increase the appropriateness of initial antimicrobial therapy and decrease timing from sepsis diagnosis to initiation of antimicrobials in a bid to reduce the mortality in patients with sepsis .